Implications of Competitive Inhibition in the Acetylene Reduction Assay for Dinitrogen Fixation

An earlier paper which analysed the implications of diffusionlimitation for acetylene reduction by intact legume noduleshas recently been criticized for ignoring competitive inhibitionof acetylene reduction by dinitrogen. Mathematical analysesof competitive inhibition show that the dependence of acetylenereduction rate on acetylene concentration under atmosphericN₂ is described adequately by an equation functionally equivalentto the Michaelis—Menten equation, despite competitiveinhibition. Therefore, no modification of the original analysisis required. However, competitive inhibition is shown to bea significant factor when experiments under atmospheric N₂ andunder argon are compared. Acetylene reduction, dinitrogen fixation, competitive inhibition, diffusion limitation, legume nodules     

The Physiology and Nitrogen-fixing Capability of Aquatically and Terrestrially Grown Neptunia plena: The Importance of Nodule Oxygen Supply

The aquatic legume Neptunia plena (L.) Benth. was grown in non-aeratedwater culture or vermiculite. Growth, nodulation, nitrogen fixationand nodule physiology were investigated. Over an 80-d period,plants grew and fixed nitrogen and carbon equally well in bothrooting media, although distribution of growth between plantparts varied. Total nodule dry weights and volumes were similarbut vermiculite-grown plants had three times as many (smaller)nodules than those grown in water. Oxygen diffusion resistanceof nodules exposed to 21% oxygen and 10% acetylene did not differsignificantly. Both treatments showed similar declines in rootrespiration and acetylene reduction activity (approx. 10%) whenroot systems were exposed to stepped decreases and increasesin rhizosphere oxygen concentration. However, nitrogenase activityof aquatically grown plants was irreversibly inhibited by rapidexposure of nodules to ambient air, whereas vermiculite-grownplants were unaffected. Aeration of water-cultured N. plenareduced stem length (but not mass) and number of nodules perplant. The concentration of nitrogen fixation by 163%. PossibleO₂ transport pathways from the shoot atmosphere to roots andnodules are discussed. Aquatic legume, diffusion resistance, Neptunia plena, nitrogen fixation, oxygen, root nodules     

Measurements of Nitrogen Fixation (C2H2), Photosynthesis and Respiration Using an Open System in the Natural Environment

A flow-through technique has been used successfully to measureacetylene reduction by an acetylene-sensitive legume in thefield. The results suggest that nodules in the field do notexhibit an acetylene-linked decline, implying that they havea much larger diffusion resistance in the field than a controlledenvironment. There was good agreement between the maximum ratesof nitrogen fixation, estimated using the acetylene reductiontechnique, and the maximum rates of nitrogen accumulation inthe biomass above ground. This suggests that the acetylene techniquecan give an accurate measurement of nitrogenase activity overtime periods of the order of hours and a useful daily measureof biological nitrogen fixation in the field; rates of nitrogenfixation below a depth of 0.2 m were negligible. When measurements of carbon dioxide were made by passing airupwards through the soil, steady state conditions were not achievedfor several hours because of the diffusive flux from the surroundingsoil. It is suggested that the unwanted inward diffusive fluxcan be prevented by ‘air-sealing’ the soil enclosure. Nitrogen fixation, acetylene reduction, carbon dioxide, diffusion resistance     

Carbon Costs of Nitrogenase Activity in Legume Root Nodules determined using Acetylene and Oxygen

There is a coupled decrease in respiration and nitrogenase activityof nodules of many legume symbioses induced by exposure to acetylenein the presence of 21% O₂. The respiratory costs of nitrogenaseactivity can be determined directly and distinguished from respiratorycosts for growth and maintenance of roots and nodules, usingthe linear regression of respiration on nitrogenase activity.The regression gradient represents the carbon costs for thetransfer of one pair of electrons by nitrogenase in terms ofmoles CO₂ released per mole of ethylene produced. The interceptof the regression is the growth and maintenance respirationof nodules or nodulated roots. Exposure to acetylene at decreasedor increased oxygen concentrations in the range from 10% to70% resulted in a wider range of values for CO₂ production andnitrogenase activity that fell on the same regression line asvalues obtained during the acetylene-induced decline at 21%oxygen. Oxygen concentrations below 10% increased significantlythe proportion of anaerobic respiration and produced changesin nitrogenase activity not correlated with CO₂ production.Provided that these limits are not exceeded, oxygen-inducedchanges in nodule activity in the presence of acetylene canbe used to measure the efficiency of those symbioses which donot exhibit an acetylene-induced decline at a fixed oxygen concentration. Respiratory cost (moles CO₂/mole ethylene) remained relativelyconstant with plant age for detached pea nodules (2.8), attachednodulated roots of lucerne (2.5) and detached nodulated rootsof field bean (4.2). However, for lucerne and field beans theproportion of total root respiration coupled to nitrogenasedeclined with time. A survey of 13 legume species gave values from 2 to 5 molesCO₂/mole C₂H₄ Rhizobium strain and host-dependent variationsin efficiency were found. Key words: Nitrogenase, Legume root nodules, Respiration, Oxygen     

Diffusion of Gases through Plant Tissues : Entry of Acetylene into Legume Nodules

I have measured acetylene diffusion through plant tissues including nodules from several species of legume—vetch, peas, soybeans, and Sesbania rostrata. The observed half-time for reequilibration of internal and external concentration is less than 1 minute for typical nodules. Inward diffusion of acetylene in air is rapid relative to the use of acetylene by nitrogenase so that diffusion of acetylene would not be a significant limiting factor for nitrogenase activity in air. However, under an atmosphere of Ar:O₂ where there is no N₂ reduction, the inward diffusion rate of acetylene into larger nodules could produce a measurable limitation of observed nitrogenase activity at low acetylene concentrations.     

A novel fix- symbiotic mutant of Lotus japonicus, Ljsym105, shows impaired development and premature deterioration of nodule infected cells and symbiosomes

Nitrogen-fixing symbiosis between legume plants and rhizobia is established through complex interactions between two symbiotic partners. To identify the host legume genes that play crucial roles in such interactions, we isolated a novel Fix- mutant, Ljsym105, from a model legume Lotus japonicus MG-20. The Ljsym105 plants displayed nitrogen-deficiency symptoms after inoculation with Mesorhizobium loti under nitrogen-free conditions, but their growth recovered when supplied with nitrogen-rich nutrients. Ljsym105 was recessive and monogenic and mapped on the upper portion of chromosome 4. The mutant Ljsym105 formed an increased number of small and pale-pink nodules. Nitrogenase (acetylene reduction) activity per nodule fresh weight was low but retained more than 50% of that of the wild-type nodules. Light and electron microscopic observations revealed that the Ljsym105 nodule infected cells were significantly smaller than those of wild-type plants, contained enlarged symbiosomes with multiple bacteroids, and underwent deterioration of the symbiosomes prematurely as well as disintegration of the whole infected cell cytoplasm. These results indicate that the ineffectiveness of the Ljsym105 nodules is primarily due to impaired growth of infected cells accompanied with the premature senescence induced at relatively early stages of nodule development. These symbiotic phenotypes are discussed in respect to possible functions of the LjSym105 locus in the symbiotic interactions required for establishment of the nitrogen-fixing symbiosis.     

Manipulation of Ethylene Synthesis in Roots Through Bacterial ACC Deaminase for Improving Nodulation in Legumes

Symbiotic association between rhizobia and legumes results in the development of unique structures on roots, called nodules. Nodulation is a very complex process involving a variety of genes that control NOD factors (bacterial signaling molecules), which are essential for the establishment, maintenance and regulation of this process and development of root nodules. Ethylene is an established potent plant hormone that is also known for its negative role in nodulation. Ethylene is produced endogenously in all plant tissues, particularly in response to both biotic and abiotic stresses. Exogenous application of ethylene and ethylene-releasing compounds are known to inhibit the formation and functioning of nodules. While inhibitors of ethylene synthesis or its physiological action enhance nodulation in legumes, some rhizobial strains also nodulate the host plant intensively, most likely by lowering endogenous ethylene levels in roots through their 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity. Co-inoculation with ACC deaminase containing plant growth promoting rhizobacteria plus rhizobia has been shown to further promote nodulation compared to rhizobia alone. Transgenic rhizobia or legume plants with expression of bacterial ACC deaminase could be another viable option to alleviate the negative effects of ethylene on nodulation. Several studies have well documented the role of ethylene and bacterial ACC deaminase in development of nodules on legume roots and will be the primary focus of this critical review.     

A Re-Evaluation of the Role of the Infected Cell in the Control of O2 Diffusion in Legume Nodules

Two different simulation models were constructed to describe O2 diffusion into the bacteria-infected cells of legume nodules: one based on a central zone of uniform spherical cells and the other on a central zone of packed, uniform cubical cells with air spaces along the edges. The cubical model more closely approximated the geometry and gas diffusion characteristics of infected cells than did the spherical model. The models relied on set values for the innermost O2 concentration in the infected cell (1-20 nM) and predicted values for the free O2 and oxygenated leghemoglobin gradients toward the cell:space interface. The cubical model but not the spherical model predicted saturation of leghemoglobin (Lb) oxygenation at or within a few micrometers of the gas-filled intercellular space and predicted that the space concentration could be as high as 1.3% O2 when the fractional oxygenation of Lb and respiration rate within the infected cell were typical of that which has been measured in vivo. In the model, the higher the space O2 concentration, the greater the saturation of Lb by O2 and the greater the collapse of Lb-facilitated diffusion near the cell:space interface. This was predicted to result in a greater resistance to O2 diffusion from the space to the bacteroids, thereby providing an intrinsic, homeostatic mechanism for controlling the rate of O2 influx into infected cells. Changes in the physiological features of the simulated cubical infected cell, such as the proportion of the cell as cytosol, the surface area of the cell exposed to a space, the maximum rate of cellular respiration, or the concentration of Lb in the cytoplasm, significantly altered the extent to which the infected cell would be able to regulate its diffusive resistance. These results demonstrate the possibility of a Lb-based mechanism for controlling the O2 concentration within the infected cells. If such a mechanism exists in legume nodules, it would give the infected cell an ability to exercise fine control over its internal environment, a process that could complement a physical diffusion barrier that may exist in the inner cortex or elsewhere in the nodule and provide coarse control over O2 diffusion.     

Effect of nitrate on acetylene reduction activity and carbohydrate composition of Phaseolus vulgaris nodules

When Phaseolus vulgaris L. cv. Kentucky Wonder plants were supplied with various levels of nitrate for 34 days, nodule weight (plant)⁻¹, acetylene reduction activity (g nodule)⁻¹, and sugar concentration in nodules were depressed >60% (7.5 m M nitrate vs nil nitrate). Starch concentration in nodules was more than double the sugar concentration and declined only slightly in response to nitrate level. At the highest level of nitrate, sugar concentration in nodules was 50% greater than that in roots and nodule starch was about 6-fold greater than root starch on a fresh weight basis. When plants were grown with 1 m M nitrate and then supplied with 12 m M nitrate for 7 days, the rapid decline in acetylene reduction activity coincided with a decline in sucrose concentration. However, glucose and fructose concentrations declined only after the largest decrease in acetylene reduction had occurred, and the quantitative decrease in glucose and fructose in nodules was small relative to sucrose. Other results showed that the magnitude of the effect of nitrate on some nodule carbohydrate compounds depends on Rhizobium phaseoli strain and on whether plants were grown with or without nitrate prior to experimental treatments. Some of the results are consistent with the carbohydrate-deprivation hypothesis for inhibition of legume nodules by nitrate. However, there are several complications involved in the interpretation of results of this type, and other possible explanations for the results are suggested.     

Respiration and nitrogen fixation in nodulated roots of soya bean and pea

Summary Oxygen uptake, carbon dioxide evolution and nitrogenase activity, measured either as hydrogen evolution (under argon 80%, oxygen 20%) or as the reduction of acetylene to ethylene, were assayed over the same time period by a direct mass-spectrometric method. When carbon dioxide evolution was used to estimate carbohydrate consumption, the results agreed with other work on whole plants. The RQ values obtained in these experiments were always less than 1.0 and thus the carbohydrate consumption calculated from oxygen uptake suggests that previous estimates, using carbon dioxide evolution as a measure of the cost of nitrogen fixation may be underestimates. Lag periods observed in the reduction of acetylene to ethylene suggest that there is a resistance to diffusion of gases in the root nodules.     

A Model of the Regulation of Nitrogenase Electron Allocation in Legume Nodules (I. The Diffusion Barrier and H2 Inhibition of N2 Fixation)

A mathematical model is presented to explain the regulation of nitrogenase electron allocation to N2 fixation (EAC) in legume nodules. The model is based on two assumptions: (a) that H2 inhibits N2 fixation in a competitive manner; and (b) that O2, H2, and N2 move into and out of nodules by diffusion and their movement is impeded by a diffusion barrier, the permeability of which is controlled to maintain a very low infected cell O2 concentration. When the model was used to simulate nodules displaying a range of values for total nitrogenase activity (TNA), maximum EAC values were predicted to be between 0.69 and 0.71, and a negative correlation was predicted to exist between EAC and TNA. These predictions were in good agreement with empirically derived values reported in the literature and support the suggestion that H2 inhibition of N2 fixation is a major determinant in the regulation of nitrogenase EAC in legume nodules. Two versions of the model were constructed. A closed-pore model assumed that the diffusion barrier consisted of a solid shell of water of variable thickness in the nodule cortex. An open-pore model assumed that a small number of gas-filled intercellular spaces connected the nodule central zone with the root atmosphere and these pores were opened or closed by water to provide variations in the nodule's permeability to gas diffusion. Because of differences in the diffusivity of gases in the gaseous and aqueous phases, the model predicted that, at a given infected cell O2 concentration, an open-pore diffusion barrier would result in less H2 accumulation in the infected cells than a closed-pore diffusion barrier. Therefore, the model may be used to test specific hypotheses about the physical structure of the barrier to gas diffusion in legume nodules.     

Analysis of acetylene reduction rates of soybean nodules at low acetylene concentrations

It has been previously proposed that acetylene reduction data at subsaturating acetylene concentrations could be interpreted by use of the Michaelis-Menten equation, based on the acetylene concentration external to the nodules. One difficulty of this view is that the assumption that the system is not diffusion limited is violated when studying intact nodules. The presence of a gas diffusion barrier in the nodule cortex leads to an alternate expression for the gas exchange rates at subsaturating gas concentrations. A theoretical comparison of the `apparent' Michaelis-Menten model and diffusion model illustrated the difficulties observed in the former model of overestimating the Michaelis-Menten coefficient and yielding a correlation between the Michaelis-Menten coefficient and the maximum rate. On the other hand, use of a diffusion model resulted in (a) estimates of the Michaelis-Menten coefficient consistent with enzyme studies, (b) stability of the estimates of the Michaelis-Menten coefficient independent of treatment, and (c) a sensitivity of the diffusion barrier conductance to plant drought stress. It was concluded that all studies of nodule gas exchange need to consider possible effects caused by the presence of a diffusion barrier.     

Lignification of cell walls of infected cells in Casuarina glauca nodules that depend on symplastic sugar supply is accompanied by reduction of plasmodesmata number and narrowing of plasmodesmata

The oxygen protection system for the bacterial nitrogen‐fixing enzyme complex nitrogenase in actinorhizal nodules of Casuarina glauca resembles that of legume nodules: infected cells contain large amounts of the oxygen‐binding protein hemoglobin and are surrounded by an oxygen diffusion barrier. However, while in legume nodules infected cells are located in the central tissue, actinorhizal nodules are composed of modified lateral roots with infected cells in the expanded cortex. Since an oxygen diffusion barrier around the entire cortex would also block oxygen access to the central vascular system where it is required to provide energy for transport processes, here each individual infected cell is surrounded with an oxygen diffusion barrier. In order to assess the effect of these oxygen diffusion barriers on oxygen supply for energy production for transport processes, apoplastic and symplastic sugar transport pathways in C. glauca nodules were examined. The results support the idea that sugar transport to and within the nodule cortex relies to a large extent on the less energy‐demanding symplastic mechanism. This is in line with the assumption that oxygen access to the nodule vascular system is substantially restricted. In spite of this dependence on symplastic transport processes to supply sugars to infected cells, plasmodesmal connections between infected cells, and to a lesser degree with uninfected cells, were reduced during the differentiation of infected cells.     

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.     

Problems of the Acetylene Reduction Technique Applied to Water-Saturated Paddy Soils

The acetylene reduction assay for the measurement of N₂ fixation in a water-saturated paddy soil is limited by the slow diffusion of acetylene and ethylene. In laboratory incubation tests, vigorous shaking after the assay period is needed to release ethylene into the gas within the assay vials. Shaking prior to the incubation is also effective for dissolving acetylene in the water-saturated soil. However, a water-saturated soil depth of less than 10 mm during incubation is recommended. In field assays, some amounts of ethylene remain in the water-saturated soil phase of the acetylene reduction assay chamber, but stirring the water-saturated soil before sampling reduces the amount of ethylene remaining in soil. Evidence of a downward movement of acetylene and an upward movement of ethylene through rice plants was obtained. Because of the rapid transfer of acetylene to rice plant roots, an in situ acetylene reduction assay covering a rice hill is likely to detect nitrogen fixation in the proximity of roots where acetylene is easily accessible. Acetylene introduction to the water-saturated soil phase prior to assay did not greatly increase the acetylene reduction rate. Carbon dioxide enrichment in the assay chamber did not enhance nitrogen fixation in a paddy including rice and algae during a 1-day cycle.     

Further Errors in the Acetylene Reduction Assay: Effects of Plant Disturbance

A flow-through gas system was used to study the effects of disturbanceon nitrogenase (acetylene reduction) activity of nodulated rootsystems of soyabean (Glycine max) and white clover (Trifoliumrepens). Detopping plus removal of the rooting medium (by shaking)produced a substantial decrease in maximum nitrogenase activity.This response is due to a reduction in oxygen flux to the bacteroidscaused by an increase in the oxygen diffusion resistance ofthe nodule. The decrease in maximum nitrogenase activity wasmuch smaller for roots subjected to detopping only. Thus, theeffect of root shaking is more important than that of shootremoval. The effect of detopping plus root shaking on nitrogenase activityoccurred whether the plants were equilibrated and assayed at25°C or 15°C. However, the effect of disturbance onthe oxygen diffusion resistance of the nodules, and thus onnitrogenase activity, was greater at the higher temperature.At the lower temperature the oxygen diffusion resistance ofthe nodules had already been increased in response to the reducedrequirement for oxygen. These nodules were less susceptibleto the effects of disturbance. Thus, comparisons of the effectsof equilibration temperature on nitrogenase activity produceddifferent results depending on whether intact or disturbed systemswere used. With intact systems activity was lower at the lowertemperature but with detopped/shaken roots the lowest activityoccurred at the higher temperature. It is concluded that the use of detopped/shaken roots can producesubstantial errors in the acetylene reduction assay, which makesthe assay invalid even when used for comparative purposes. However,comparisons with rates of ¹⁵N₂ fixation and H₂ production showthat accurate measurements of nitrogenase activity can be obtainedfrom maximum rates of acetylene reduction by intact plants ina flow-through gas system. The continued use of assay proceduresin which cumulated ethylene production from disturbed systemsis measured in closed vessels must be questioned. Key words: Nodules, acetylene, nitrogenase activity     

Estimation of nitrogenase using a colorimetric determination for ethylene

Ethylene is measured by oxidizing it to formaldehyde and determining the formaldehyde colorimetrically. The assay is applied to estimation of nitrogenase in nodulated legume roots by measuring the ethylene produced from acetylene.     

Ethylene inhibits the Nod factor signal transduction pathway of Medicago truncatula

Legumes form a mutualistic symbiosis with bacteria collectively referred to as rhizobia. The bacteria induce the formation of nodules on the roots of the appropriate host plant, and this process requires the bacterial signaling molecule Nod factor. Although the interaction is beneficial to the plant, the number of nodules is tightly regulated. The gaseous plant hormone ethylene has been shown to be involved in the regulation of nodule number. The mechanism of the ethylene inhibition on nodulation is unclear, and the position at which ethylene acts in this complex developmental process is unknown. Here, we used direct and indirect ethylene application and inhibition of ethylene biosynthesis, together with comparison of wild-type plants and an ethylene-insensitive supernodulating mutant, to assess the effect of ethylene at multiple stages of this interaction in the model legume Medicago truncatula. We show that ethylene inhibited all of the early plant responses tested, including the initiation of calcium spiking. This finding suggests that ethylene acts upstream or at the point of calcium spiking in the Nod factor signal transduction pathway, either directly or through feedback from ethylene effects on downstream events. Furthermore, ethylene appears to regulate the frequency of calcium spiking, suggesting that it can modulate both the degree and the nature of Nod factor pathway activation.     

Nodulation of legume trees from South East Brazil

Summary An extended survey of nodulation of legume trees from South-East Brazilian forests was conducted. Six new species from the Caesalpinioideae, 23 from the Mimosoideae and 27 from the Papilionoideae are reported to have nodules. Nitrogenase activity (acetylene reduction) was tested for all nodules and rhizobia were isolated from the most active.     

What triggers the regulation of nitrogenase activity in forage legume nodules after defoliation?

The dramatic decrease in nitrogenase activity after the defoliation of forage legumes has been recognized for a long time; however, the underlying mechanisms are not understood yet. The impact of current photosynthesis can be excluded. The precise role of carbohydrate availability is still unclear and remains to be established. From current knowledge we can conclude that, after defoliation, nitrogenase activity in legume nodules is down-regulated by a variable oxygen diffusion resistance. The triggering elements are not known; there is, however, increasing evidence that the plant's demand for symbiotically fixed nitrogen plays an important role. The possibility is here discussed that, after defoliation, a nitrogen feedback mechanism regulates nitrogenase activity through a variable oxygen diffusion resistance in the nodules.