Measurement of the fractional oxygenation of leghemoglobin in intact detached pea nodules by reflectance spectroscopy

A method is presented for the rapid measurement of the spectral properties of detached nodules of pea (Pisum sativum L. cv “Sparkle”) by diffuse reflectance spectroscopy. After correcting the spectra for surface light scattering, the spectrum of leghemoglobin is obtained. From this, the fractional oxygenation of leghemoglobin and the internal O₂ concentration can be calculated. With this method, we determined internal O₂ while measuring nitrogenase activity (C₂H₂) in detached pea nodules over a range of external O₂ concentrations. Nitrogenase activity was maximum when leghemoglobin was 25% oxygenated, corresponding to a calculated free O₂ concentration of 45 nanomolar in infected cells. Advantages of this method over previous methods which employed transmitted light are: (a) many nodules can be assayed simultaneously, (b) nitrogenase activity (C₂H₂) can be determined at the same time as spectra are recorded, and (c) spectra can be obtained from nodules submerged in buffer containing metabolic effectors.     

Control of Seed Respiration and Growth in Vicia faba by Oxygen and Temperature: No Evidence for an Oxygen Diffusion Barrier

The rate of dry matter accumulation by seeds of Vicia faba L. cv. Minica increases with temperature in the range of 16 to 26°C. The duration of dry matter accumulation decreases with temperature, resulting in a decrease of final seed dry weight. In this study we test the hypothesis that a diffusion barrier for O₂, located in the seed coat, inhibits seed respiration and growth. The rate of O₂ uptake of intact seeds and of excised embryos and seed coats (separated seeds) was measured in air and buffer at 16, 20, and/or 26°C at various O₂ concentrations and developmental stages. Oxygen uptake rates of intact seeds in buffer were only 9 to 15% of those in air. In buffer, the respiration rate of intact seeds decreased at a pO₂ below air saturation (21 kilopascals), whereas separated seeds showed a decline of O₂ uptake only below 80% of air saturation. In air, embryo excision had no effect on the sensitivity of seed respiration to pO₂, at both 20 and 26°C. In air at 20°C, separated and intact seeds showed similar rates of O₂ uptake. Oxygen uptake by intact seeds, both halfway and beyond the linear growth phase, showed a temperature coefficient Q₁₀ of 2.3 and was insensitive to pO₂ in the range of 80 to 100% of ambient. These results indicate that V. faba seed respiration in air is not limited by the diffusion of O₂ into the seed.     

Nitrogen Fixation (C(2)H(2) Reduction) by Broad Bean (Vicia faba L.) Nodules and Bacteroids under Water-Restricted Conditions

Water potentials of leaves and nodules of broad bean (Vicia faba L.) cultivated on a sandy mixture were linearly and highly (r² = 0.99) correlated throughout a water deprivation of plants. A decrease of 0.2 megapascal of the nodule water potential (Ψ_nod) induced an immediate 25% inhibition of the highest level of acetylene reduction of broad bean nodules attached to roots. This activity continued to be depressed when water stress increased, but the effect was less pronounced. Partial recovery of optimal C₂H₂ reduction capacity of mildly water stressed nodules (Ψ_nod = −1.2 megapascals) was possible by increasing the external O₂ partial pressure up to 60 kilopascals. The dense packing of the cortical cells of nodules may be responsible for the limitation of O₂ diffusion to the central tissue. Bacteroids isolated from broad bean nodules exhibited higher N₂ fixation activity with glucose than with succinate as an energy-yielding substrate. Bacteroids from stressed nodules appeared more sensitive to O₂, and their optimal activity declined with increasing nodule water deprivation. This effect could be partly due to decreased bacteroid respiration capacity with water stress. Water stress was also responsible for a decrease of the cytosolic protein content of the nodule and more specifically of leghemoglobin. The alteration of the bacteroid environment appears to contribute to the decline in N₂ fixation under water restricted conditions.     

Effects of carbohydrate on the internal oxygen concentration, oxygen uptake, and nitrogenase activity in detached pea nodules

The interaction between carbon substrates and O₂ and their effects on nitrogenase activity (C₂H₂) were examined in detached nodules of pea (Pisum sativum L. cv “Sparkle”). The internal O₂ concentration was estimated from the fractional oxygenation of leghemoglobin measured by reflectance spectroscopy. Lowering the endogenous carbohydrate content of nodules by excising the shoots 16 hours before nodule harvest or by incubating detached nodules at 100 kPa O₂ for 2 hours resulted in a 2- to 10-fold increase in internal O₂, and a decline in nitrogenase activity. Conversely, when detached nodules were supplied with 100 millimolar succinate, the internal O₂ was lowered. Nitrogenase activity was stimulated by succinate but only at high external O₂. Oxygen uptake increased linearly with external O₂ but was affected only slightly by the carbon treatments. The apparent diffusion resistance in the nodule cortex was similar in all of the treatments. Carbon substrates can thus affect nitrogenase activity indirectly by affecting the O₂ concentration within detached nodules.     

Effects of ambient oxygen and of fixed nitrogen on concentrations of glutathione, ascrobate, and associated enzymes in soybean root nodules

Soybean (Glycine max [L.] Merr.) root nodules contain the enzymes of the ascorbate-glutathione cycle for defense against activated forms of oxygen. Nodulated roots of hydroponically grown soybean plants were exposed to atmospheres containing 2, 21, 50, or alternating 21 and 50 kilopascals of O₂. The activities of ascorbate (ASC) peroxidase, monodehydroascorbate (MDHA) reductase, dehydroascorbate (DHA) reductase, and glutathione (GSSG) reductase were higher in nodules exposed to high pO₂. Nodule contents of ascorbate and reduced glutathione were also greater in the high pO₂ treatments. Treatment of nodulated plants with fixed nitrogen (urea) led to concomitant decreases in acetylene reduction activity, in leghemoglobin content, and in activities of ASC peroxidase, DHA reductase, and GSSG reductase. Activity of MDHA reductase and glutathione concentrations in nodules were not affected by treatment with urea. The enzymes of the ascorbate-glutathione cycle were also detected in uninfected soybean roots, although at levels substantially below those in nodules. These observations indicate that the ascorbate-glutathione cycle can adjust to varying physiological conditions in nodules and that there is a key link between N₂ fixation and defenses against activated forms of oxygen.     

Effect of pO(2) on the Formation and Status of Leghemoglobin in Nodules of Cowpea and Soybean

Nodulated cowpea (Vigna unguiculata [L.] Walp. cv Vita 3: Bradyrhizobium strain CB756) and soybean (Glycine max [L.] Merr. cv White Eye: Bradyrhizobium strain CB1809) were grown with their root systems maintained in a flowing gas stream containing a range of pO₂ (1-80%, v/v) in N₂ for up to 28 days after planting. At the extremes of sub- and supra-ambient pO₂, the levels of leghemoglobin (Lb) in nodules were reduced. However, neither the proportional composition of Lb component proteins (eight in soybean, three in cowpea) nor their oxidation state was affected by pO₂. Short-term changes in pO₂ (transferring plants grown with sub- or supra-ambient pO₂ in the rhizosphere to air or vice versa) caused a significant decline in Lb content and, in cowpea but not soybean, where pO₂ was increased, a higher percentage of oxidation of Lb. Combining data on changes in Lb level of cowpea nodules grown in sub-ambient pO₂ with those for their structural adaptation to an under supply of O₂ indicated that, despite the nodules having a lower level of Lb, the amount per infected cell was increased by up to twofold and per bacteroid up to fivefold (in those from 1% O₂) compared to those grown in air. Progressive decline in pO₂ resulted in a progressive increase on this basis, indicating a close relationship between Lb content and the adaptation of nodule functioning to external O₂ level.     

Effect of pO(2) during Growth on the Gaseous Diffusional Properties of Nodules of Cowpea (Vigna unguiculata L. Walp.)

Adaptations of nodules of cowpea (Vigna unguiculata L. Walp. cv Vita 3: Bradyrhizobium CB 756) to growth in pO₂ ranging from 1 to 80% O₂ (volume/volume) involved both readily reversible mechanisms of adjustment and more stable alterations which together resulted in nodules with widely ranging resistance to diffusion of gases. Those grown in subambient pO₂ (1-5% O₂ were altered such that rapid diffusional adjustment was unable to prevent irreversible loss of nitrogenase on their transfer to higher levels of O₂. Those cultured in 80% had adapted to over-supply of O₂ such that their transfer to lower levels of O₂ limited both nitrogenase and respiratory CO₂ release. There was also some evidence for `protective respiration.' Measurement of diffusional properties based on gas exchange kinetics indicated that gaseous permeability values for nodules from 5 to 40% O₂ were relatively constant around 20 × 10⁻³ millimeters per second, while those for nodules from 1% O₂ were as high as 67.7 × 10⁻³ millimeter per second and from 80% as low as 6.8 × 10⁻³ millimeters per second. Estimates of the thickness of the diffusion barrier ranged from 7.5 micrometers for nodules from 1% O₂ to 71.9 micrometers in those from 80% O₂.     

Effect of Oxygen and Malate on NO(3) Inhibition of Nitrogenase in Soybean Nodules

Soybean (Glycine max cv Hodgson) nitrogenase activity (C₂H₂ reduction) in the presence or absence of nitrate was studied at various external O₂ tensions. Nitrogenase activity increased with oxygen partial pressure up to 30 kilopascals, which appeared to be the optimum. A parallel increase in ATP/ADP ratios indicated a limitation of respiration rate by low O₂ tensions in the nodule, and the values found for adenine nucleotide ratios suggested that the nitrogenase activity was limited by the rate of ATP regeneration. In the presence of nitrate, the nitrogenase activity was low and less stimulated by increased pO₂, although the nitrite content per gram of nodules decreased from 0.05 to 0.02 micromole when pO₂ increased from 10 to 30 kilopascals. Therefore, the accumulation of nitrite inside the nodule was probably not the major cause of the inhibition. Instead, inhibition by nitrate could be due to competition for reducing power between nitrate reduction and bacteroid or mitochondrial respiration inside the nodule. This is supported by the observation of decrease in ATP/ADP ratios from 1.65, in absence of nitrate, to 0.93 in the presence of this anion at 30 kilopascals O₂. Furthermore, the inhibition was suppressed by the addition, to the plant nutrient solution, of 15 millimolar l-malate, a carbon substrate that is considered to be the major source of reductant for the bacteroids in the symbiosis.     

Reversible o(2) inhibition of nitrogenase activity in attached soybean nodules

Various forms of stress result in decreased O₂ permeability or decreased capacity to consume O₂ in legume root nodules. These changes alter the nodule interior O₂ concentration (O_i). To determine the relationship between O_i and nitrogenase activity in attached soybean (Glycine max) nodules, we controlled O_i by varying external pO₂ while monitoring internal H₂ concentration (H_i) with microelectrodes. O_i was monitored by noninvasive leghemoglobin spectrophotometry (nodule oximetry). After each step-change in O_i, H_i approached a new steady state, with a time constant averaging 23 s. The rate of H₂ production by nitrogenase was calculated as the product of H_i, nodule surface area, and nodule H₂ permeability. H₂ permeability was estimated from O₂ permeability (measured by nodule oximetry) by assuming diffusion through air-filled pores; support for this assumption is presented. O_i was nearly optimal for nitrogenase activity (H₂ production) between 15 and 150 nm. A 1- to 2-min exposure to elevated external pO₂ (40-100 kPa) reduced H_i to zero, but nitrogenase activity recovered quickly under air, often in <20 min. This rapid recovery contrasts with previous reports of much slower recovery with longer exposures to elevated pO₂. The mechanism of nitrogenase inhibition may differ between brief and prolonged O₂ exposures.     

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 pO₂ without nitrogenase inhibition and to determine whether the nitrogenase activity of the nodules is limited by pO₂ under ambient conditions. A computer-controlled gas blending apparatus was used to produce linear increases (ramps) in pO₂ around attached nodulated roots of soybean plants in an open gas exchange system. Nitrogenase activity (H₂ production in N₂:O₂ and Ar:O₂) and respiration (CO₂ evolution) were monitored continuously as pO₂ 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 O₂-based limitation to nitrogenase activity occurs in soybean nodules under ambient conditions.     

Elicitor-Inducible Caffeoyl-Coenzyme A 3-O-Methyltransferase from Petroselinum crispum Cell Suspensions : Purification, Partial Sequence, and Antigenicity

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 O₂ permeability, based on noninvasive spectrophotometry of leghemoglobin, was evaluated using intact, attached nodules of Lotus corniculatus. First, the relationship between nodule respiration (O₂ consumption) rate and internal O₂ concentration was determined from the rate of decrease in fractional oxygenation of leghemoglobin (FOL) under N₂. The rate of increase of FOL under 100% O₂ was then used to calculate nodule O₂ permeability, after correcting for respiration. Inactivation of nitrogenase by exposure to 100% O₂ for 15 minutes led to decreases in both permeability and O₂-saturated respiration (V_max), but the brief (<15 seconds) exposures to 100% O₂ required by the assay itself had little effect on either parameter. A gradual increase in external O₂ 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 O₂ 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 O₂ in the infected cells of the nodule (O_i). The system was used to show that inhibition of nitrogenase activity in soybean nodules by NO₃⁻ 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 H₂ evolution in Ar:O₂) 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.     

Steady and nonsteady state gas exchange characteristics of soybean nodules in relation to the oxygen diffusion barrier

An open gas exchange system was used to monitor the nonsteady state and steady state changes in nitrogenase activity (H₂ evolution in N₂:O₂ and Ar:O₂) and respiration (CO₂ evolution) in attached, excised, and sliced nodules of soybean (Glycine max L. Merr.) exposed to external pO₂ of 5 to 100%. In attached nodules, increases in external pO₂ in steps of 10 or 20% resulted in sharp declines in the rates of H₂ and CO₂ evolution. Recovery of these rates to values equal to or greater than their initial rates occurred within 10 to 60 minutes of exposure to the higher pO₂. Recovery was more rapid at higher initial pO₂ and in Ar:O₂ compared to N₂:O₂. Sequential 10% increments in pO₂ to 100% O₂ resulted in rates of H₂ evolution which were 1.4 to 1.7 times the steady state rate at 20% O₂ in Ar. This was attributed to a relief at high pO₂ from the 40% decline in nitrogenase activity that was induced by Ar at a pO₂ of 20%. Changes in nodule respiration rate could not account for the nodules' ability to adjust to high external pO₂, supporting the hypothesis that soybean nodules have a variable barrier to O₂ diffusion which responds slowly (within minutes) to changes in pO₂. Nodule excision and slicing resulted in 45 and 78% declines, respectively, in total specific nitrogenase activity at 20% O₂. In contrast with the result obtained with intact nodules, subsequent 10% increases in pO₂ in Ar:O₂ did not result in transient declines in H₂ evolution rates, but in the rapid attainment of new steady state rates. Also, distinct optima in nitrogenase activity were observed at about 60% O₂. These results were consistent with an increase in the diffusive resistance of the nodule cortex following nodule excision or nodule slicing. This work also shows the importance of using intact plants and continuous measurements of gas exchange in studies of O₂ diffusion and nitrogenase activity in legume nodules.     

Adaptation of Nodulated Soybean (Glycine max L. Merr.) to Growth in Rhizospheres Containing Nonambient pO(2)

Nodulated soybean (Glycine max L. Merr. cv White Eye inoculated with Bradyrhizobium japonicum strain CB 1809) plants were cultured in the absence of combined N from 8 to 28 days with their root systems maintained continuously in 1, 2.5, 5, 10, 20, 40, 60, or 80% O₂ (volume/volume) in N₂. Plant dry matter yield was unaffected by partial pressure of oxygen (pO₂) and N₂ fixation showed a broad plateau of maximum activity from 2.5 to 40 or 60% O₂. Slight inhibition of nitrogenase activity occurred at 1% O₂ and as much as 50% inhibition occurred at 80% O₂. Low pO₂ (less than 10%) decreased nodule mass on plants, but this was compensated for by those nodules having higher specific nitrogenase activities. Synthesis and export of ureides in xylem was maintained at a high level (70-95% of total soluble N in exudate) over the range of pO₂ used. Measurements of nitrogenase (EC 1.7.99.2) activity by acetylene reduction indicated that adaptation of nodules to low pO₂ was largely due to changes in ventilation characteristics and involved increased permeability to gases in those grown in subambient pO₂ and decreased permeability in those from plants cultured with their roots in pO₂ greater than ambient. A range of structural alterations in nodules resulting from low pO₂ were identified. These included increased frequency of lenticels, decreased nodule size, increased volume of cortex relative to the infected central tissue of the nodule, as well as changes in the size and frequency of extracellular voids in all tissues. In nodules grown in air, the inner cortex differentiated a layer of four or five cells which formed a band, 40 to 50 micrometers thick, lacking extracellular voids. This was reduced in nodules grown in low pO₂ comprising one or two cell layers and being 10 to 20 micrometers thick in those from 1% O₂. Long-term adaptation to different external pO₂ involved changes which modify diffusive resistance and are additional to adjustments in the variable diffusion barrier.     

Response of the Endophytic Diazotroph Gluconacetobacter diazotrophicus on Solid Media to Changes in Atmospheric Partial O2 Pressure

Gluconacetobacter diazotrophicus is an N₂-fixing endophyte isolated from sugarcane. G. diazotrophicus was grown on solid medium at atmospheric partial O₂ pressures (pO₂) of 10, 20, and 30 kPa for 5 to 6 days. Using a flowthrough gas exchange system, nitrogenase activity and respiration rate were then measured at a range of atmospheric pO₂ (5 to 60 kPa). Nitrogenase activity was measured by H₂ evolution in N₂-O₂ and in Ar-O₂, and respiration rate was measured by CO₂ evolution in N₂-O₂. To validate the use of H₂ production as an assay for nitrogenase activity, a non-N₂-fixing (Nif⁻) mutant of G. diazotrophicus was tested and found to have a low rate of uptake hydrogenase (Hup⁺) activity (0.016± 0.009 μmol of H₂ 10¹⁰ cells⁻¹ h⁻¹) when incubated in an atmosphere enriched in H₂. However, Hup⁺ activity was not detectable under the normal assay conditions used in our experiments. G. diazotrophicus fixed nitrogen at all atmospheric pO₂ tested. However, when the assay atmospheric pO₂ was below the level at which the colonies had been grown, nitrogenase activity was decreased. Optimal atmospheric pO₂ for nitrogenase activity was 0 to 20 kPa above the pO₂ at which the bacteria had been grown. As atmospheric pO₂ was increased in 10-kPa steps to the highest levels (40 to 60 kPa), nitrogenase activity decreased in a stepwise manner. Despite the decrease in nitrogenase activity as atmospheric pO₂ was increased, respiration rate increased marginally. A large single-step increase in atmospheric pO₂ from 20 to 60 kPa caused a rapid 84% decrease in nitrogenase activity. However, upon returning to 20 kPa of O₂, 80% of nitrogenase activity was recovered within 10 min, indicating a “switch-off/switch-on” O₂ protection mechanism of nitrogenase activity. Our study demonstrates that colonies of G. diazotrophicus can fix N₂ at a wide range of atmospheric pO₂ and can adapt to maintain nitrogenase activity in response to both long-term and short-term changes in atmospheric pO₂.     

Frankia vesicles provide inducible and absolute oxygen protection for nitrogenase

When Frankia HFPCcI3 was grown in culture at oxygen O₂ levels ranging from 2 to 70 kilopascals O₂, under nitrogen fixing conditions, nitrogenase activity adapted to ambient pO₂ and showed a marked optimum close to growth pO₂. Vesicles were thin walled at low pO₂ and very thick walled at high pO₂. Freeze fracture transmission electron microscopy confirmed that Frankia produces vesicles with outer walls thickened by multiple lipid-like monolayers, in proportion to ambient pO₂.     

Appearance of purine-catabolizing enzymes in fix and fix root nodules on soybean and effect of oxygen on the expression of the enzymes in callus tissue

The appearance of enzymes involved in the formation of ureides, allantoin, and allantoic acid, from inosine 5′-monophosphate was analyzed in developing root nodules of soybean (Glycine max). Concomitant with development of effective nodules, a substantial increase in specific activities of the enzymes 5′-nucleotidase (35-fold), purine nucleosidase (10-fold), xanthine dehydrogenase (25-fold), and uricase (200-fold), over root levels was observed. The specific activity of allantoinase remained constant during nodule development. With ineffective nodules the activities were generally lower than in effective nodules; however, the activities of 5′-nucleotidase and allantoinase were 2-fold higher in ineffective nodules unable to synthesize leghemoglobin than in effective nodules. Since the expression of uricase has been shown to be regulated by oxygen (K Larsen, BU Jochimsen 1986 EMBO J 5: 15-19), the expression of the remaining enzymes in the purine catabolic pathway were tested in response to variations in O₂ concentration in sterile soybean callus tissue. Purine nucleosidase responded to this treatment, exhibiting a 4-fold increase in activity around 2% O₂. 5′-Nucleotidase, xanthine dehydrogenase, and allantoinase remained unaffected by variations in the O₂ concentration. Hence, the expression of two enzymes involved in ureide formation, purine nucleosidase and uricase, has been demonstrated to be influenced by O₂ concentration.     

Nicotinate, nicotinamide, and the reactivity of leghemoglobin in soybean root nodules

Nicotinate has been postulated to interfere with the binding of O₂ to ferrous leghemoglobin in soybean (Glycine max) root nodules. For such a function, the levels of nicotinate in nodules must be sufficiently high to bind a significant amount of leghemoglobin. We have measured levels of nicotinate, nicotinamide, and leghemoglobin in soybean nodules from plants 34 to 73 days after planting in a glasshouse. On a per gram nodule fresh weight basis, levels between 10.4 and 21 nanomoles for nicotinate, 19.2 and 37.8 nanomoles for nicotinamide, and 170 to 280 nanomoles for leghemoglobin were measured. Even if all the nicotinate were bound to ferrous leghemoglobin, only 11% or less of the total leghemoglobin would be unavailable for binding O₂. Using the measured levels of nicotinate and a pH of 6.8 in the cytosol of presenescent soybean nodules, we estimate that the proportion of ferrous leghemoglobin bound to nicotinate in such nodules would be less than 1%. These levels of nicotinate are too low to interfere with the reaction between ferrous leghemoglobin and O₂ in soybean root nodules.     

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 H₂ evolution in Ar:O₂ (80:20), decreased to <50% of the initial rate within 1 hour after detopping, and net CO₂ production decreased to about 65% of the initial value. In a separate experiment in which nodule oximetry was used, nodule O₂ permeability decreased 50% within 5 hours in each species. A similar decrease in the O₂-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 O₂ inactivation of nitrogenase.     

Nodule structure and nitrogenase activity ofCoriaria arborea in response to varying pO2

When excised root nodules ofCoriaria arborea are assayed for nitrogenase activity at various pO₂ they show a broad optimum between 20 and 40 kPa O₂, with some evidence for adaptation. Continuous flow assays of nodulated root systems of intact plants indicate that Coriaria shows an acetylene induced decline in nitrogenase activity. When root systems were subject to step changes in pO₂ nitrogenase activity responded with a steep decline followed by a slower rise in activity both at lower and higher than ambient pO₂. Thus Coriaria nodules are able to adapt rapidly to oxygen levels well above and well below ambient. Measurement of nodule diffusion resistance showed that the adaptation is accompanied by rapid increase in resistance at above ambient pO₂ and decrease in resistance at below ambient pO₂. Plants grown with root systems at pO₂ from 5–40 kPa O₂ did not differ in growth or nodulation. The anatomy of Coriaria nodules shows they have a dense periderm which encircles the nodule and also closely invests the infected zone. The periderm is both thicker and more heavily suberised in nodules grown at high pO₂ than at low pO₂. Vacuum infiltration of India ink indicates that oxygen diffusion is entirely through the lenticel and via a small gap adjacent to the stele.