Leaf O(2) uptake in the dark is independent of coincident CO(2) partial pressure.

Elevated CO(2), in the dark, is sometimes reported to inhibit leaf respiration, with respiration usually measured as CO(2) efflux. Oxygen uptake may be a better gauge of respiration because non-respiratory processes can affect dark CO(2) efflux in elevated CO(2). Two methods of quantifying O(2) uptake indicated that leaf respiration was unaffected by coincident CO(2) level in the dark.

Whole-plant gas exchange and reductive biosynthesis in white lupin.

Simultaneous measurements of CO(2) (CER) and O(2) (OER) exchange in roots and shoots of vegetative white lupin (Lupinus albus) were used to calculate the flow of reducing power to the synthesis of biomass that was more reduced per unit of carbon than carbohydrate. On a whole-plant basis, the diverted reductant utilization rate (DRUR which is: 4 x [CER + OER]) of shoot tissue was consistently higher than that of roots, and values obtained in the light were greater than those in the dark. An analysis of the biomass being synthesized over a 24-h period provided an estimate of whole-plant DRUR (3.5 mmol e(-) plant(-1) d(-1)), which was similar to that measured by gas exchange (3.2 mmol e(-) plant(-1) d(-1)). Given that nitrate reduction to ammonia makes up about 74% of whole-plant DRUR, root nitrate reduction in white lupin was estimated to account for less than 43% of whole-plant nitrate reduction. The approach developed here should offer a powerful tool for the noninvasive study of metabolic regulation in intact plants or plant organs.

Oxygen regulation of a nodule-located carbonic anhydrase in alfalfa.

Control of the permeability to oxygen is critical for the function of symbiotic nitrogen fixation in legume nodules. The inner cortex (IC) seems to be a primary site for this regulation. In alfalfa (Medicago sativa) nodules, expression of the Msca1 gene encoding a carbonic anhydrase (CA) was previously found to be restricted to the IC. We have now raised antibodies against recombinant Msca1 protein and used them, together with antibodies raised against potato leaf CA, to demonstrate the presence of two forms of CA in mature nodules. Each antibody recognizes a different CA isoform in nodule tissues. Immunolocalization revealed that leaf-related CAs were localized primarily in the nitrogen-fixing zone, whereas the Msca1 protein was restricted exclusively to the IC region, in indeterminate and determinate nodules. In alfalfa nodules grown at various O(2) concentrations, an inverse correlation was observed between the external oxygen pressure and Msca1 protein content in the IC, the site of the putative diffusion barrier. Thus Msca1 is a molecular target of physiological processes occurring in the IC cells involved in gas exchange in the nodule.

A simplified approach for modeling diffusion into cells.

Regulation of the intracellular concentration of substrates is essential for the maintenance of a stable cellular environment. Diffusion and reaction processes supply and consume substrates within cells and determine their steady-state concentrations. To realistically represent these processes by computer simulation they must be modeled in three dimensions. Yet three-dimensional models are inherently computing intensive. This study describes a method, which substantially simplifies the modeling of diffusion into a polyhedral body (a cube), that was used as a model representation of a cell. The method is applied to a case study of oxygen diffusion into nitrogen-fixing, rhizobia-infected cells in legume nodules. The method involved generating a one-dimensional representation of the three-dimensional problem to provide a "surface area profile" of three-dimensional diffusion. The one-dimensional models were significantly easier to program, several orders of magnitude faster to solve and in this study were validated by assessing their results against those of comparable three-dimensional models of diffusion into the same body. The results show the one-dimensional method to be a close approximation of a three-dimensional source-sink problem with systematic differences below 10% for fractional oxygenation of leghemoglobin, cell respiration and nitrogenase activity. Larger differences between models (up to 45%) in the predicted average and innermost O(2)concentrations had no effects on the physiological conclusions of the study, but were attributed to the poorer resolution of the three- than the one-dimensional model, and to an inherent simplification in the derivation of the one-dimensional surface area profiles. The one-dimensional modeling approach was found to be a simple, yet powerful tool for the study of diffusion and reaction in biological systems.

Phloem Glutamine and the Regulation of O2 Diffusion in Legume Nodules.

The aim of the present study was to test the hypothesis that the N content or the composition of the phloem sap that supplies nodulated roots may play a role in the feedback regulation of nitrogenase activity by increasing nodule resistance to O2 diffusion. Treating shoots of lupin (Lupinus albus cv Manitoba) or soybean (Glycine max L. Merr. cv Maple Arrow) with 100 [mu]L L-1 NH3 caused a 1.3-fold (lupin) and 2.6-fold (soybean) increase in the total N content of phloem sap without altering its C content. The increase in phloem N was due primarily to a 4.8-fold (lupin) and 10.5-fold (soybean) increase in the concentration of glutamine N. In addition, there was a decline in both the apparent nitrogenase activity and total nitrogenase activity that began within 4 h and reached about 54% of its initial activity within 6 h of the start of the NH3 treatment. However, the potential nitrogenase activity values in the treated plants were not significantly different from those of the control plants. These results provide evidence that changes in the N composition of the phloem sap, particularly the glutamine content, may increase nodule resistance to O2 diffusion and, thereby, down-regulate nodule metabolism and nitrogenase activity by controlling the supply of O2 to the bacteria-infected cells.

The simultaneous measurement of low rates of CO2 and O2 exchange in biological systems.

An instrument for measuring low rates of biological O2 exchange using an open-flow gas analysis system is described. A novel differential O2 sensor that is capable of measuring as little as 0.4 Pa O2 against a back-ground of ambient air (20,900 Pa O2), yet has a dynamic range of +/- 2000 Pa O2 (i.e., +/- ca. 2% O2) is described. Baseline drift was typically less than 0.025 Pa min-1. The differential O2 sensor was incorporated into a respiratory quotient/photosynthetic quotient analyzer that contained other environmental sensors for atmospheric pressure, absolute O2 and CO2 concentration, temperature of the differential O2 sensor block, and differential pressure between reference and sample streams. Protocols for how these sensors can be used to calibrate the differential O2 sensor and to improve its stability with time are described. Together, the differential O2 sensor, the environmental sensors, and the simple calibration techniques allow for simultaneous, noninvasive, and accurate measurements of O2 and CO2 exchange in tissues with metabolic rates as low as about 0.1 mumol O2 or CO2 h-1. Example data are provided in which O2 differentials of 3 to 41 Pa O2 were measured in an open-flow system.

Drought Stress, Permeability to O2 Diffusion, and the Respiratory Kinetics of Soybean Root Nodules.

In legume nodules, treatments such as detopping or nitrate fertilization inhibit nodule metabolism and N2 fixation by decreasing the nodule's permeability to O2 diffusion, thereby decreasing the infected cell O2 concentration (Oi) and increasing the degree to which nodule metabolism is limited by O2 availability. In the present study we used nodule oximetry to assess and compare the role of O2 limitation in soybean (Glycine max L. Merr) nodules inhibited by either drought or detopping. Compared to detopping, drought caused only minor decreases in Oi, and when the external O2 concentration was increased to raise Oi, the infected cell respiration rate in the drought-stressed plants was not stimulated as much as it was in the nodules of the detopped plants. Unlike those in detopped plants, nodules exposed to moderate drought stress displayed an O2-sufficient respiration rate that was significantly lower than that in control nodules. Despite possible side effects of oximetry in altering nodule metabolism, these results provided direct evidence that, compared to detopping, O2 limitation plays a minor role in the inhibition of nodule metabolism during drought stress and changes in nodule permeability are the effect, not the cause, of a drought-induced inhibition of nodule metabolism and the O2-suffiecient rate of respiration.

Effects of Temperature on Infected Cell O2 Concentration and Adenylate Levels in Attached Soybean Nodules.

To assess the role of O2 in the regulation of nodule metabolism following a decrease or an increase in temperature, the fractional oxygenation of leghemoglobin (FOL) was measured in soybean (Glycine max L. Merr.) nodules during rapid and gradual changes in temperature from 20[deg]C to either 15 or 25[deg]C. The affinity of leghemoglobin for O2 was also measured at each temperature and the values were used to calculate the infected cell O2 concentration (Oi). After nodules were transferred to 15[deg]C, FOL and Oi increased and adenylate energy charge (AEC = [ATP + 0.5ADP]/[ATP + ADP + AMP]) increased from 0.70 to 0.78. The temperature increase was associated with a decrease in FOL and Oi. We concluded that changes in nodule temperature alter the respiratory demand of the nodules for O2, resulting in a change in Oi and a shift in the balance between ATP consumption and ATP production within the nodule tissue.

The Role of Oxygen in the Regulation of Nitrogenase Activity in Drought-Stressed Soybean Nodules.

The aim of this study was to investigate the mechanism of nitrogenase inhibition in drought-stressed soybean (Glycine max L.) nodules to determine whether this stress was similar to other inhibitory treatments (e.g. detopping) known to cause an O2 limitation of nodule metabolism. Nodulated soybean plants were either detopped or subjected to mild, moderate, or severe drought stress by growth in different media and by withholding water for different periods. All treatments caused a decline in nitrogenase activity, and in the drought-stressed nodules, the decline was correlated with more negative nodule water potentials. Increases in rhizosphere O2 concentration stimulated nitrogenase activity much more in detopped plants than in drought-stressed plants, reflecting a greater degree of O2 limitation with the detopped treatment than with the drought-stressed treatment. These results indicated that drought stress differs from many other inhibitory treatments, such as detopping, in that its primary cause is not a decrease in nodule permeability and a greater O2 limitation of nodule metabolism. Rather, drought stress seems to cause a decrease in the maximum O2-sufficient rate of nodule respiration or nitrogenase activity, and the changes in nodule permeability reported to occur in drought-stressed nodules may be a response to elevated O2 concentrations in the infected cell that may occur as nodule respiration declines.

Acclimation of Soybean Nodules to Changes in Temperature.

This study examines how O2 status, respiration rate, and nitrogenase activity of soybean (Glycine max) nodules acclimate to short-term (<30 min) temperature change from 20 to 15[deg]C or from 20 to 25[deg]C. Acclimation responses were compared between nodules on uninhibited plants and nodules that were severely O2 limited by exposure to Ar:O2. In uninhibited nodules the decrease in temperature caused a rapid inhibition of nitrogenase activity followed by partial recovery, whereas in Ar:O2-inhibited nodules the temperature decrease caused a minor stimulation followed by a gradual decline in nitrogenase activity. In contrast, the temperature increase caused a gradual increase in nitrogenase activity in uninhibited nodules, and an initial inhibition followed by a rapid rise in Ar:O2-inhibited nodules. In both uninhibited and Ar:O2-inhibited nodules, temperature had only minor effects on the degree to which nitrogenase activity was limited by O2 supply, but nodule permeability to O2 diffusion was greater at 25[deg]C, and less at 15[deg]C, than that measured at 20[deg]C. On the basis of these data, we propose that temperature change alters the nodule's respiratory demand and that the observed changes in nodule permeability occur to maintain control over the infected cell O2 concentration as the O2 demand increases at high temperature or decreases at low temperature.

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.

A Model of the Regulation of Nitrogenase Electron Allocation in Legume Nodules (II. Comparison of Empirical and Theoretical Studies in Soybean).

In N2-fixing legumes, the proportion of total electron flow through nitrogenase (total nitrogenase activity, TNA) that is used for N2 fixation is called the electron allocation coefficient (EAC). Previous studies have proposed that EAC is regulated by the competitive inhibition of H2 on N2 fixation and that the degree of H2 inhibition can be affected by a nodule's permeability to gas diffusion. To test this hypothesis, EAC was measured in soybean (Glycine max L. Merr.) nodules exposed to various partial pressures of H2 and N2, with or without changes in TNA or nodule permeability to gas diffusion, and the results were compared with the predictions of a mathematical model that combined equations for gas diffusion and competitive inhibition of N2 fixation (A. Moloney and D.B. Layzell [1993] Plant Physiol 103: 421-428). The empirical data clearly showed that decreases in EAC were associated with increases in external pH2, decreases in external pN2, and decreases in nodule permeability to O2 diffusion. The model predicted similar trends in EAC, and the small deviations that occurred between measured and predicted values could be readily accounted for by altering one or more of the following model assumptions: K1(H2) of nitrogenase (range from 2-4% H2), Km(N2) of nitrogenase (range from 4-5% N2), the allocation of less than 100% of whole-nodule respiration to tissues within the diffusion barrier, and the presence of a diffusion pathway that is open pore versus closed pore. The differences in the open-pore and closed-pore versions of the model suggest that it may be possible to use EAC measurements as a tool for the study of legume nodule diffusion barrier structure and function. The ability of the model to predict EAC provided strong support for the hypothesis that H2 inhibition of N2 fixation plays a major role in the in vivo control of EAC and that the presence of a variable barrier to gas diffusion affects the H2 and N2 concentration in the infected cell and, therefore, the degree of H2 inhibition.

Composition and Distribution of Adenylates in Soybean (Glycine max L.) Nodule Tissue.

Adenylates (ATP, ADP, and AMP) may play a central role in the regulation of the O2-limited C and N metabolism of soybean nodules. To be able to interpret measurements of adenylate levels in whole nodules and to appreciate the significance of observed changes in adenylates associated with changes in O2-limited metabolism, methods were developed for measuring in vivo levels of adenylate pools in the cortex, plant central zone, and bacteroid fractions of soybean (Glycine max L. Merr cv Maple Arrow x Bradyrhizobium japonicum strain USDA 16) nodules. Intact nodulated roots were either frozen in situ by flushing with prechilled Freon-113(-156[deg]C) or by rapidly (<1 s) uprooting plants and plunging them into liquid N2. The adenylate energy charge (AEC = [ATP + 0.5 x ADP]/[ATP + ADP + AMP]) of whole-nodule tissue (0.65 [plus or minus] 0.01, n = 4) was low compared to that of subtending roots (0.80 [plus or minus] 0.03, n = 4), a finding indicative of hypoxic metabolism in nodules. The cortex and central zone tissues were dissected apart in lyophilized nodules, and AEC values were 0.84 [plus or minus] 0.04 and 0.61 [plus or minus] 0.03, respectively. Although the total adenylate pool in the lyophilized nodules was only 41% of that measured in hydrated tissues, the AEC values were similar, and the lyophilized nodules were assumed to provide useful material for assessing adenylate distribution. The nodule cortex contained 4.4% of whole-nodule adenylates, with 95.6% being located in the central zone. Aqueous fractionation of bacteroids from the plant fraction of whole nodules and the use of marker enzymes or compounds to correct for recovery of bacteroids and cross-contamination of the bacteroid and plant fractions resulted in estimates that 36.2% of the total adenylate pool was in bacteroids, and 59.4% was in the plant fraction of the central zone. These are the first quantitative assessments of adenylate distribution in the plant and bacteroid fractions of legume nodules. These estimates were combined with theoretical calculations of rates of ATP consumption in the cortex (9.5 nmol g-1 fresh weight of nodule s-1), plant central zone (38 nmol g-1 fresh weight of nodule s-1), and bacteroids (62 nmol g-1 fresh weight of nodule s-1) of soybean nodules to estimate the time constants for turnover of the total adenylate pool and the ATP pool within each nodule fraction. The low values for time constant (1.6-5.8 s for total adenylate, 0.9-2.5 s for ATP only) in each fraction reflect the high metabolic activity of soybean nodules and provide a background for further studies of the role of adenylates in O2-limited nodule metabolism.

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.

Role of Oxygen in the Limitation and Inhibition of Nitrogenase Activity and Respiration Rate in Individual Soybean Nodules.

Although infected cell O2 concentration (Oi) is known to limit respiration and nitrogenase activity in legume nodules, techniques have not been available to measure both processes simultaneously in an individual legume nodule. Consequently, details of the relationship between nitrogenase activity and Oi are not fully appreciated. For the present study, a probe was designed that allowed open circuit measurements of H2 evolution (nitrogenase activity) and CO2 evolution (respiration rate) in a single attached soybean nodule while simultaneously monitoring fractional oxygenation of leghemoglobin (and thereby Oi) with a nodule oximeter. Compared to measurements of whole nodulated roots, use of the probe led to inhibition of nitrogenase activity in the single nodules. During oximetry measurements, total nitrogenase activity (TNA; peak H2 evolution in Ar/O2) in the single nodules was 16% of that in whole nodulated roots and 48% of nodulated root activity when Oi was not being measured simultaneously. This inhibition did not affect the nodules' ability to regulate Oi, because exposure to Ar/O2 (80:20, v/v) caused nitrogenase activity and respiration rate to decline, and this decline was linearly correlated with a concurrent decrease in Oi. When the nodules were subsequently exposed to a linear increase in external pO2 from 20 to 100% O2 at 2.7% O2/min, fractional leghemoglobin oxygenation first increased gradually and then more rapidly, reaching saturation at a pO2 between 76 and 100% O2. Plots of nitrogenase activity and respiration rate against Oi showed that rates increased with Oi up to a value of 57 nM, with half-maximal rates being attained at Oi values between 10 and 14 nM O2. The maximum nitrogenase activity achieved during the increase in pO2 (potential nitrogenase activity) was 30 to 57% of that measured in intact nodulated roots, showing that O2 limitation of nitrogenase activity could account for a significant proportion of the inhibition of TNA associated with the use of the probe. However, some factor(s) in addition to O2 must have limited the activity of single nodules at both subsaturating and saturating Oi. At Oi values greater than about 57 nM, nitrogenase activity and nodule respiration were inhibited, but, because this inhibition has been shown previously to be readily reversible when the Oi was lowered, it was not attributed to direct O2 inactivation of the nitrogenase protein. These results indicate that maximum nitrogenase activity in legume nodules is supported by a narrow range of Oi values. Possible biochemical mechanisms are discussed for both O2 limitation of nitrogenase activity at low Oi and inhibition of nitrogenase activity at high Oi.

Nitrogenase activity, nodule respiration, and o(2) permeability following detopping of alfalfa and birdsfoot trefoil.

Gas exchange measurements and noninvasive leghemoglobin (Lb) spectrophotometry (nodule oximetry) were used to monitor nodule responses to shoot removal in alfalfa (Medicago sativa L. cv Weevlchek) and birdsfoot trefoil (Lotus corniculatus L. cv Fergus). In each species, total nitrogenase activity, measured as H(2) evolution in Ar:O(2) (80:20), decreased to <50% of the initial rate within 1 hour after detopping, and net CO(2) production decreased to about 65% of the initial value. In a separate experiment in which nodule oximetry was used, nodule O(2) permeability decreased 50% within 5 hours in each species. A similar decrease in the O(2)-saturated respiration rate (V(max)) for the nodule central zone occurred within 5 hours in birdsfoot trefoil, but only after 24 hours in alfalfa. Lb concentration, also measured by oximetry, decreased after 48 to 72 hours. The decrease in permeability preceded the decrease in V(max) in each species. V(max) may depend mainly on carbohydrate availability in the nodule. If so, then the decrease in permeability could not have been triggered by decreasing carbohydrate availability. Both oximetry and gas exchange data were consistent with the hypothesis that, for the cultivars tested, carbohydrate availability decreased more rapidly in birdsfoot trefoil than in alfalfa nodules. Fractional Lb oxygenation (initially about 0.15) decreased during the first 24 hours after detopping but subsequently increased to >0.65 for a majority of nodules of each species. This increase could lead to O(2) inactivation of nitrogenase.

Effect of Increases in Oxygen Concentration during the Argon-Induced Decline in Nitrogenase Activity in Root Nodules of Soybean.

When intact nodulated roots of soybean (Glycine max L. Merr. nodulated with Bradyrhizobium japonicum strain USDA 16) were exposed to an atmosphere lacking N(2) gas (Ar:O(2) 80:20), total nitrogenase activity (measured as H(2) evolution) and respiration (CO(2) evolution) declined with time of exposure. In Ar-inhibited nodules, when the O(2) concentration in the rhizosphere was increased in a linear ;ramp' of 2.7% per minute, 93% of the original H(2) evolution and 99% of the CO(2) evolution could be recovered. The internal nodule O(2) concentration (estimated from leghemoglobin oxygenation) declined to 56% of its initial value after 60 minutes of Ar:O(2) exposure and could be partially recovered by the linear increases in O(2) concentration. Nodule gas permeability, as estimated from the lag in ethylene production following exposure of nodules to acetylene, decreased to 26% of its initial value during the Ar-induced decline. Collectively, the results provide direct evidence that the Ar-induced decline results from decreased nodule gas permeability and indicate that the decline in permeability, rather than being immediate, occurs gradually over the period of Ar:O(2) exposure.

Measurement of legume nodule respiration and o(2) permeability by noninvasive spectrophotometry of leghemoglobin.

Physiological regulation of nodule gas permeability has a central role in the response of legumes to such diverse factors as drought, defoliation, and soil nitrate. A new method for quantifying nodule respiration and O(2) permeability, based on noninvasive spectrophotometry of leghemoglobin, was evaluated using intact, attached nodules of Lotus corniculatus. First, the relationship between nodule respiration (O(2) consumption) rate and internal O(2) concentration was determined from the rate of decrease in fractional oxygenation of leghemoglobin (FOL) under N(2). The rate of increase of FOL under 100% O(2) was then used to calculate nodule O(2) permeability, after correcting for respiration. Inactivation of nitrogenase by exposure to 100% O(2) for 15 minutes led to decreases in both permeability and O(2)-saturated respiration (V(max)), but the brief (<15 seconds) exposures to 100% O(2) required by the assay itself had little effect on either parameter. A gradual increase in external O(2) concentration from 20 to 40% resulted in a reversible decrease in permeability, but no change in V(max). The new method is likely to be useful for research on nodule physiology and might also be applicable to agronomic research and crop improvement programs.

Mechanism of Nitrogenase Inhibition in Soybean Nodules : Pulse-Modulated Spectroscopy Indicates that Nitrogenase Activity Is Limited by O(2).

A novel, pulse-modulated spectroscopic system for measuring fractional leghemoglobin oxygenation and infected cell O(2) concentration (O(i)) in intact attached nodules of soybean (Glycine max) is described. The system is noninvasive and uses a pulsed (1000 Hertz) light-emitting diode coupled to an optical fiber to illuminate the nodule with light at 660 nanometer. A second optical fiber receives a portion of the light reflected from the nodule and directs this to a photodiode. A lock-in amplifier measures only the signal from the photodiode which is in phase with the pulsed light from the light-emitting diode, and the voltage output from the amplifier, proportional to reflectance, is used to calculate fractional leghemoglobin oxygenation and the nanomolar concentration of free O(2) in the infected cells of the nodule (O(i)). The system was used to show that inhibition of nitrogenase activity in soybean nodules by NO(3) (-) treatment, stem-girdling, continuous darkness, or nodule disturbance is caused by a reduction in O(i) and limitation of respiration in support of nitrogenase activity. A plot of nitrogenase activity (measured as peak H(2) evolution in Ar:O(2)) versus O(i) for the various treatments was consistent with the concept that O(i) limits in vivo nitrogenase activity in legume nodules under adverse conditions. The potential for using O(i) to estimate nitrogenase activity in laboratory and field-grown legumes is discussed.

Effects of gradual increases in o(2) concentration on nodule activity in soybean.

The objectives of this study were to determine whether attached nodules of soybean (Glycine max L. Merr.) could adjust to gradual increases in rhizosphere pO(2) without nitrogenase inhibition and to determine whether the nitrogenase activity of the nodules is limited by pO(2) under ambient conditions. A computer-controlled gas blending apparatus was used to produce linear increases (ramps) in pO(2) around attached nodulated roots of soybean plants in an open gas exchange system. Nitrogenase activity (H(2) production in N(2):O(2) and Ar:O(2)) and respiration (CO(2) evolution) were monitored continuously as pO(2) was ramped from 20 to 30 kilopascals over periods of 0, 5, 10, 15, and 30 minutes. The 0, 5, and 10 minute ramps caused inhibitions of nitrogenase and respiration rates followed by recoveries of these rates to their initial values within 30 minutes. Distinct oscillations in nitrogenase activity and respiration were observed during the recovery period, and the possible basis for these oscillations is discussed. The 15 and 30 minute ramps did not inhibit nitrogenase activity, suggesting that such inhibition is not a factor in the regulation of nodule diffusion resistance. During the 30 minute ramp, a stimulation of nitrogenase activity was observed, indicating that an O(2)-based limitation to nitrogenase activity occurs in soybean nodules under ambient conditions.