Polyamines in Rhizobium, Bradyrhizobium, Azorhizobium and Argobacterium

Abstract Eighteen strains of Rhizobium including four species, R. leguminosarum, R. meliloti, R. loti and R. fredii , nine strains of Bradyrhizobium japonicum and three strains of Azorhizobium caulinodans contained putrescine and honospermidine as major polyamines. All these nodulating N₂-fixing rhizobia lack spermidine. Spermidine and cadaverine were present only in a limited number of R. meliloti and B. japonicum . Polymanine-synthetic activity was not affected by the differences in ability to produce phytoxine (rhizobitoxine and dihydrorhizobitoxine) H₂-uptake-hydrogenation in the organisms. Putrescine and homospermidine were major polyamined in a strain of Agrobacterium rhizogenes . All the eight strains of Agrobacterium tumefaciens as well as A. rubi, A. radiobacter and two other strains of A. rhizogenes contained putrescine and spermidine as major polyamines and homospermidine and spermine (and thermospermine) as minor polyamines.     

Isolation and identification of a N2-fixing zoogloea-forming bacterium from kallar grass histoplane

Semi-solid medium was used to isolate an aerobic, N₂-fixing (C₂H₂-reducing), H₂-utilizing bacterium from the roots of kallar grass ( Leptochloa fusca ). The organism was identified by morphological, cultural and biochemical characteristics. The N₂-fixing, zoogloeal floc-forming isolate described here is a new species.     

New type of N2-fixing unicellular cyanobacterium (blue-green alga)

Abstract A new N₂-fixing unicellular cyanobacterium identified as a Synechococcus sp. was isolated and purified as an axenic culture. It fixed N₂ aerobically either under continuous illumination or in alternating light-dark cycle. The N₂-fixing properties of the new isolate and Gloeocapsa are discussed.     

Inorganic nitrogen regulation of glutamate uptake in the cyanobacterium Nostoc muscorum

In Nostoc muscorum (Anabaena ATCC 27893) glutamate was not metabolised as a fixed nitrogen source, rather it functioned as an inhibitor of growth. The latter effect was nitrogen source specific and occurred in N₂-fixing cultures but not in cultures assimilating nitrate or ammonium. NO₃^--grown cultures lacked heterocysts and nitrogenase activity and showed a nearly 50% reduction in glutamate uptake rates, as well as in the final extent of glutamate taken up, compared to N₂-fixing or nitrogen-limited control cultures. NH₄⁺-grown cultures showed a similar response, except that the reduction in glutamate uptake rates and the final exten of glutamate taken up was over 80%. The present results suggest a relation between nitrate/ammounium nitrogen-dependent inhibition of glutamate uptake, probably via repression of the glutamate transport system, and glutamate toxicity.     

Carbon Costs Associated with N2 Fixation in Vicia faba L and Pisum sativum 1. over a 14-Day Period

Abstract: Long-term (14 days) carbon costs of N₂ fixation were studied in pot trials. For this purpose the CO₂ release from the root space of nodulated and non-nodulated (urea nourished) Vicia faba L. and Pisum sativum L. plants was compared and related to the amount of fixed or assimilated N. Additional measurements of shoot CO₂ exchange and dry matter increment were carried out in order to calculate the overall carbon balance. The carbon costs for N₂ fixation in Vicia faba 1. (2.87 mg C/mg N_fiX) were higher than in Pisum sativum L. (2.03 mg C/mg N_fix). However, the better carbon efficiency in Pisum sativum 1. did not lead to a better growth performance compared to Vicia faba L. Vicia faba L. compensated for the carbon and energy expenditure by more intensive photosynthesis in the N₂-fixing treatment. This was not the case with Pisum sativum L., where the carbon balance indicates that the carbon costs of N₂ fixation restricted root growth. It is proposed that low carbon costs for N₂ fixation indicate an adaptation to a critical carbon supply of roots and nodules, e.g., during the pod-filling of grain legumes.     

Photosynthetic oxygen evolution within Sesbania rostrata stem nodules

The tropical wetland legume, Sesbania rostrata Brem. forms N₂-fixing nodules along its stem and on its roots after infection by Azorhizobium caulinodans . The N₂-fixing tissue is surrounded by a cortex of uninfected cells which, in the stem nodules (but not the root nodules), contain chloroplasts. The photosynthetic competence of these chloroplasts was assessed through a novel technique involving image analysis of chlorophyll a fluorescence. Calculation of the quantum efficiency of photosystem II (PS II) photochemistry from these images indicated that most of the chloroplasts with potential for non-cyclic photosynthetic electron transport were concentrated within the mid- and inner-cortex, close to the edge of the N₂-fixing tissue. PS II activity in the cortical cells was confirmed in vivo using O₂-specific microelectrodes which showed that the concentration of O₂ (pO₂) in the outer cortex could rise from less than 1% up to 23.4% upon increased irradiance of the nodule, but that the pO₂ of the inner cortex and infected tissue remained less than 0.0025%. Nitrogenase activity of stem nodules, as measured using a flow-through acetylene reduction assay (no H₂ evolution was evident), showed a reversible increase of 28% upon exposure of the nodules to supplemental light. This increase resembled that obtained with stem nodules upon their exposure to an external pO₂ of 40%.     

Nitrogen-fixing legumes and silvigenesis in a rain forest in French Guiana: a taxonomic and ecological approach

The nodulation status and nodule morphology of 62 taxa of Leguminosae in a rain forest in French Guiana are reported according to the taxonomy of the family. The N₂-fixing species are then fitted into 'functional groups' according to their behaviour towards illumination, in order to evaluate their importance in the global dynamics of the stand. The results showed that 67% of the observed species were nodulated (50, 71 and 77% of the Caesalpiniaceae, Mimosaceae and Papilionaceae, respectively). In the Caesalpiniaceae, nodule-like structures were reported in the genus Crudia and in the species Senna quinquangulata , although this needs to be confirmed. All the nodules studied in this subfamily were astragaloid and mucunoid. In the Mimosaceae, the ability of a new genus ( Balizia ) to form nodules was reported, as well as nodulation on aerial roots in Inga stipularis . The nodules studied were mainly mucunoid. In the Papilionaceae, nodulation on aerial roots in Poecilanthe hostmannii and on conventional roots of the genus Paramachaerium were reported for the first time. All types of nodular structures were found in this subfamily but the structures were quite uniform at the tribal level. These are consistent with suggestions that nodule morphology has a taxonomic value. Eight functional groups of N₂-fixing species are proposed, ranging from light dependance to shade tolerance. These results indicate the important role played by N₂-fixing species in the global dynamics of the stand and that N inputs by N₂ fixation were continuous along the gradient of energetic resources that characterizes the silvigenetic process. The interactions between the photosynthetic capacities of the species and the ability to fix N₂ in low light conditions are discussed.     

Differences in nitrogen metabolism of Faidherbia albida and other N2-fixing tropical woody acacias reflect habitat water availability

The activities of nitrate reductase and glutamine synthetase were evaluated in young plants of Faidherbia albida , a tropical woody legume, fed with different N sources under hydroponic conditions. Results showed that assimilation of both NO₃⁻ and NH₄⁺ preferentially took place in shoots. A basal amount of nitrate reductase activity was detected in shoots of plants grown with an NO₃⁻-free solution or placed under N₂-fixing conditions, and also in nodules of N₂-fixing plants. This strongly suggests that constitutive nitrate reductase activity is present in these organs. Analyses of the soluble nitrogenous content showed that the major form of N in the different organs was α-amino acids (particularly amides), irrespective of the N status of the culture conditions. The same result was obtained for nodulated plants grown in local sandy soil. In this case, amide-N generally accounted for more than 40% of the total soluble N. This was especially true in nodules. Ureide-N never exceeded 9% of the total soluble N and did not appear to increase with increasing nodule nitrogenase activity. Amides were also predominant in three N₂-fixing Sahelian acacias ( Acacia seyal , A. nilotica and A. tortilis ), showing that F. albida does not differ from Sahelian Acacia in terms of the metabolism of fixed N. However, like another Sahelian acacia growing preferentially near water ( A. nilotica ), F. albida can be distinguished from acacias growing strictly in arid zones ( A. seyal and A. tortilis ) in terms of initial growth, water and nitrate management.     

Development and N2-Fixing Activity of the Benthic Microbial Community in Transplanted Spartina alterniflora Marshes in North Carolina

Growth and maturation of transplanted salt marshes is often limited by the availability of nitrogen (N). We examined the role of N₂-fixing benthic microbial assemblages (microalgae and associated bacteria) in two restored marshes (1-year-old and 6-year-old marsh) and a natural salt marsh in the Newport River Estuary, North Carolina. Benthic N₂ fixation (nitrogenase activity, NA), chlorophyll a (Chl a ) concentration, Spartina alterniflora (smooth cordgrass) stem counts, and sediment organic matter content were determined in the three marshes. Significant differences were observed between sites for both Chl a and NA. The 1-year-old marsh always exhibited the highest levels of NA and Chl a . Sediment organic matter content was lowest in the 1-year-old marsh (∼2%), intermediate in the 6-year-old marsh (∼5%), and highest in the natural marsh (∼10%). Carbon and nitrogen analyses were also performed on the 1-year-old marsh sediments, which were depleted in N. A positive correlation was observed between surface sediment N and Chl a . Remineralized, microbially derived N may provide growth-limiting inorganic N to Spartina transplants. N₂-fixing microbial assemblages in the 1-year-old marsh may also be an important food source for marsh infauna. Benthic N₂-fixing microbial assemblages play a key role in the N economy of restored salt marshes.     

15N natural abundance during early and late succession in a middle-European dry acidic grassland

δ¹⁵N and total nitrogen content of above- and belowground tissues of 13 plant species from two successional stages (open pioneer community and ruderal grass stage) of a dry acidic grassland in Southern Germany were analysed, in order to evaluate whether resource use partitioning by niche separation and N input by N₂-fixing legumes are potential determinants for species coexistence and successional changes. Within each stage, plants from plots with different legume cover were compared. Soil inorganic N content, total plant biomass and δ¹⁵N values of bulk plant material were significantly lower in the pioneer stage than in the ruderal grass community. The observed δ¹⁵N differences were rather species- than site-specific. Within both stages, there were also species-specific differences in isotopic composition between above- and belowground plant dry matter. Species-specific δ¹⁵N signatures may theoretically be explained by (i) isotopic fractionation during microbial-mediated soil N transformations; (ii) isotopic fractionation during plant N uptake or fractionation during plant–mycorrhiza transfer processes; (iii) differences in metabolic pathways and isotopic fractionation within the plant; or (iv) partitioning of available N resources (or pools) among plant groups or differential use of the same resources by different species, which seems to be the most probable route in the present case. A significant influence of N₂-fixing legumes on the N balance of the surrounding plant community was not detectable. This was confirmed by the results of an independent in situ removal experiment, showing that after 3 years there were no measurable differences in the frequency distribution between plots with and without N₂-fixing legumes.     

Immunolocalization of the uptake hydrogenase in the marine cyanobacterium Lyngbya majuscula CCAP 1446/4 and two Nostoc strains

In N₂-fixing cyanobacteria, the reduction of N₂ to NH₃ is coupled with the production of molecular hydrogen, which is rapidly consumed by an uptake hydrogenase, an enzyme that is present in almost all diazotrophic cyanobacteria. The cellular and subcellular localization of the cyanobacterial uptake hydrogenase remains uncertain, and it is definitely strain dependent. Previous studies focused mainly on heterocystous cyanobacteria and used heterologous antisera. The present work represents the first effort to establish the subcellular localization of the uptake hydrogenase in a N₂-fixing filamentous nonheterocystous cyanobacterium, Lyngbya majuscula CCAP 1446/4, using the first antiserum produced against a cyanobacterial uptake hydrogenase. The data obtained revealed higher specific labelling associated with the thylakoid membranes of L. majuscula , reinforcing the idea that the cyanobacterial uptake hydrogenase is indeed a membrane-bound protein. For comparative purposes, the localization of the uptake hydrogenase was also investigated in two distinct heterocystous cyanobacterial strains, and while in Nostoc sp. PCC 7120 the labelling was only observed in the heterocysts, in Nostoc punctiforme , the presence of uptake hydrogenase antigens was detected in both the vegetative cells and heterocysts, corresponding most probably to an inactive and an active form of the enzyme.     

Aerobic nitrogen fixation is confined to a subset of cells in the non-heterocystous cyanobacterium Symploca PCC 8002

Symploca PCC 8002 Kützing is a filamentous cyanobacterium that lacks the specialized cells, known as heterocysts, that protect nitrogenase from O₂ in most aerobic N₂-fixing cyanobacteria. Nevertheless, Symploca is able to carry out N₂ fixation in the light under aerobic conditions. When cultures were grown under light/dark cycles, nitrogenase activity commenced and increased in the light phase and declined towards zero in the dark. Immunolocalization of dinitrogenase reductase in sectioned Symploca trichomes showed that the enzyme was present only in 9% of the cells. These cells lacked any obvious mechanical protection against atmospheric O₂ and their ultrastructural characteristics were similar to those of cells that did not contain any dinitrogenase reductase. The nitrogenase-containing cells possessed carboxysomes that were rich in ribulose-1,5-bisphosphate carboxylase/oxygenase and phycoerythrin, a light harvesting pigment of PS II. This indicates that these cells had a capacity for both N₂ fixation and photosynthesis. The significance of the localization pattern for dinitrogenase reductase is discussed in the context of N₂ fixation in Symploca PCC 8002.     

Effects of mycorrhizal colonization on biomass production and nitrogen fixation of black locust (Robinia pseudoacacia) seedlings grown under elevated atmospheric carbon dioxide

Interactive effects of elevated atmospheric CO₂ and arbuscular mycorrhizal (AM) fungi on biomass production and N₂ fixation were investigated using black locust ( Robinia pseudoacacia ). Seedlings were grown in growth chambers maintained at either 350 μmol mol⁻¹ or 710 μmol mol⁻¹ CO₂. Seedlings were inoculated with Rhizobium spp. and were grown with or without AM fungi. The ¹⁵N isotope dilution method was used to determine N source partitioning between N₂ fixation and inorganic fertilizer uptake. Elevated atmospheric CO₂ significantly increased the percentage of fine roots that were colonized by AM fungi. Mycorrhizal seedlings grown under elevated CO₂ had the greatest overall plant biomass production, nodulation, N and P content, and root N absorption. Additionally, elevated CO₂ levels enhanced nodule and root mass production, as well as N₂ fixation rates, of non- mycorrhizal seedlings. However, the relative response of biomass production to CO₂ enrichment was greater in non-mycorrhizal seedlings than in mycorrhizal seedlings. This study provides strong evidence that arbuscular mycorrhizal fungi play an important role in the extent to which plant nutrition of symbiotic N₂-fixing tree species is affected by enriched atmospheric CO₂.     

Oxygen and the regulation of nitrogen fixation in legume nodules

In N₂-fixing legume nodules, O₂ is required in large amounts for aerobic respiration, yet nitrogenase, the bacterial enzyme that fixes N₂, is O₂ labile. A high rate of O₂ consumptition and a cortical barrier to gas diffusion work together to maintain a low, non-inhibitory O₂ concentration in the central, infected zone of the nodule. At this low O₂ concentration, cytosolic leghemoglobin is required to facilitate the diffusion of O₂ through the infected cell to the bacteria. The resistance of the cortical diffusion barrier is variable and is used by legume nodules to regulate the O₂ concentration in the infected cells such that it limits aerobic respiration and N₂ fixation at all times. The resistance of the diffusion barrier and therefore the degree of O₂ limitation seems to be regulated in response to changes in the O₂ concentration of the central infected zone, the supply of phloem sap to the nodule, and the rate of N assimilation into the end products of fixation.     

Nitrogen fixation (N-15 dilution) with soybeans under Thai field conditions. IV. Effect of N addition and Bradyrhizobium japonicum inoculation in soils with indigenous B. japonicum populations

The effects of Bradyrhizobium japonicum inoculation and pre-plant additions of N fertilizer on soybean ( Glycine max L. Merrill) yields and levels of N₂ fixation were studied under field conditions at two sites in Thailand. Bacterial inoculants were composed of B. japonicum strains selected for high N₂ fixation levels in Thai soils. Nitrogen fertilizer addition rates used were from 0 to 250 kg N/ha in 50 kg N/ha increments. At the Chiang Mai site in northern Thailand, bacterial inoculation increased nodule weights on plants receiving 100 kg N/ha or less. Increases in nodule parameters due to inoculation were evident at 45 d after planting (DAP) but disappeared by 60 DAP. Addition of N fertilizers decreased the incidence of nodulation and sap ureide contents and decreased the contribution of N₂ fixation to the N content of plants at maturity as measured by N-15 isotope dilution methods. At the Kampang Saen site in central Thailand, bacterial inoculation had significant positive effects on nodule numbers and weights, ARA, sap ureide contents and levels of N₂ fixed as measured by N-15 isotope dilution methods. Addition of N fertilizers at this site also reduced the effectiveness of N₂-fixing symbioses. It was concluded that small additions of N fertilizer added before planting did not significantly decrease N₂ fixation levels, but did have a significant positive effect on plant growth. Larger N additions would reduce N₂ fixation levels in excess of the benefits of adding more N in chemical form.     

Fixation of molecular nitrogen by Methanosarcina barkeri

Abstract Methanosarcina barkeri cells were observed in ammonia-free anaerobic acetate enrichments for sulfate-reducing bacteria. The capacity of Methanosarcina to grow diazotrophically was proved with a pure culture in mineral media with methanol. The cell yields with N₂ or NH₄⁺ ions as nitrogen source were 2.2 g and 6.1 g dry weight, respectively, per mol of methanol. Growth experiments with ¹⁵N₂ revealed that 84% of the cell nitrogen was derived from N₂. Acetylene was highly toxic to Methanosarcina and only reduced at concentrations lower than 100 μmol dissolved per 1 of medium. Assimilation of N₂ and reduction of acetylene were inhibited by NH₄⁺ ions. The experiments show that N₂ fixation occurs not only in eubacteria but also in archaebacteria. The ecological significance of diazotrophic growth of Methanosarcina is discussed.     

Effects of exogenous application and stem infusion of ascorbate on soybean (Glycine max) root nodules

Numerous biochemical and physiological studies have demonstrated the importance of ascorbate (ASC) as a reducing agent and antioxidant in higher plant metabolism. Of special note is the capacity of ASC to eliminate damaging activated oxygen species (AOS) including O₂^−· and H₂O₂. N₂-fixing legume nodules are especially vulnerable to oxidative damage because they contain large amounts of leghaemoglobin which produces AOS through spontaneous autoxidation; thus, ASC and other components of the ascorbate–reduced glutathione (ASC–GSH) pathway are critical antioxidants in nodules. In order to establish a meaningful correlation between concentrations of ASC and capacity for N₂ fixation in legume root nodules, soybean ( Glycine max ) plants were treated with excess ASC via exogenous irrigation or continuous intravascular infusion through needles inserted directly into plant stems. Treatment with ASC led to striking increases in nitrogenase activity (acetylene reduction), nodule leghaemoglobin content, and activity of ASC peroxidase, a key antioxidant enzyme. The concentration of lipid peroxides, which are indicators of oxidative damage and onset of senescence, was decreased in ASC-treated nodules. These results support the conclusion that ASC is critical for N₂ fixation and that elevated ASC allows nodules to maintain a greater capacity to fix N₂ over longer periods.     

Growth and the efficiency of root respiration of Pisum sativum as dependent on the source of nitrogen

Growth and efficiency of root respiration were investigated in Pisum sativum L. cv. Alaska and cv. Rondo. Plants were grown in culture solutions, either in symbiosis with Rhizobium leguminosanm , or with an abundant supply of nitrate or ammonium and completely lacking nodules. In comparison with plants utilizing nitrate or ammonium, Ni-fixing plants showed lower rates of dry matter and nitrogen accumulation, as well as lower rates of total and cytochrome-mediated root respiration. Rates of shoot dry matter accumulation and root respiration in plants utilizing ammonium were lower than in plants utilizing nitrate. The efficiency of root respiration was high in N₂-fixing plants, as indicated by a low activity of the SHAM-sensitive, alternative, non-phosphorylating pathway. In nitrate and ammonium grown plants of cv. Alaska, the efficiency of root respiration was about the same, and in both cases lower than in N₂-fixing plants. The efficiency of root respiration in non-symbiotically grown pea plants was generally higher than in many non-legumes. Comparison of the ATP costs of synthesis of root dry matter for different N-sources was complicated by large differences in relative growth rate of the root and in shoot to root ratio between N-treatments. A quantitative correction of the ATP production during synthesis of root dry matter for differences in shoot to root ratio and root maintenance respiration has been made. It is concluded that ATP costs of root dry matter production are highest in the case of N₂-fixing plants. In plants utilizing ammonium, ATP costs of synthesis of root dry matter were slightly lower than in plants utilizing nitrate. The physiological significance of the alternative pathway in root metabolism is discussed in relation to the assimilation of different sources of nitrogen.