Phosphorus deficiency increases the argon-induced decline of nodule nitrogenase activity in soybean and alfalfa

Open-flow assays of H₂ evolution in Ar:O₂ (80:20, v/v) by nodulated roots were performed in situ with soybean [Glycine max (L.) Merr.] and alfalfa [Medicago sativa L.) grown in sand with orthophosphate (Pi) nutrition either limiting (low-P) or non-limiting (control) for plant growth. Nodule growth was more limited than shoot growth by P deficiency. Phosphorus concentration was less affected in nodules than in other parts of the low-P plants. During assays, nitrogenase activity declined a few minutes after exposure of the nodulated roots to Ar. The magnitude of this argon-induced decline (Ar-ID) was less in alfalfa than in soybean. In both symbioses the magnitude of the Ar-ID was larger in low-P than control plants. Moreover, the minimum H₂ evolution after the Ar-ID, was reached earlier in low-P plants. The Ar-ID was partly reversed by raising the external partial pressure of O₂ in the rhizosphere. The magnitude of the Ar-ID in soybean was correlated negatively to nodule and shoot mass per plant, individual nodule mass, H₂ evolution in air prior to the assay, and nodule N and P concentrations. Possible reasons, including nodule size and nodule O₂ permeability, for the increase in Ar-ID in P-deficient plants are discussed and an interpretation of the P effect on nodule respiration and energetic metabolism is proposed. Received: 17 May 1996 / Accepted: 16 September 1996     

Nonphotosynthetic CO(2) Fixation by Alfalfa (Medicago sativa L.) Roots and Nodules

The dependence of alfalfa (Medicago sativa L.) root and nodule nonphotosynthetic CO₂ fixation on the supply of currently produced photosynthate and nodule nitrogenase activity was examined at various times after phloem-girdling and exposure of nodules to Ar:O₂. Phloemgirdling was effected 20 hours and exposure to Ar:O₂ was effected 2 to 3 hours before initiation of experiments. Nodule and root CO₂ fixation rates of phloem-girdled plants were reduced to 38 and 50%, respectively, of those of control plants. Exposure to Ar:O₂ decreased nodule CO₂ fixation rates to 45%, respiration rates to 55%, and nitrogenase activities to 51% of those of the controls. The products of nodule CO₂ fixation were exported through the xylem to the shoot mainly as amino acids within 30 to 60 minutes after exposure to ¹⁴CO₂. In contrast to nodules, roots exported very little radioactivity, and most of the ¹⁴C was exported as organic acids. The nonphotosynthetic CO₂ fixation rate of roots and nodules averaged 26% of the gross respiration rate, i.e. the sum of net respiration and nonphotosynthetic CO₂ assimilation. Nodules fixed CO₂ at a rate 5.6 times that of roots, but since nodules comprised a small portion of root system mass, roots accounted for 76% of the nodulated root system CO₂ fixation. The results of this study showed that exposure of nodules to Ar:O₂ reduced nodule-specific respiration and nitrogenase activity by similar amounts, and that phloem-girdling significantly reduced nodule CO₂ fixation, nitrogenase activity, nodule-specific respiration, and transport of ¹⁴C photoassimilate to nodules. These results indicate that nodule CO₂ fixation in alfalfa is associated with N assimilation.     

The effect of excision on O2 diffusion and metabolism in soybean nodules

Nitrogen-fixing nodules of soybean [Glycine max (L.) Merr. cv. Maple Arrow inoculated with Bradyrhizobium japonicum USDA 16] were studied before and after excision from the root to determine the role the O₂ regulation plays in the inhibition of nodule activity and the potential for using excised nodules nodules in studies of nodule metabolism. Relative nitrogenase (EC 1.7.99.2) activity (H₂ evolution in N₂:O₂) and nodule respiration (CO₂ evolution) were monitored first in intact nodulated roots and then in freshly excised nodules of the same plant to determine the time course of the decline in nodule metabolism. Folowing excision, nitrogenase activity and respiration declined rapidly in the first minute and then more gradually. After 40 min the rate of H₂ evolution was only 14–28% of that in the intact plant. In some nodules activity declined steadily, and in others there was a partial recovery in activity ca 10 min after detachment. Infected cell O₂ concentration (O_i), measured by a spectro-photometric technique, also declined after nodule detachment with a time course similar to the declines in nitrogenase activity and respiration. Following excision, O_i levels declined rapidly from ca 21 nM in attached nodules to 8–12 nM at 4–10 min after excision and then more gradually to 2–3 nM O₂ at 30–40 min after excision. These results show that the nodules' permeability to gas diffusion continued to be regulated for up to 40 min after detachement. At 40 min after detachment, when excised nodules displayed steady-state rates of gas exchange, linear increases in pO₂ from 20 to 100% at 4% min^?1 resulted in recoveries of H² and CO² evolution, indicating that O_i limited nitrogenase activity durig this period, and that energy reserves were greatly in excess of the O₂ available for respiration. When detached nodules were equilibrated for 12 h at 20, 30 and 50% O₂, O_i values measured at supra-ambient pO₂ were greater than those at 20% O₂ and were linked with a more rapid decline in nitrogenase activity. Also, increases in external pO₂ (O_c) failed to stimulate nodule metabolism, suggesting that the nodules' energy reserves were no longer greatly in excess of their respiratory demands. It was concluded that soybean nodules could provide useful material for steady-state studies of nodule metabolism between 40 and 240 min after detachment, but to attain metabolic rates equivalent to in vivo rates the nodules must be exposed to above-ambient pO₂.     

O2 regulation and O2‐limitation of nitrogenase activity in root nodules of pea and lupin

The gas exchange characteristics of intact attached nodulated roots of pea (Pisum sativum cv. Finale X) and lupin (Lupinus albus cv. Ultra) were studied under a number of environmental conditions to determine whether or not the nodules regulate resistance to oxygen diffusion. Nitrogenase activity (H₂ evolution) in both species was inhibited by an increase in rhizosphere pO₂ from 20% to 30%, but recovered within 30 min without a significant increase in nodulated root respiration (CO₂ evolution). These data suggest that the nodules possess a variable barrier to O₂ diffusion. Also, nitrogenase activity in both species declined when the roots were either exposed to an atmosphere of Ar:O₂ or when the shoots of the plants were excised. These declines could be reversed by elevating rhizosphere pO₂, indicating that the inhibition of nitrogenase activity resulted from an increase in gas diffusion resistance and consequent O₂-limitation of nitrogenase-linked respiration. These results indicate that nodules of pea and lupin regulate their internal O₂ concentration in a manner similar to nodules of soybean, despite the distinct morphological and biochemical differences that exist between the nodules of the 3 species. Experiments in which total nitrogenase activity (TNA = H₂ production in Ar:O₂) in pea and lupin nodules was monitored while rhizosphere pO₂ was increased gradually to 100%, showed that the resistance of the nodules to O₂ diffusion maintains nitrogenase activity at about 80% of its potential activity (PNA) under normal atmospheric conditions. The O₂-limitation coefficient of nitrogenase (OLC_N= TNA/PNA) declined significantly with prolonged exposure to Ar:O₂ or with shoot excision. Together, these results indicate a significant degree of O₂-limitation of nitrogenase activity in pea and lupin nodules, and that yields may be increased by realizing full potential activity.     

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.     

Carbohydrate supply and n(2) fixation in soybean : the effect of varied daylength and stem girdling

When arrival of shoot supplied carbohydrate to the nodulated root system of soybean was interrupted by stem girdling, stem chilling, or leaf removal, nodule carbohydrate pools were utilized, and a marked decline in the rates of CO₂ and H₂ evolution was observed within approximately 30 minutes of treatment. Nodule excision studies demonstrated that the decline in nodulated root respiration was associated with nodule rather than root metabolism, since within 3.5 hours of treatment, nodules respired at less than 10% of the initial rates. Apparently, a continuous supply of carbohydrate from the shoot is required to support nodule, but not root, function. Depletion of nodular carbohydrate pools was sufficient to account for the (diminishing) nodule respiration of girdled plants. Of starch and soluble sugar pools within the whole plant, only leaf starch exhibited a diurnal variation which was sufficient to account for the respiratory carbon loss of nodules over an 8 hour night. Under 16 hour nights, or in continuous dark, first the leaf starch pools were depleted, and then nodule starch reserves declined concomitant with a decrease in the rates of CO₂ and H₂ evolution from the nodules. Nodule soluble sugar levels were maintained in dark treated plants but declined in girdled plants. The depletion of starch in root nodules is an indicator of carbohydrate limitation of nodule function.     

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.     

Nitrogenase activity and root nodule metabolism in response to O2 and short-term N2 deprivation in dark-treated Frankia-Alnus incana plants

Inhibition of nitrogenase (EC 1.18.6.1) activity by O₂ has been suggested to be an early response to disturbance in carbon supply to root nodules in the Frankia‐Alnus incana symbiosis. Intact nodulated root systems of plants kept in prolonged darkness of 22 h were used to test responses to O₂ and short‐term N₂ deprivation (1 h in Ar:O₂). By using a Frankia lacking uptake hydrogenase it was possible to follow nitrogenase activity over time as H₂ evolution in a gas exchange system. Respiration was simultaneously recorded as CO₂ evolution. Dark‐treated plants had lower initial nitrogenase activity in N₂:O₂ (68% of controls), which declined further during a 1‐h period in the assay system in N₂:O₂ at 21 and 17% O₂, but not at 13% O₂. When dark‐treated plants were deprived of N₂ at 21 and 17% O₂ nitrogenase activity declined rapidly to 61 and 74%, respectively, after 20 min, compared with control plants continuously kept in their normal light regime. In contrast, there was no decline in dark‐treated plants at 13% O₂, and only a smaller and temporary decline in control plants at 21% O₂. When dark‐treated plants were kept at 21% O₂ during 45 min prior to N₂ deprivation at 17% O₂ the decline was abolished. This supports the idea that the decline in nitrogenase activity observed in N₂:O₂ at 21% O₂ and during N₂ deprivation was caused by O₂, which affected a sensitive nodule fraction. Nodule contents of the amino acids Gln and Cit decreased during N₂ deprivation, suggesting decreased assimilation of NH₄⁺. Contents of ATP and ADP in nodules were not affected by short‐term N₂ deprivation. ATP/ADP ratios were about 5 indicating a highly aerobic metabolism in the root nodule. We conclude that nitrogenase activity of Alnus plants exposed to prolonged darkness becomes more sensitive to inactivation by O₂. It seemed that dark‐treated plants could not adjust their nodule metabolism at higher perceived pO₂ and during cessation of NH₄⁺ production.     

The role of dark carbon dioxide fixation in root nodules of soybean

The magnitude and role of dark CO₂ fixation were examined in nodules of intact soybean plants (Harosoy 63 × Rhizobium japonicum strain USDA 16). The estimated rate of nodule dark CO₂ fixation, based on a 2 minute pulse-feed with ¹⁴CO₂ under saturating conditions, was 102 micromoles per gram dry weight per hour. This was equivalent to 14% of net nodule respiration. Only 18% of this CO₂ fixation was estimated to be required for organic and amino acid synthesis for growth and export processes. The major portion (75-92%) of fixed label was released as CO₂ within 60 minutes. The labeling pattern during pulse-chase experiments was consistent with CO₂ fixation by phosphoenolpyruvate carboxylase. During the chase, the greatest loss of label occurred in organic acids. Exposure of nodulated roots to Ar:O₂ (80:20) did not affect dark CO₂ fixation, while exposure to O₂:CO₂ (95:5) resulted in 54% inhibition. From these results, it was concluded that at least 66% of dark CO₂ fixation in soybean may be involved with the production of organic acids, which when oxidized would be capable of providing at least 48% of the requirement for ATP equivalents to support nitrogenase activity.     

Experimental determination of the respiration associated with soybean/rhizobium nitrogenase function, nodule maintenance, and total nodule nitrogen fixation

The total metabolic cost of soybean (Glycine max L. Mer Clark) nodule nitrogen fixation was empirically separated into respiration associated with electron flow through nitrogenase and respiration associated with maintenance of nodule function.

Rates of CO₂ evolution and H₂ evolution from intact, nodulated root systems under Ar:O₂ atmospheres decreased in parallel when plants were maintained in an extended dark period. While H₂ evolution approached zero after 36 hours of darkness at 22°C, CO₂ evolution rate remained at 38° of the rate measured in light. Of the remaining CO₂ evolution, 62% was estimated to originate from the nodules and represents a measure of nodule maintenance respiration. The nodule maintenance requirement was temperature dependent and was estimated at 79 and 137 micromoles CO₂ (per gram dry weight nodule) per hour at 22°C and 30°C, respectively.

The cost of N₂ fixation in terms of CO₂ evolved per electron pair utilized by nitrogenase was estimated from the slope of H₂ evolution rate versus CO₂ evolution rate. The cost was 2 moles CO₂ evolved per mole H₂ evolved and was independent of temperature.

In this symbiosis, nodule maintenance consumed 22% of total respiratory energy while the functioning of nitrogenase consumed a further 52%. The remaining respiratory energy was calculated to be associated with ammonia assimilation, transport of reduced N, and H₂ evolution.

     

Effects of n(2) deficiency on transport and partitioning of C and N in a nodulated legume

Nodulated root systems of white lupin (Lupinus albus L. cv Ultra: Rhizobium strain WU425) were exposed to Ar:O₂ (80:20, v/v) or Ar:N₂:O₂ (70:10:20, v/v/v) and C and N partitioning were examined over a 9- or 10-day period in comparison with control plants with nodulated roots retained in air. Accumulation of N ceased in plants exposed to Ar:O₂ or was much reduced in plants exposed to Ar:N₂:O₂, but net C assimilation rates and profiles of C utilization remained similar to those of control N₂-fixing plants. There was, however, a proportional reduction in CO₂ evolution from nodulated roots of the Ar:O₂ treatment. Xylem N levels fell rapidly after application of Ar:O₂. C:N ratios of phloem sap of petioles and of stem base rose during the first day of Ar:O₂ treatment and then fell progressively back to levels close to that of control plants as leaf reserves of N became available for loading of phloem. Stem top phloem sap increased progressively in C:N ratio throughout Ar:O₂ treatment, presumably due to increasing shortage of xylem derived N for xylem to phloem exchange. Reexposure of Ar:O₂-treated nodulated root systems to air prompted a rapid recovery of N₂ fixation and restoration of plant N status. Rates of N₂ fixation in plants whose roots were exposed to a range of N₂ concentrations indicated an apparent K_m of 10% N₂ for the attached intact white lupin nodule.     

Nitrogen Fixation by Subterranean Clover at Varying Stages of Nodule Dehydration: I. CARBOHYDRATE STATUS AND SHORT-TERM RECOVERY OF NODULATED ROOT RESPIRATION

The nodule water potential (_nod) of subterranean clover (Trifoliumsubterraneum L.) cv. Seaton Park incubated in a flow-throughgas-exchange system was induced to decline independently ofleaf water potential (₁) by passing a continuous dry airstreamover the nodulated roots of intact well-watered plants. Reducedtranspiration by plants whose nodules had become dehydratedwas hypothesized to be related to the decline in nitrogen fixation.Whole-plant and nodule soluble carbohydrates increased as _noddeclined. Throughout an 8 d period of continual nodule dehydration,the gaseous diffusion resistance of nodules increased and theoptimum pO₂ for nitrogenase activity declined from 52 to 28kPa. Following rehydration of the nodulated roots between days4 and 5 and between days 7 and 8, nodulated root respirationincreased to or above pre-stress levels whereas nitrogenaseactivity did not recover. Re-establishment of initial ratesof nodulated root respiration was due to the stimulation ofgrowth and maintenance respiration, not to the respiration coupledto nitrogenase activity. Although no recovery of nitrogenaseactivity occurred, the elapsed time from the introduction ofacetylene into the gas stream flowing past the nodules untilmeasurement of the acetylene-induced decline in nitrogenaseactivity, decreased substantially. This was characteristic ofan increase in the permeability of the nodules to gaseous diffusionupon rehydration. However, calculated values of nodule diffusionresistance after the 24 h periods of rehydration did not indicateany recovery of gaseous diffusion resistance based on measurementsof the respiration coupled to nitrogenase activity. Hence, useof a diffusion analogue (i.e. Fick's Law) in conjunction withnodule respiratory CO₂ efflux was unable to predict changesin permeability of the variable barrier of legume nodules followingnodule dehydration and recovery. Key words: Subterranean clover, gaseous diffusion, respiration, carbohydrates, drought     

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.     

Effect of localized nitrogen availability to soybean half-root systems on photosynthate partitioning to roots and nodules

Soybean (Glycine max [L.] Merr. cv Davis) was grown in a split-root growth system designed to maintain control of the root atmosphere. Two experiments were conducted to examine how 80% Ar:20% O₂ (Ar:O₂) and air (Air) atmospheres affected N assimilation (NH₄NO₃ and N₂ fixation) and the partitioning of photosynthate to roots and nodules. Application of NH₄NO₃ to nonnodulated half-root systems enhanced root growth and root respiration at the site of application. A second experiment applied Ar:O₂ or air to the two sides of nodulated soybean half-root systems for 11 days in the following combinations: (a) Air to both sides (Air/Air); (b) Air to one side, Ar:O₂ to the other (Air/Ar:O₂), and (c) Ar:O₂ to both sides (Ar:O₂/Ar:O₂). Results indicated that dry matter and current photosynthate (¹⁴C) were selectively partitioned to nodules and roots where N₂ was available. Both root and nodule growth on the Air side of Air/Ar:O₂ plants was significantly greater than the Ar:O₂ side. The relative partitioning of carbon and current photosynthate between roots and nodules on a half-root system was also affected by N₂ availability. The Ar:O₂ sides partitioned relatively more current photosynthate to roots (57%) than nodules (43%), while N₂-fixing root systems partitioned 36 and 64% of the carbon to roots and nodules, respectively. The Ar:O₂ atmosphere decreased root and nodule respiration by 80% and nitrogenase activity by 85% compared to half-root systems in Air while specific nitrogenase activity of nodules in Ar:O₂ was 50% of nodules supplied Air. Results indicated that nitrogen assimilation, whether from N₂ fixation or inorganic sources, had a localized effect on root development. Nodule development accounted for the major decrease in total photosynthate partitioning to non-N₂-fixing nodules. Soybean compensates for ineffective nodulation by controlling the flux of carbon to ineffective nodules and their associated roots.     

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.     

C2H2 and Ar induce rapid declines in nitrogenase activity and CO2 evolution in nodules of Datisca glomerata

Preliminary studies have indicated that after addition of C₂H₂ there is a rapid decline in nitrogenase activity in the nodules of Datisca glomerata . The present work was undertaken to determine whether (1) there is also a decline in respiration and (2) the decline is associated with the cessation of ammonia production. The rates of C₂H₄ and CO₂ evolution by nodulated root systems of Datisca were measured as a function of time after exposure to C₂H₂. The peak rate of C₂H₄ evolution occurred at 30 s after C₂H₂ exposure, while the rate of CO₂ evolution started to decline at 60 s after exposure to C₂H₂. Incubation of nodules in a gas mixture containing Ar also caused a decline in CO₂ evolution. Further, pretreatment with Ar eliminated most of the C₂H₂-induced decline in nitrogenase activity and CO₂ evolution. These C₂H₂- and Ar-induced declines in Datisca nodules are more rapid than those reported in any other nodules. They are evidence that continued ammonia formation is essential for maintenance of normal nitrogenase activity in Datisca nodules.     

Can a limitation in pholem supply to nodules account for the inhibitory effect of nitrate on nitrogenase activity in soybean?

Within 48 h of exposure of nodulated soybean [Glycine max (L.) Merr. cv. Harosoy 63 x Bradyrhizobium japonicum USDA 16] to 10 mM NO₃, significant decreases were observed in nodule-specific nitrogenase (EC 1.7.99.2) activity and CO₂ evolution and in the proportion of [¹⁴C]-labeled photosynthate partitioned to nodule biomass and respiration. These trends continued over the subsequent 3 days of the study period. Concomitant with these events was an 137% increase in the relative growth rate of the whole plant and a cessation in nodule growth. Although the concentration of total soluble sugar in nodules was not affected by NO₃ treatment, the concentration of starch declined to 13% of the control level after 2 days exposure to NO₃^?. In contrast to the effects of NO₃^?, nodules in which nitrogenase activity was partially inhibited by a 30 min exposure to 100% O₂, showed a 52% increase over control in the starch pool over a 72 h period. The results were compared with recent studies of NO₃^? inhibition of nitrogenase activity in legumes, and in contrast to these studies it was concluded that the inhibitory effects of NO₃^? could be accounted for by alterations in photosynthate partitioning to nodules. A hypothesis is proposed which attempts to account for the recent observation (J. K. Vessey, K. B. Walsh, and D. B. Layzell 1988. Physiol. Plant. 73: 113–121) that nitrogenase activity in phloem-limited and nitrate-inhibited nodules is limited by O₂ diffusion. This hypothesis separates the concepts of photosynthate partitioning and phloem supply from that of carbohydrate deprivation and related effects on the size of the carbohydrate pools in nodules.     

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     

Effect of acetylene on root respiration and acetylene reducing activity in nodulated soya bean

Acetylene decreased root and nodule respiration, as measured by CO₂ evolution of nodulated or non-nodulated Glycine max. An inhibition of 25 to 35% in 15 to 30 minutes occurred when 13% C₂H₂ was introduced in the gas flux which aerated the root nutrient solution. When the light intensity was doubled to 800 microeinsteins per square meter per second, the inhibition increased to 50% and nodule acetylene reduction activity was inhibited 50%.     

Glycolytic Flux Is Adjusted to Nitrogenase Activity in Nodules of Detopped and Argon-Treated Alfalfa Plants

To investigate the short-term (30–240 min) interactions among nitrogenase activity, NH₄⁺ assimilation, and plant glycolysis, we measured the concentrations of selected C and N metabolites in alfalfa (Medicago sativa L.) root nodules after detopping and during continuous exposure of the nodulated roots to Ar:O₂ (80:20, v/v). Both treatments caused an increase in the ratios of glucose-6-phosphate to fructose-1,6-bisphosphate, fructose-6-phosphate to fructose-1,6-bisphosphate, phosphoenolpyruvate (PEP) to pyruvate, and PEP to malate. This suggested that glycolytic flux was inhibited at the steps catalyzed by phosphofructokinase, pyruvate kinase, and PEP carboxylase. In the Ar:O₂-treated plants the apparent inhibition of glycolytic flux was reversible, whereas in the detopped plants it was not. In both groups of plants the apparent inhibition of glycolytic flux was delayed relative to the decline in nitrogenase activity. The decline in nitrogenase activity was followed by a dramatic increase in the nodular glutamate to glutamine ratio. In the detopped plants this was coincident with the apparent inhibition of glycolytic flux, whereas in the Ar:O₂-treated plants it preceded the apparent inhibition of glycolytic flux. We propose that the increase in the nodular glutamate to glutamine ratio, which occurs as a result of the decline in nitrogenase activity, may act as a signal to decrease plant glycolytic flux in legume root nodules.