Nitrogen Fixation System Research Articles

Trials to create artificial nitrogen-fixing symbioses and associations using in vitro methods: An outlook

Biological nitrogen fixation is the most important process in which some prokaryotic organisms fix N2 into ammonium. From an agricultural standpoint, biological nitrogen fixation (BNF) is critical because industrial production of nitrogen fertilizers seldom meets agricultural demands. To increase the BNF is one of the main challenges for the future. There are different possibilities for extending biological nitrogen fixation to the economically important plants. One of the possibilities is to create new artificial systems between diazotrophic bacteria and different higher plants. This is the main topic of the present review article which discusses the establishment of new associative and/or symbiotic systems, via introduction of diazotrophic bacteria into the roots by different methods; and incorporation of nitrogen-fixing bacteria in the entire plant by in vitro methods, through the establishment of intracellular endosymbioses via induced uptake of bacteria by plant protoplasts (endocytobiosis), and establishment of intercellular associations by forced introduction of bacteria into the plant tissues (exocytobiosis). The common characteristic of the methods to create artificial plant-microbe systems for atmospheric nitrogen fixation is the use of in vitro plant systems: cells, tissues and organ cultures. The review pays particular attention to new bacterial inoculation procedures for introduction of the diazotrophic bacteria inside the plant tissues.

Reaction of Nitrogen(I) Oxide with Nitrogen-fixing Systems on the Basis of Lithium, Chlorotrimethylsilane, and Transition Metal Compounds

Reaction of nitrogen(I) oxide with nitrogen-fixing systems Li/Me3SiCl/MCln (MCl4n = CrCl3, CoCl2, Cp2TiCl2, FeCl3, CuCl2). In these systems nitrogen(I) oxide, molecular nitrogen, and air nitrogen undergo reductive silylation to tris(trimethylsilyl)amine. The efficiency of the process was estimated by the molar ratio of the tris(trimethylsilyl)amine formed to metal chloride MClnn. The reaction of N2O with the nitrogen-fixing systems including CoCl2 and Cp2TiCl2 is not exhausted by the reduction of the former to molecular nitrogen and its subsequent fixation by transition metal complexes.

Review: Dark-induced changes in legume nodule functioning

Exposure of nodulated leguminous plants to prolonged periods of continuous darkness has been used as a convenient tool to investigate host plant control on nitrogen fixing systems in legume root nodules. Foliar dark treatment of plants results in a rapid decline in N2 -fixation in terms of acetylene reduction activity (ARA) and predisposes the nodules to metabolic and structural senescence. After 2 d of darkness, a significant decrease is seen in nitrogenase (N2 -ase) proteins of common bean nodules. The effect of dark treatments on nodule respiration varies with plant species. A variable decrease in nodule carbohydrates is observed in different plant species under dark treatments, but no direct correlation between N2 -ase activity and gross levels of carbohydrates present in the nodules has been detected. Usually nodule leghemoglobin (Lb) shows a decrease of variable intensity depending on plant species. The mRNA of Lb, sucrose synthase and glutamine synthetase shows a significant decline within 24 h of complete darkness. Dark-induced acceleration of proteolytic activity and decreased plant growth are reflected in decreased nodule proteins and accumulation of free amino acids following a drop in ARA. Antioxidants such as ascorbic acid and glutathione, along with the enzymes of their oxidation–reduction cycle, show a considerable decrease in their content and activity in nodules from dark-treated plants. Among H2 O2 scavengers, nodule catalase activity decreases in most of the plants studied, but peroxidase activity shows an increase. Dark-induced adverse effects on N2 -fixation are completely or partially reversible on shifting the plants back to a normal light/dark regime. Significant changes in nodule ultrastructure are induced by dark treatment. Attempts have been made to explain the mechanisms underlying dark-induced changes in nodule functioning.

Polyamine-induced changes in symbiotic parameters of the Galega orientalis-Rhizobium galegae nitrogen-fixing system

Plants of goat's rue (Galega orientalis) inoculated with Rhizobium galegae strain HAMBI 540 were grown in the presence of putrescine (Put), spermidine (Spd) or spermine (Spm), and several symbiotic characteristics were investigated to delineate the influence of polyamines (PA) on this nitrogen-fixing system. All three PA exerted a concentration-dependent effect on the nodule parameters tested. The increment of nodulation ability and nodule biomass accumulation was extreme (from 2.4- to 4.0-fold) when plants were subjected to 10 and 50 μM of various PA. However, at 100 μM a negative effect was observed. The acetylene-reduction activity of nodulated roots was increased also in response to treatment with the lower PA concentrations. The level of nitrogenase activity supported by succinate was significantly higher in bacteroids isolated from PA-treated nodules than in bacteroids from control nodules. The symbiotic parameters were also dependent on the type of PA used; the most effective being the diamine Put, while Spm showed a smaller physiological effect with respect to the others. Polyamines altered the ultrastructure of Galega nodule infected cells. After treatment with these substances, pronounced changes in the relative volume of the main components of infected cells and their compartments were observed. The significance of the structural observations and morphometric analyses, their relationship to differences in nitrogen fixation and possible modes of action are discussed.

Long-term effects of nitrate on lucerne (Medicago sativa L.) nitrogen fixation is not influenced by the denitrification status of the microsymbiont

Studies on the inhibitory effects of combined nitrogen on biological nitrogen fixation in legume crops have been usually carried out after short-term nitrate treatments at high concentrations. As these treatments are quite different from field conditions, a study was conducted to evaluate the effects of the continuous presence of nitrate (0, 1, 5 and 10 mM) throughout three months on lucerne (Medicago sativa L.). Plants were grown in a greenhouse with perlite as substrate and were inoculated with a denitrifying Sinorhizobium meliloti strain (102-F-51) and a non-denitrifying strain (102-F-65). During the first 60 days of growth, the highest nitrate treatment resulted in a complete inhibition of the main symbiotic parameters (nodule initiation and development and specific nitrogen fixation) in plants inoculated with either strain. However, after 3 months of growth in the presence of nitrate, this inhibition was partly abolished, with a high number of new functioning nodules being formed. Acetylene reduction activity (ARA) of these plants was 70% of the control plants. As this process was observed in plants nodulated with either strain, it is concluded that this was not related to the denitrifying ability of the strain, but is an intrinsic property of the lucerne nitrogen fixing system. As legume plants usually grow under natural field conditions in the continuous presence of nitrate, the ability to use simultaneously nitrate and atmospheric nitrogen could be of adaptive and agronomic importance.

Reduction of carbon monoxide by molybdenum-containing hydroxide systems

The reduction of CO by the nitrogen-fixing systems Ti(OH)3−Mo(OH)3 and MgTi2O4−Mo(OH)3 was studied in aqueous and water-methanol media; in the latter,14CO was used as the reagent. The main reaction product is methanol, whose yield in the H2O−MeOH−KOH mixture is almost an, order of magnitude higher than that in an aqueous alkaline solution. The data obtained were compared to those for the reduction of N2

On the path to catalysts for the low-temperature ammonia synthesis

A nontraditional approach to the development of catalysts for low-temperature ammonia synthesis is considered. The approach is characterized by application of catalysts representing heterogeneous analogs of the known homogeneous nitrogen-fixing systems based on transition metal compounds and strong electron donors. The use of this approach led to the development of catalysts that considerably surpass in their activity (at atmospheric pressure) the industrial catalyst for the ammonia synthesis. Some of the developed catalysts are active in the formation of ammonia from dinitrogen and dihidrogen even at 110–150°C. The mechanisms of activation and hydrogenation of dinitrogen over these new catalysts are discussed.

Nitrogen fixation and the mass balances of carbon and nitrogen in ecosystems

Ecosystems with high rates of nitrogen fixation often have high loss rates through leaching or possibly denitrification. However, there is no formal theoretical context to examine why this should be the case nor of how nitrogen accumulates in such open systems. Here, we propose a simple model coupling nitrogen inputs and losses to carbon inputs and losses. The nitrogen balance of this model system depends on plant (nitrogen fixer) growth rate, its carrying capacity, N fixed/C fixed, residence time of nitrogen and carbon in biomass, litter decay rate, litter N/C, and fractional loss rate of mineralized nitrogen. The model predicts the requirements for equilibrium in a nitrogen-fixing system, and the conditions on nitrogen fixation and losses in order for the system to accumulate nitrogen and carbon. In particular, the accumulation of nitrogen and carbon in a nitrogen-fixing system depend on an interaction between residence time in vegetation and litter decay rate in soil. To reflect a possible increased uptake of soil nitrogen and decreased respiratory cost of symbiotic nitrogen fixers, the model was then modified so that fixation rate decreased and growth rate increased as nitrogen capital accumulated. These modifications had only small effects on carbon and nitrogen accumulation. This suggests that switching from uptake of atmospheric nitrogen to mineral soil nitrogen as nitrogen capital accumulates simply results in a trade-off between energetic limitations and soil nitrogen limitations to carbon and nitrogen accumulation. Experimental tests of the model are suggested.

Isolation and Characterization of an Oxygen Sensitive Mutant of Azorhizobium caulinodans

An oxygen sensitive mutant of Azorhizobium caulinodans strain IRBG 46 was isolated by NTG mutagenesis. It was defective in N2 fixation under 3% O2 level, while under 1% O2 it was almost as active as the parent strain IRBG 46. The mutant was also found to be a slow grower with reduced respiratory activity, low azide tolerance and no catalase activity. However, it did not differ from its parent strain with respect to nitrate respiration. Under symbiotic condition the mutant formed smaller, light green nodules as compared to bigger, dark green nodules formed by the wild type strain. The mutant was also defective in N2 fixation under symbiotic condition. Complementation analysis showed that the mutation might be in either fixL or fixJ gene which are involved in O2 regulation of nif/fix gene expression. A possible role of all these factors in conferring a highly O2 tolerant nitrogen fixing system in the organism, has been discussed.

ChemInform Abstract: New Chemistry of Vanadium(II)

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Detection of the in vivo incorporation of a metal cluster into a protein. The FeMo cofactor is inserted into the FeFe protein of the alternative nitrogenase of Rhodobacter capsulatus.

The photosynthetic bacterium Rhodobacter capsulatus has, in addition to the Mo nitrogenase, a second Mo-independent nitrogen-fixing system, an 'iron-only' nitrogenase which is strongly repressed by molybdate. The MoO4(2-) concentration causing 50% repression of the alternative nitrogenase in nifHDK- cells was 6 nM. If MoO4(2-) was added to a growing nifHDK- culture which had already expressed the alternative nitrogenase, the production of ethane from acetylene, by whole cells, was stimulated dramatically. In spite of the fact that C2H4 formation decreased continuously during the duration of the experiment (3 days), the total C2H6 production increased about twofold within the first 24 h, whereas the relative yield of C2H6 increased from 2% (C2H6/C2H4 x 100) in the absence of MoO4(2-), to a maximal value of 69% in the presence of MoO4(2-) (1 mM) after 72 h incubation. This 'Mo effect' appeared to be stronger the higher the MoO4(2-) concentration in the medium and the longer the incubation time. In the presence of ReO4-, WO4(2-) or VO4(3-), a similar effect did not occur. The 'Mo effect' was not observed in a nifHDK- nifE- double mutant which is unable to synthesize the FeMo cofactor and was diminished in a nifHDK- nifQ- mutant. Crude extracts from nifHDK- cells cultivated in the presence of MoO4(2-), also showed enhanced production of ethane. Component 1, purified from those extracts, displayed an S = 3/2 EPR signal which was relatively weak but characteristic for the FeMoco. These results strongly support the suggestion that the 'Mo effect' is a consequence of the formation of a hybrid enzyme consisting of the apoprotein of the alternative nitrogenase and the FeMo cofactor of the conventional nitrogenase. The 'Mo effect' was not influenced by the addition of chloramphenicol to the cultures. The occurrence of the 'Mo effect' appeared, therefore, to be independent of de-novo protein synthesis. The analysis of nifE-lacZ and nifN-lacZ fusions proved that both genes necessary for the FeMo cofactor synthesis are also expressed under conditions of MoO4(2-) deficiency. The possible explanations for incorporation of the FeMoco into component 1 of the alternative nitrogenase are discussed.

Improving nitrogen-fixing systems and integrating them into sustainable rice farming

This paper summarizes recent achievements in exploiting new biological nitrogen fixation (BNF) systems in rice fields, improving their management, and integrating them into rice farming systems. The inoculation of cyanobacteria has been long recommended, but its effect is erratic and unpredictable. Azolla has a long history of use as a green manure, but a number of biological constraints limited its use in tropical Asia. To overcome these constraints, the Azolla-Anabaena system as well as the growing methods were improved. Hybrids between A. microphylla and A. filiculoides (male) produced higher annual biomass than either parent. When Anabaena from high temperature-tolerant A. microphylla was transferred to Anabaena-free A. filiculoides, A. filiculoides became tolerant of high temperature. Azolla can have multiple purposes in addition to being a N source. An integrated Azolla-fish-rice system developed in Fujian, China, could increase farmers' income, reduce expenses, and increase ecological stability. A study using Azolla labeled with 15N showed the reduction of N losses by fish uptake of N. The Azolla mat could also reduce losses of urea N by lowering floodwater-pH and storing a part of applied N in Azolla. Agronomically useful aquatic legumes have been explored within Sesbania and Aeschynomene. S. rostrata can accumulate more than 100kg N ha-1 in 45 d. Its N2 fixation by stem nodules is more tolerant of mineral N than that by root nodules, but the flowering of S. rostrata is sensitive to photoperiod. Aquatic legumes can be used in rainfed rice fields as N scavengers and N2 fixers. The general principle of integrated uses of BNF in rice-farming systems is shown.

Biological nitrogen fixation for sustainable agriculture: A perspective

The economic and environmental costs of the heavy use of chemical N fertilizers in agriculture are a global concern. Sustainability considerations mandate that alternatives to N fertilizers must be urgently sought. Biological nitrogen fixation (BNF), a microbiological process which converts atmospheric nitrogen into a plant-usable form, offers this alternative. Nitrogen-fixing systems offer an economically attractive and ecologically sound means of reducing external inputs and improving internal resources. Symbiotic systems such as that of legumes and Rhizobium can be a major source of N in most cropping systems and that of Azolla and Anabaena can be of particular value to flooded rice crop. Nitrogen fixation by associative and free-living microorganisms can also be important. However, scientific and socio-cultural constraints limit the utilization of BNF systems in agriculture. While several environmental factors that affect BNF have been studied, uncertainties still remain on how organisms respond to a given situation. In the case of legumes, ecological models that predict the likelihood and the magnitude of response to rhizobial inoculation are now becoming available. Molecular biology has made it possible to introduce choice attributes into nitrogen-fixing organisms but limited knowledge on how they interact with the environment makes it difficult to tailor organisms to order. The difficulty in detecting introduced organisms in the field is still a major obstacle to assessing the success or failure of inoculation. Production-level problems and socio-cultural factors also limit the integration of BNF systems into actual farming situations. Maximum benefit can be realized only through analysis and resolution of major constraints to BNF performance in the field and adoption and use of the technology by farmers.

Involvement of GroEL in nif gene regulation and nitrogenase assembly

Several approaches were used to study the role of GroEL, the prototype chaperonin, in the nitrogen fixation (nif) system. An Escherichia coli groEL mutant transformed with the Klebsiella pneumoniae nif gene cluster accumulated very low to nondetectable levels of nitrogenase components compared with the isogenic wild-type strain or the mutant cotransformed with the wild-type groE operon. In K. pneumoniae, overexpression of the E. coli groE operon markedly accelerated the rate of appearance of the MoFe protein and its constituent polypeptides after the start of derepression. The groEL mutation in E. coli decreased NifA-dependent beta-galactosidase expression from the nifH promoter but did not affect the constitutive expression of nifA from the tet promoter of ntr-controlled expression from the nifLA promoter. The possibility that GroEL is required for the correct folding of NifA was supported by coimmunoprecipitation of NifA with anti-GroEL antibodies. Kinetic analyses of nitrogenase assembly in 35S pulse-chased K. pneumoniae pointed to the existence of high-molecular-weight intermediates in MoFe protein assembly and demonstrated the transient binding of newly synthesized NifH and NifDK to GroEL. Overall, these results indicate that GroEL fulfills both regulatory and structural functions in the nif system.

Synthesis and molecular structures of chromium-iron-sulfide and [Cp 3Cr 3(μ 3-S) 4Fe] 2Fe(CO) 3 · C 6H 6 clusters with tetrahedral metal frameworks

Oxidative decarbonylation of Fe 2(CO) 6(μ-SPh) 2 under the action of Cp 2Cr 2(SCMe 3) 2S produced the tetrahedral Cp 3Cr 3(μ 3-S) 4Fe(Sph) cluster (III) and the unique nonanuclear [Cp 3Cr 3(μ 3-S) 4Fe] 2Fe(CO) 3S·C 6H 6 cluster (IV), depending on the molecular ratio of the reagents. Both complexes were characterized by X-ray structural analysis. In IV two heterometallotetrahedra are connected via a bridging Fe(CO) 3S group and FeFe bond (3.110 Å). Cluser III may be considered as a model of ferredoxin while cluster IV could be seen as a model nitrogen fixing system.

Growth, photosynthesis and nitrogen fixation ofAnabaena doliolum exposed to Assam crude extract

Petroleum contaminants pose serious threat to the structure and functioning of aquatic ecosystems. Although their effects on various algal species have been studied before, heterocystous blue-green algae (cyanobacteria) have been little explored in this regard. Inadequate information concerning the effects of petroleum oils on them, especially on their nitrogen fixing system, prompted the authors to undertake this study. In this communication the authors report the influence of Assam crude on growth, photosynthesis ({sup 14}C incorporation), nitrogen fixation (nitrogenase activity) and heterocyst differentiation in Anabaena doliolum. This species and other heterocystous cyanobacteria occur widely in soil and aquatic ecosystems.

Similarities among nitrogenase proteins and amongnif-HDK genes ofMethanosarcina barkeri and of other diazotrophic bacteria

Similarities between the nitrogen-fixing systems of the archaebacteriumMethanosarcina barkeri (strain Fusaro) and a number of eubacteria were investigated. Using antibodies againstRhizobium leguminosarum nitrogenase and a probe of clonednif-HDK genes of this species, homology withM. barkeri was demonstrated on the protein level and to a greater extent on the DNA level.

Chemical models and nitrogenase

This paper discusses four aspects of the nitrogen-fixation problem: the structure of nitrogenase; the mechanism of the function of nitrogenase; the chemistry of dinitrogen; and the function of nitrogenase at an atomic level. It is concluded that the chemistry of singly-bound end-on dinitrogen complexes supplies precedents for many of the features of the function of nitrogenase, such as the stoichiometry, the formation of hydrazine upon quenching and the evolution of dihydrogen. Whether the protic nitrogen-fixing systems are as consistent with the chemistry of nitrogenase is yet to be demonstrated. This does not prove that an end-on species must be involved in nitrogenase function, but it is probably the best model thus far.

Future directions for biological nitrogen fixation research

Enormous progress has been made in the last decade in understanding the physiology and genetics of nitrogen-fixing microorganisms, but relatively little has been done to translate theory into practice. The object of this note is to define strategies for research on applied nitrogen fixation and thus to help increase food and energy production and contribute to the restoration of soil fertility. Attention is given here to the symbiotic nitrogenfixing systems, legumes and actinorhizal plants, because of their unquestionable importance in tropical agriculture, agroforestry and forestry (National Research Council, 1979; Dawson, 1986). No attempt is made to evaluate the priorities for research on Azolla, non-symbiotic Cyanobacteria or non-symbiotic nitrogen-fixing bacteria in plant litter (Hill et al., 1988) or in the rhizosphere of cereals and pasture grasses (associative nitrogen fixation). Problems related to nitrogen fixation by Cyanobacteria have already been extensively explored and carefully reviewed (IRRI, 1979; Lumpkin and Plucknett, 1980; Roger and Kulasooriya, 1980; Roger and Reynaud, 1982; Roger and Watanabe, 1986). Generally the ecosystem received little nitrogen from non-symbiotic rhizospheric nitrogen-fixing microorganisms because they do not get enough energy to fix appreciable amounts of nitrogen and are often hindered in their activity in the field (Alexander, 1982; Barraquio et al., 1984; Beringer and Hirsch, 1984). However recent experiments based on the use of "N suggest that the nitrogen-fixing bacteria associated with the roots of several tropical crops, including wetland rice, sugar cane and some forage grasses, can fix significant amounts of nitrogen (Boddey and Dobereiner, 1987). Rhizospheric microorganisms can also contribute to plant growth through other

Where to now?

This paper is concerned with looking into the future and trying to discern the shape of the directions nitrogen fixation research will take. Accordingly, much of it may be proved incorrect or impracticable; this is the danger for anyone who makes forecasts. It seems clear that, although rapid progress is being made in our theoretical understanding of the nitrogen fixation process, little of that progress has yet been applied in a practical sense to improve crop production. Our future directions need to encompass this phase of application. One of the dilemmas is to decide how to use our techniques: to forge new nitrogen-fixing systems or associations, or to improve existing ones, or to pursue some combinations of the two. In the legume systems, there is still much slack in technology to be taken up across the world. Simple problems in production, such as widespread boron deficiency in Thailand, remain to be corrected. Some questions to be considered include the following: (i) The ability to manipulate expression of sym and nif genes exists; what are we going to do with it? (ii) Acid tolerance in legume bacteria remains a major problem. What conditions such tolerance, and how can it be recognized and exploited? (iii) Nitrogen fixation in legume nodules depends on dicarboxylate supplies from the plant, apparently because the legume controls what the nodule bacteroids receive. Would a greater supply of dicarboxylates improve nitrogen fixation? Would making other classes of substrates available to bacteroids in larger amounts have beneficial effects? (iv) ‘Alternative’ nitrogenases are now known; can they be used beneficially in existing or new systems?