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.     

The relative efficiency of nitrogen fixation in pea root nodules

Summary Experiments were performed to investigate the causes of low relative efficiency, RE, in legume root nodules. Nitrogen fixing activity and RE varied with time of incubation of nodules and with different temperatures and oxygen concentrations. The effects of nitrogen concentration and carbon dioxide concentration were also examined. In each case the RE was inversely related to nitrogen fixing activity; measured by acetylene reduction. Increasing the nitrogen concentration had no effect on either nitrogen fixing activity or RE. Experiments with isolated bacteroids gave higher RE values than the whole nodules from which they were isolated. All the results were consistent with hydrogen inhibition of nitrogenase within the nodule being the cause of low RE.     

Translocation of nitrogen and expression of nodule-specific uricase (nodulin-35) in Robinia pseudoacacia

Uricase (urate oxidase, EC 1.7.3.3) activity and nodule-specific uricase II (nodulin-35) were detected in the nodules from a number of legume: Rhizobium symbioses ( Vigna unguiculata (L.) Walp., Phaseolus vulgaris L., and Kennedia coccinea Vent.) in the Phaseoleae, as well as in those of Robinia pseudoacacia L. which belongs to the tribe Robineae. Neither uricase activity nor nodulin-35 was detected in nodules from Lupinus angustifolius L., an amide-forming symbiosis of the tribe Genisteae. Nodules of R. pseudoacacia also showed high levels of allantoinase (EC 3.5.2.5) activity but activity of enzymes earlier in the pathway of ureide synthesis (xanthine dehydrogenase, EC 1.2.1.37; inosine monophosphate dehydrogenase, EC 1.2.1.14; and xanthosine nucleosidase, EC 3.2.2.1) could not be detected. Analysis of transport fluids (xylem, phloem and nodule exudates) from R. pseudoacacia found that asparagine, and, to a lesser extent, glutamine were the major translocated nitrogenous solutes. Ureides accounted for, at most, 2.6% of the N in transport fluids (tracheal xylem sap) and in nodule exudate, 0.1%. In common with nodules of the ureide-forming symbioses, those of R. pseudoacacia contained a high proportion of uninfected interstitial cells (53.7 ± 2.3%) in the central N₂-fixing tissue whereas in L. angustifolius only 2.5 ± 0.4% of cells in this tissue were uninfected. These data have been interpreted to indicate that expression of nodule-specific uricase is related to the differentiation of uninfected interstitial cells in nodules and not to the synthesis of ureides.     

Metabolism and translocation of fixed nitrogen in the nodulated legume

Summary Nitrogen (N₂) fixed by Rhizobium bacteroids in the legume nodule is excreted as ammonia to the surrounding host cell where it is efficiently assimilated into the amide group of glutamine. Generally glutamine is a minor exported solute of nitrogen, being further metabolised to asparagine in temperate species and to the ureides, allantoin and allantoic acid in tropical species. These solutes serve as the principal translocated forms of nitrogen in xylem. Compartmentalisation of the pathways of nitrogen metabolism and the role of ammonia in regulation of their activity is examined in nodules of both asparagine-forming (Lupinus albus L.) and ureide-forming (Vigna unguiculata L. Walp) symbioses.     

Biological nitrogen fixation in mixed legume/grass pastures

Biological nitrogen fixation (BNF) in mixed legume/grass pastures is reviewed along with the importance of transfer of fixed nitrogen (N) to associated grasses. Estimates of BNF depend on the method of measurement and some of the advantages and limitations of the main methods are outlined. The amounts of N fixed from atmospheric N₂ in legume/grass pastures throughout the world is summarised and range from 13 to 682 kg N ha^-1 yr^-1. the corresponding range for grazed pastures, which have been assessed for white clover pastures only, is 55 to 296 kg N ha^-1 yr^-1.Biological nitrogen fixation by legumes in mixed pastures is influenced by three primary factors; legume persistence and production, soil N status, and competition with the associated grass(es). These factors and the interactions between them are discussed. Legume persistence, production and BNF is also influenced by many factors and this review centres on the important effects of soil moisture status, soil acidity, nutrition, and pests and disease.Soil N status interacts directly with BNF in the short and long term. In the short-term, increases in soil inorganic N occurs during dry conditions and where N fertiliser is used, and these will reduce BNF. In the long-term, BNF leads to accumulation of soil N, grass dominance, and reduced BNF. However, cyclical patterns of legume and grass dominance can occur due, at least in part, to temporal changes in plant-available N levels in soil. Thus, there is a dynamic relationship between legumes and grasses whereby uptake of soil N by grass reduces the inhibitory effect of soil N on BNF and competition by grasses reduces legume production and BNF. Factors affecting the competition between legumes and grasses are considered including grass species, grazing animals, and grazing or cutting management.Some fixed N is transferred from legumes to associated grasses. The amount of N transferred below-ground, predominantly through decomposition of legume roots and nodules, has been estimated at 3 to 102 kg N ha^-1 yr^-1 or 2 to 26% of BNF. In grazed pasture, N is also transferred above-ground via return in animal excreta and this can be of a similar magnitude to below-ground transfer.Increased BNF in mixed legume/grass pastures is being obtained through selection or breeding of legumes for increased productivity and/or to minimise effects of nutrient limitations, low soil moisture, soil acidity, and pests and disease. Ultimately, this will reduce the need to modify the pasture environment and increase the role of legumes in low-input, sustainable agriculture.     

Nitrogen fixation and growth of non-crop legume species in diverse environments

Of the 643 legume genera, few have been exploited in agriculture and 40% have not even been evaluated for their ability to nodulate and fix nitrogen. Most of these are in tropical/subtropical regions, with habitats ranging from extremely dry to flooded. Recent work in some of these areas shows that plants can nodulate under conditions previously thought to be disadvantageous. The accepted dogma that nitrogen fixing legumes have a high demand for P is challenged and examples of how legumes can extract P from soils with low available P described. Species tolerant to shading and high soil Al are cited, although the mechanisms of adaptation are not yet clear. Some tropical soils have high nitrate levels and, contrary to perceived wisdom, there are legumes which can nodulate under such conditions. Many tropical tree legumes prefer ammonium to nitrate and are able to fix nitrogen and assimilate ammonium at the same time. In all these cases, there are genotypic differences, both within and among species.

Large areas of tropical fresh water, such as the Brazilian Pantanal and the Orinoco floodplain have nodulated legumes predominant in their flora. The ecological potential of these has not been evaluated. One of the sites of nodule evolution is likely to have been in such areas. Modes of infection of legumes by rhizobia vary with taxonomic tribe and may represent evolution for survival in different environments. As more legumes, from more ecosystems are studied, a wider range of adaptations is likely to be found. Work is urgently needed to study these, especially in areas being cleared for agriculture or by logging.     

Immunolocalization of ferritin in determinate and indeterminate legume root nodules

Summary In eukaryotic organisms ferritin is a protein involved in the storage of iron. The occurrence of ferritin and its relationship to the effectiveness of the nitrogen-fixing activity have been previously studied during the early stages of the nodule development by biochemical methods. We have used immunocytochemistry techniques to determine the precise location of ferritin and the behavior of this protein along the nodule development. The major localization was found in plastids and amyloplasts of infected and uninfected cells of the three legume nodules studied. A decrease of the immunolabelling was observed in infected cells of lupin and soybean senescing nodules and in the senescent zone of indeterminate alfalfa nodules. In the cortex of soybean and lupin nodules, ferritin increased during nodule ageing and the immunogold particles were mainly located in crystalline structures. The putative role of ferritin and plastids during nodule development is discussed.     

几种生态因素对西北干旱地区豆科植物结瘤固氮的影响

通过对西北干旱地区栽培和野生豆科植物不同环境条件固氮状况的调查表明,栽培豆科植物一般能自然结瘤,野生豆科植物种的结瘤率极低。根瘤颜色栽培植物多为粉红色,而野生植物多为白色、黄色或棕色。通过对水分、光照强度和温度等不同条件下根瘤ARA测定,表明根瘤固氮活性与生态条件关系密切,而土壤水分是限制根瘤固氮活性表达的主要因素。     

Localization of carbonic anhydrase in legume nodules

Extracts of the central infected zone and the surrounding cortex of nodules from Lupinus angustifolius L., Vigna unguiculata L. (Walp), Pisum sativum L., Phaseolus vulgaris L., Vicia faba L. and Medicago sativa L. contained significant activities of carbonic anhydrase (CA). Immunoassay of extracts using antisera to a putative nodule CA (Msca1) cloned from M. sativa also indicated expression in both tissue types. Quantitative confocal microscopy using laser scanning imaging and a fluorescent CA‐specific probe (5‐dimethylaminonaphthalene‐1‐sulfonamide [DNSA]) localized expression to the infected cells in the central zone tissue and a narrow band of 2–3 files of cells in the cortical tissue that corresponded to the inner cortex. In the infected cells, the enzyme activity was distributed evenly in the cytosol, but in the inner cortical cells, it was restricted to the periphery – possibly to the plasma membrane or cell wall. The functions of CA in these two tissues are considered in relation to the carbon metabolism of nodules and the participation of the inner cortex in the regulation of gaseous diffusive resistance.     

Correlations between the ascorbate-glutathione pathway and effectiveness in legume root nodules

Legume root nodules use the ascorbate-glutathione pathway to remove harmful H₂O₂. In the present study. effective and ineffective nodules from soybean and alfalfa were compared with regard to this pathway. Effective nodules had higher activity of all 4 enzymes (ascorbate peroxidase, EC 1. 11. 1. 11: monodehydroascorbate reductase, EC 1. 6. 5. 4: dehydroascorbate reductase, EC 1. 8. 5. 1: and glutathione reductase, EC 1. 6. 4. 2). The concentration of thiol tripeptides (primarily homoglutathione) was about 1 m M in effective nodules – a level 3–4-fold higher than in ineffective nodules. Effective nodules contained higher levels of NAD⁺. NADP⁺ and NADPH. but not of NADH or ascorbate. The increased capacity for peroxide scavenging in effective nodules as compared to ineffective nodules emphasizes the important protective role that this pathway may play in processes related to nitrogen fixation.     

Proteolysis and nitrogen fixation in faba-bean (Vicia faba) nodules under water stress

Nodulated faba-beans ( Vicia faba L. var. minor) exhibiting high rates of N₂ fixation (133 μmol C₂H₄ g⁻¹ dry weight h⁻¹), were subjected to water restriction. A loss of C₂H₂ reduction due to water stress was always associated with a decline of the leghemoglobin content for each of the 4 decreasing values of Ψ_mod. Electron micrographs showed ultrastructural alterations of the fixing tissue, which affected both partners and increased with the severity of water stress. In the nodule cytosol, the alkaline proteolysis approximately doubled when Ψ_mod decreased from −0.55 MPa to −1.55 MPa. Concomitantly, an increase of the nodule intracellular pH from 6.3 to 7.0 was observed. Proteolysis was due to serine proteases, exhibiting a pH-optimum of 8 and which actively degraded purified leghemoglobin in vitro (K_m=100 μ M ). The degradation of leghemoglobin during water stress may contribute to the loss of C₂H₂ reduction and may affect the pattern of recovery upon rewatering.     

Relative efficiency of nitrogen fixation and the occurrence of hydrogenase in pea root nodules

Summary The apparent K_m(hydrogen) for uptake of hydrogen by pea root nodules was determined. This enabled the concentration gradient necessary for the evolution of hydrogen to be calculated for nodules with no hydrogenase activity. This indicated that hydrogen inhibition of nitrogenase is not likely to be the cause of the low relative efficiency of legume root nodules. The factors that affect electron allocation between protons and nitrogen in nitrogenase are reviewed and it is concluded that there must be some as yet unknown factor that affects electron distribution inRhizobium nitrogenase. One possibility is put forward and considered. A strain ofRhizobium was used that was found to possess hydrogenase activity in combination with pea variety Feltham First but not with variety Meteor. The control of this enzyme is briefly discussed.     

On the efficiency of legume supernodulating mutants

The supernodulating mutants of legumes lack the internal regulation of the number of symbiotic root nodules that harbour N₂-fixing nodule bacteria. On one hand, these mutants represent an efficient tool for dramatic increase in the degree of rhizobial symbiosis development. The trait of released nodulation is often associated with the desirable resistance of nodule initiation and functioning to the inhibition by ambient nitrate. On the other hand, the more intense and stable atmospheric nitrogen fixation of supernodulated plants is devalued by plant growth depression that results from the disproportion between the photosynthetic capacity of the shoot and the catabolic demands of symbiotic nodules. The deleterious effects of excessive nodulation can be neutralised or alleviated by a breeding strategy aimed at creating an ideotype of N₂-fixing legume. The growth depression can be diminished by the reduction in the nodule number typical for supernodulators, that is, 6–10-fold of the wild type, to the level found permissive for the particular crop. This shift should be accompanied with breeding aimed at the increased photosynthetic capacity of the shoot. Forage varieties of legumes represent a reserve of high photosynthetic and shoot growth capacity, thanks to a long-term breeding history for green biomass accumulation. Moreover, the deleterious effects of supernodulation are less perceived after introgression into the background of forage varieties in view of different criteria in their evaluation, such as nitrogen accumulation and biomass production per crop area unit. The growth of supernodulators can be further corrected by breeding for auxiliary traits such as long-vine shoot architecture, a longer vegetation period and late flowering. The same strategy is applicable to the compensation for inherent pleiotropic changes in plant development, which are often associated with primarily symbiotic mutations. Supporting evidence for the efficiency of the described approach has already been reported.     

Relationships between nitrogen fixation and photosynthesis as the main components of the productivity in alfalfa

Alfalfa (Medicago sativa L.) plants were inoculated with Sinorhizobium meliloti Tn-5 mutants featuring various nitrogen-fixing effectiveness and then grown in sand culture to study relations between CO₂ exchange, plant productivity, and nitrogen fixation. At the flowering stage, the relationship between nitrogen fixation and photosynthesis of whole alfalfa plants was described with the logarithmic curve. At the same stage of plant development, a close relationship was observed between nitrogen fixation rate and plant weight; this relationship showed a trend toward saturation at high rates of nitrogen fixation. The increase in nitrogenase activity of root nodules was accompanied by stimulation of root respiration; the relation of respiration to nitrogen-fixing activity was manifested stronger than its relation to the total root weight. It is concluded that highly effective strains of root nodule bacteria can realize their potential only in combination with complementary plant genotypes featuring active photosynthesis that provides a balanced supply of assimilates for both the symbiotic apparatus and growth processes in the macrosymbiont.     

A high-affinity iron transport system of Rhizobium meliloti may be required for efficient nitrogen fixation in planta

We have analyzed the ability of single site insertion mutants of Rhizobium meliloti 1021 defective in various components of a high-affinity iron transport system to produce nodules, fix nitogen and promote plant growth. Our results indicate that a high-affinity iron transport system may significantly increase the ability of the differentiated form of the bacterium to fix nitrogen and induce an increase in plant growth.Abbreviations EDDA ethylenediamine-N,N-bis(2-hydroxyphenylacetic acid) - CAS chrome azurol S     

Relationship between growth,nitrogen fixation and assimilation in a legume (Medicago sativa L.)

Summary Symbiotic N₂ fixation, NO ₃ ⁻ assimilation and protein accumulation in the shoots were measured simultaneously in alfalfa (Medicago sativa L.) grown in the field or in pots, in order to study how the balance between the two modes of nitrogen nutrition could be influenced by agronomic factors, such as harvest, mineral nitrogen supply and drought stress. During periods of rapid growth, fixation and assimilation may function simultaneously; they are antagonistic at the beginning and at the end of the growth cycle, when the nitrogen requirement of the plant is lower. When nitrogen nutrition does not limit growth, mineral nitrogen supply favours assimilation at the expense of fixation, but does not modify the amount of nitrogen accumulated, which is adjusted to the growth capacity of the plant. After cutting, nitrate assimilation compensated for the decrease in fixation and supplied the plant with the nitrogen required by the regrowth, the proliferation of which determined the fixation recovery. Drought stress decreased N₂ fixation much more than NO ₃ ⁻ assimilation. The latter made growth recovery possible when water supply conditions became normal again. These results suggested the existence of an optimum level of nitrate assimilation, which differed depending on the age of the plants and allowed both maximum growth and fixing activity.     

Model of gas exchange and diffusion in legume nodules

A mathematical model is described which allows the estimation of rates of O₂, CO₂, N₂, and H₂ exchange from legume nodules under steady state conditions of N₂ fixation. Calculated rates of gas exchange under defined conditions of nodule size, relative growth rate (RGR), specific total nitrogenase activity (TNA), nitrogenase electron allocation coefficient (EAC), uptake-hydrogenase activity (HUP) and nature of the N export product compared favorably with experimentally-obtained rates reported in the literature. Therefore the model was used to predict the effects of varying each of these nodule characteristics on the rates of gas exchange, and on the apparent respiratory cost (CO₂/NH₃) and sucrose cost (sucrose consumed/NH₃) of N₂ fixation.The model predicted that, all other characters being equal, ureide-producing nodules would consume 8% less sucrose per N fixed than asparagine-producing nodules, but would display an apparent respiratory cost which would be 5% higher than that in asparagine-producing nodules. In both ureide-producing and asparagine-producing nodules, the major factor affecting the apparent respiratory cost of N₂ fixation was predicted to be EAC, followed by TNA, nodule RGR and nodule size. The relative importance of HUP in improving the apparent respiratory cost of N₂ fixation was predicted to be largely dependent upon its potential role in the regulation of EAC. Abbreviations: See Appendix 1.