Plant Physiol. (1987) 84, 900-9050032-0889/87/84/0900/06/$0 1.00/0

Regulation of Soybean Nitrogen Fixation in Response toRhizosphere OxygenI. ROLE OF NODULE RESPIRATION

Received for publication November 3, 1986 and in revised form April 20, 1987

P. RANDALL WEISZ AND THOMAS R. SINCLAIR*United States Department ofAgriculture, Agricultural Research Service, Agonomy Physiology Laboratory,University ofFlorida, Gainesville, Florida 32611

ABSTRACT

Nitrogen fixation (acetylene reduction) rates of nodules on intact field-grown soybean (Glycine max) subjected to altered oxygen concentration(0.06-0A cubic millimeter per cubic millimeter) returned to initial ratesduring an 8-hour transitory period. Hydroponically grown soybean plantsalso displayed a transitory (1-4 hours) response to changes in therhizosphere oxygen concentration after which the fixation rates returnedto those observed under ambient oxygen concentrations. It was hypothe-sized that soybean nodules contain a regulatory mechanism which main-tains a stable oxygen concentration inside nodules at a sufficiently lowconcentration to allow nitrogenase to function. A possible physiologicalmechanism which could account for this regulation is adjustment innodule respiration activity such that nodule oxygen concentration andnitrogen fixation are maintained at stable levels. Experiments designedto characterize the non-steady-state oxygen response and to test for thepresence of nodule respiratory control are presented. Non-steady-stateacetylene reduction and nodule respiration (oxygen uptake) rates meas-ured after alterations in the external oxygen concentration indicated thatthe regulatory mechanism required 1 to 4 hours to completely adjust tochanges in the external oxygen concentration. Steady-state nodule res-piration, however, did not respond to alterations in the rhizosphere oxygenconcentration. It was concluded that soybean nodules can adjust to a widerange of rhizosphere oxygen concentrations, but the mechanism whichcontrols nitrogen fixation rates does not involve changes in the nodulerespiration rate.

Symbiotic nitrogen fixation rates are affected by the oxygenconcentration in the rhizosphere around plant roots and nodules.Short-term experiments in which nitrogen fixation rates of de-tached soybean nodules were assayed as either acetylene reduc-tion or "5N2 uptake, demonstrated that after exposure to alteredoxygen concentrations nodule activity responded proportionallyto the change in oxygen (1, 17, 19). Similar findings have beenreported for intact nodule and root systems in a wide range oflegumes including white clover (Trifolium repens), pea (Pisumsativum), chickpea (Cicer arietinum), cowpea ( Vigna unguicu-lata), peanut (Arachis hypogea), and lupin (Lupinus albus) (28,29). This oxygen enhancement of nitrogen fixation has beenattributed by some to an artifact of nodule disturbance, detach-ment, or the use of saturating concentrations of acetylene (15,29). Oxygen effects have, however, been reported for attached,intact undisturbed nodules of field-grown soybean in the absenceof saturating acetylene (25). In this latter study, increasing therhizosphere oxygen concentration from ambient to 0.4 mm3

mm-3 resulted in a nearly twofold increase in nitrogen fixationrates.

Respiration, measured as oxygen uptake by both detachednodules and intact nodulated root systems, has also been shownto be sensitive to oxygen concentration (17, 24, 30). Since bothnodule respiration and nitrogenase activity are sensitive to theoxygen concentration at which they are assayed, it has beensuggested that the restriction ofoxygen flux by a diffusion barrierin the nodule cortex may limit nodule respiration and thereforeenergy production necessary for nitrogen fixation (11, 23, 24).

All of the experiments cited above were typically completedwithin an hour after the oxygen concentration around the nod-ules was altered, and thus only the short-term effects of alteredoxygen concentration were considered. In contrast to these ex-periments, long-term exposures to elevated or decreased rhizo-sphere oxygen concentration have failed to show a response in(a) field-grown soybean nodule activity as measured by acetylenereduction (5, 6), (b) plant growth and nitrogen content ofsoybeanand pea (14) or (c) plant and nodule mass in field-grown soybean(23). These results lead to the conclusion that rhizosphere oxygenhas little or no effect on long-term nitrogen fixation rates ofintact plants. In light of this apparent discrepancy between theshort-term and long-term studies, the first objective of this re-search was to re-evaluate the long-term effects of altered rhizo-sphere oxygen concentration on nitrogenase activity of intactfield-grown soybean plants.

Results from the experiments reported below indicated that 8h after altering the rhizosphere oxygen concentration of intactfield-grown soybean plants the nitrogen fixation rates were equiv-alent to the initial rates. This is in agreement with the findingsof Criswell et al. (5, 6) who speculated that soybean nodules maycontain a regulatory mechanism which allows steady-state nitro-genase activity to remain constant over a range of oxygen con-centrations. Thus, the oxygen effects observed in all of the short-term experiments cited above may have only been a transitoryresponse to the altered rhizosphere oxygen concentration. Thishypothesis predicts that, during the time this non-steady-stateresponse is observable, a regulatory mechanism is being activatedto return nodule activity rates back to levels comparable to thoseoriginally observed under ambient oxygen concentrations.At least two possible regulatory mechanisms might exist which

could adjust nitrogen fixation rates when the external oxygenconcentration is altered. It has been proposed that the permea-bility of the oxygen diffusion barrier in nodules might be adjust-ing in response to oxygen concentration (21). This possibility ofchanging nodule permeability to oxygen is examined experimen-tally in detail in a subsequent paper (26). However, an alternativemechanism for stabilizing nitrogen fixation rates when externaloxygen concentrations are changed is to have adjustments in

900 https://plantphysiol.orgDownloaded on January 8, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

NODULE RESPIRATION AND NITROGEN FIXATION RESPONSE TO OXYGEN

nodule respiration rates (18). Bergersen and Turner (3, 4) re-ported that isolated bacteroids from soybean nodules containfrom two to four different cytochrome oxidase systems whichmay function at different internal nodule oxygen concentrations.They demonstrated that bacteroid respiration rates can vary overa range of dissolved oxygen concentrations from approximately3. 10-7 mm3 mm-3 to 3.10` mm' mm3 and yet maintain afairly constant supply ofATP to nitrogenase for nitrogen fixation.Such a mechanism could provide the stable, low internal noduleoxygen environment necessary for nitrogenase to function byaltering the nodule respiration rate. At the same time the supplyrate of ATP to nitrogenase would remain nearly constant, re-sulting in stable nodule nitrogen fixation rates. This hypothesisimplies that nodule respiration rates vary in response to alteredoxygen concentrations and yet nitrogenase activity remains con-stant.Oxygen enrichment experiments have not been reported where

nodule respiration has been monitored through the initial 4 to 8h period during which the hypothesized respiratory regulationmechanism might be induced. Thus, it is not currently knownwhether respiratory control of nitrogen fixation rates can accountfor the adjustment in nitrogenase activity after long exposures toaltered oxygen. Short-term experiments have, however, indicatedthat respiratory control may function in response to changes inthe external oxygen concentration. Pankhurst and Sprent (17)showed that soybean acetylene reduction rates were not respon-sive to oxygen concentrations above 0.3 mm3 mm-3, whereasnodule oxygen uptake increased linearly between 0 and 1 mm3mm-3 oxygen. Similar findings were reported for intact Alnusrubra nodules (30). Sheehy et al. (21) estimated intact whiteclover nodule oxygen uptake as CO2 evolution and failed to findevidence of respiratory control at elevated oxygen concentra-tions. In their experiments (their Fig. 1B, Ref. 21) CO2 evolutionand nodule acetylene reduction both responded in a 'Michaelis-Menten' type relationship to the external oxygen concentrations.Sheehy et al. (21), however, assumed that the respiratory quotientfor these nodules was constant and equal to unity, an assumptionwhich has been reported to be incorrect (2, 17, 30) for oxygenconcentrations ranging from 0.1 to 0.4 mm3 mm3 . A decreasein the nodule respiratory quotient at elevated oxygen concentra-tions results in nodule respiration rates erroneously appearing tobe independent of oxygen concentration when estimated ascarbon dioxide evolution, even though nodule respiration asoxygen uptake continues to increase more linearly (17, 30). Theseshort-term experiments indicated that nodules may be able toadjust to long-term exposures to altered oxygen concentrationsthrough respiratory control, and that to test this hypothesisnodule respiration must be assayed as oxygen uptake. Conse-quently, the second objective of this research was to measure thenonsteady-state, long-term response of intact nodule oxygenuptake rates and nitrogen fixation rates to altered oxygen con-centrations and to test specifically for respiratory involvement inthe adjustments of nitrogen fixation rates.

MATERIALS AND METHODSField Experiments: Steady-State Response to Altered Oxygen

Concentrations. The cultivar 'Biloxi' was field grown in Gaines-ville, FL, on Arredondo fine sand soil (loamy, siliceous, hyper-thermic Grossarenic Paleudult). Biloxi is an indeterminate, ma-turity group VIII cultivar which in this experiment remainedvegetative until June 14 when the first flowers appeared. Theexperiment was terminated on June 19, at which time the plantswere at growth stage RI ( 12).

Field preparation included application of 55 g m-2 of 1-10-20(N-P205-K20) fertilizer and the incorporation of 0.3 ml m-2 oftrifluralin (a,a,a,trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine)herbicide. On April 2, 1984, rows spaced 0.9 m apart were seeded

with 30 seeds mn'. Open-ended root chambers used for theacetylene-reduction assay (9) were installed in the rows at 1-mintervals immediately after sowing. Alachlor [2-chloro-2',-6'-diethyl-N-(methyoxymethyl)acetanilide] and chlorpyrifos [0,0-diethyl-043,5,6-trichloro-2-pyridyl) phosphorothioate] were thenboth applied to the plot at a rate of 0.4 ml m-2. Sprinklerirrigation was applied to the plot to assure well-watered condi-tions.

In situ ethylene production rates of intact plants in the open-ended assay chambers were measured at four sub-saturatingacetylene concentrations (0.001, 0.004, 0.008, and 0.01 mm3mm-3) at both midday and midnight over a period of 5 to 7 d.These low acetylene concentrations do not result in the inhibitionof nitrogenase activity (9) reported for acetylene concentrationstwo orders of magnitude higher (16). The technique describedby Denison et al. (10) was used to analyze these data and todetermine the mean apparent Km for nitrogenase for all theassays performed (n = 247). The mean and standard error forthe Km was found to be 0.00380 and 0.000285 mm3 mm-3,respectively. This mean value was used to calculate the maxi-mum ethylene production rate at saturating acetylene concentra-tions (Vm.) for each assay as described by Denison and Sinclair(8).To test the long-term effects of altered oxygen concentration

around the nodules, the oxygen concentration in the rhizosphereof one set of 5 plants was altered and compared to another setof 5 plants left under ambient conditions. Each treatment wasassayed twice daily as described above for 2 to 3 d, and then onetreatment was switched from ambient oxygen to either 0.06, 0.1,0.3, 0.35, or 0.4 mm3 mm-3 in mid-afternoon approximately 8h before the next acetylene-reduction assay. Plants in both treat-ment groups were then further assayed twice a day for 2 to 6more d. To eliminate variability in the Vm., data due to differ-ences among plants in nodule mass, each Vmax estimate wasconverted to a percentage of the first value observed for a givenplant under ambient oxygen conditions. A t-test was used to testfor differences between treatment means for normalized Vmax oneach day of the experiments.Hydroponic Experiments: Non-Steady-State Response to Al-

tered Oxygen Concentrations. To be consistent with the fieldstudies, the cultivar 'Biloxi' was used for these experiments. Seedswere surface sterilized with 2% sodium hypochlorite and germi-nated on moist filter paper. After gernmination the seedlings weretransferred to growth pouches until the primary root was ap-proximately 50 mm long. The seedlings were then individuallytransferred to bored No. 3 rubber stoppers which were plf-r d inthe lid of a 1.5-L hydroponic chamber made from 102 mmdiameter PVC pipe and inoculated with a commercial Brady-rhizobium japonicum inoculum (Nitragin Corporation'). Plantswere maintained in half-strength nitrogen-free nutrient solution(13) which was continuously aerated by flowing 33 ml s-' of airthrough an aquarium glass bead bubbler in the bottom of eachhydroponic chamber. The hydroponic chambers were submergedin a water bath which regulated the temperature around the rootsand nodules to 26°C. Illumination was provided by a 'Sun-Brella'(Environmental Growth Chambers, Chagrin Falls, OH) whichconsisted of a multi-vapor metal halide lamp (General ElectricNo. E-37) in combination with one high-pressure sodium lamp(General Electric No. E- 18) in a water-cooled jacket and whichprovided 950 to 1400 iE m-2 s-' of photosynthetically activeradiation depending on position in the canopy. The photoperiodwas adjusted to a 16-h day to assure that plants remainedvegetative throughout the assay period. The air temperature

'Mention of company names or commercial products does not implyrecommendation or endorsement by the United States Department ofAgriculture over others not mentioned.

901

https://plantphysiol.orgDownloaded on January 8, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

WEISZ AND SINCLAIR

around the soybean shoots varied throughout the day averagingapproximately23°C.The day before an individual plant was to be assayed it was

transferred from the hydroponic chamber to a stainless-steel flow-through assay chamber (Fig. 1). The volume of the empty assaychamber was 20 ml. An intact plant, including the rubber stopperthrough which the stem grew in the hydroponic chamber, wasused by inserting the nodulated root system through the bore ofthe assay chamber until the rubber stopper sealed the top of thechamber (Fig. 1). An air-tight seal at the lower opening of thechamber was made by impregnating the roots in silicon greaseinside a split and bored No. 3 rubber stopper. The assay chamberwas then placed on top of a modified hydroponic chamber.Moisturized air was continuously flowed through the assay cham-ber at 0.33 ml s-' to assure that the nodules were well aerated.The assay chamber was maintained in the same water bath andunder the same lighting conditions as the growth chambersdescribed above.The oxygen concentration in the gas phase was measured with

C-

FIG. 1. Stainless steel flow through assay chamber (A) with hydro-ponic support chamber (B). Moisturized gas was supplied to the assay

chamber through port D and exited the assay chamber from port C. Airentered the hydroponic chamber through port E. The lower opening ofthe assay chamber was sealed with a split and bored No. 3 rubber stopper(G). Roots passing through the stopper were impregnated in silicongrease (F), and the stopper was further capped with putty (H).

a Walker-type Clark-style oxygen electrode (Decogon Devices,Inc.) (7). The electrode polarizing voltage was controlled by aChemical Microsensor (Diamond Electro-Tech) and the outputsignal monitored by a minicomputer. The electrode was cali-brated with N2 gas and air to determine the zero base line andambient oxygen signal, respectively. After calibration, a four-wayvalve was used to expose the electrode to either the gas supplyentering the assay chamber or to the gas exiting the chamber.Root plus nodule respiration was estimated as the difference inoxygen concentration in the gas supply and in that exiting thechamber (typically about 0.0024 mm3 mm-3) multiplied by thevolumetric flow rate (0.33 ml s-'). Switching the electrode be-tween the supply and exit gas in this manner allowed respirationrates to be determined at least once every ten minutes. When anexperiment was completed, the plant in the assay chamber wasremoved, the nodules were harvested from the root system, andthe plant was then returned to the assay chamber. Typically, bareroot respiration rates were stable after 30 min and assayed underthe experimental conditions used for that plant. Bare root respi-ration rate was subtracted from the root plus nodule respirationrates to yield nodule respiration.

Nitrogenase activity of the intact nodules in the assay chamberwas determined using the acetylene-reduction technique. To dothis, the air supply rate to the assay chamber was increased to3.33 ml s-' and acetylene added to a final saturating concentra-tion of 0.10 mm3mm3. To determine the nodule ethyleneproduction rate, 4 min after the acetylene flow was begun, a 1ml sample was drawn from the gas exiting the assay chamberand injected into a gas chromatograph fitted with a flame ioni-zation detector. The acetylene was then removed from the airsupply which was returned to the normal flow rate and anyresidual acetylene flushed from the chamber.

Saturating concentrations of acetylene can have an inhibitoryeffect on nitrogenase activity in which the ethylene productionrate begins to decline after a period of several minutes of exposure(16). This inhibitory effect can cause a serious underestimationof nitrogenase activity depending on the amount of time thenodules are exposed to high acetylene concentrations before theethylene production rate is assayed. To determine the timedependency of this acetylene response for these hydroponicallygrown soybean, the acetylene concentration of the gas suppliedto the assay chamber was set at 0.10 mm3 mm-3. After theaddition of acetylene, the gas exiting the chamber was sampledevery 2 min for a total of 15 min. Nine assays were run in thisfashion.The day before a plant was to be assayed, it was transferred

from a hydroponic growth chamber to the assay chamber. Thefollowing day acetylene reduction and nodule respiration wereassayed at ambient oxygen. The oxygen concentration in the gassupply was then altered to either 0.1 (for 10 plants) or 0.4 mm3mm3 (for 4 plants). The time required for the step change inoxygen concentration to be completed in these experiments wasapproximately 5 min. Nodule respiration and acetylene-reduc-tion were assayed at regular intervals until steady-state valueswere obtained. When the experiment was terminated, the nod-ules were harvested and the root respiration was measured at theoxygen concentrations used for the individual experiment.

RESULTSField Experiments: Steady-State Response to Altered Oxygen

Concentrations. Acetylene reduction rates at midnight were typ-ically lower than those observed during the day. These diurnalcycles in acetylene reduction are consistent with the effects thatdaily cycles in soil temperature have previously been reported tohave on nitrogen fixation rate (8, 22). On 16 June, plants assayedfor 3 d at ambient oxygen concentration were switched to 0.1mm3mm-3 oxygen and assayed for another 3 d. Treatment

902 Plant Physiol. Vol. 84, 1987

https://plantphysiol.orgDownloaded on January 8, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

NODULE RESPIRATION AND NITROGEN FIXATION RESPONSE TO OXYGEN

means for VmL, did not differ at the midnight assay following theafternoon when the oxygen concentration in the 4 experimentalchambers was altered (Fig. 2). The following noon, treatmentmeans appeared to differ with the plants at 0.1 mm3 mm-3oxygen having lower acetylene-reduction rates; however, theapparent difference was not statistically significant (a = 0.05).Treatment mean values for Vmax did not differ significantly onany date of this experiment.

Further confirmation of the lack of a long-term response tosubambient oxygen concentrations was obtained on June 9. Fiveplants which had been continuously assayed for 2 d were switchedfrom ambient to 0.06 mm3 mm-' oxygen and assayed for 2 mored. After the oxygen concentration was reduced, the mean Vmavalues did not differ significantly (a = 0.05) between the subam-bient and ambient oxygen treatments on any date.The results from supra-ambient oxygen concentrations also

failed to show a long-term response. On May 28, five plantsassayed for 2 d were switched from ambient to 0.4 mm3 mm 3oxygen and assayed for 6 more d. Treatment means for normal-ized acetylene reduction did not differ between ambient andelevated oxygen treatments except on one occasion 6 d after theoxygen treatment began (Fig. 3).On May 17, five plants assayed continuously for 2 d were

switched from ambient oxygen to 0.3 mm mm-3 . Acetylene

13C

xO 12CE

- 11c

c 10

% 9c

14 16June

Date18 20

FIG. 2. Mean normalized maximum acetylene reduction (percent ofinitial value) versus date for two treatments, ambient oxygen (0), and an

oxygen regulated treatment switched from ambient to 0.1 mm3 mm-3oxygen (U) at the time indicated by the arrow. Treatment means did notdiffer significantly (a=0.05) on any date. Error bars indicate the standarderror of the mean (n=5). Initial mean and standard error acetylenereduction values were 0.27±0.05 mm3 s-' and 0.26±0.05 mm3 s-' forthe ambient and oxygen regulated treatments, respectively.

140

E120-

-100-

80 oAmbient OxygenmOxygen Regulated

27 29 312 4

May JuneDate

FIG. 3. Mean normalized maximum acetylene reduction (percent ofinitial value) versus date for two treatments, ambient oxygen (0), and an

oxygen regulated treatment switched from ambient to 0.4 mm3 mm-3oxygen (U) at the time indicated by the arrow. An "a" indicates treatmentmeans differ significantly at the a = 0.05 level. Error bars indicate theSEM (n = 5). Initial mean and standard error acetylene reduction valueswere 0.20 ± 0.02 mm3 s-' and 0.13 ± 0.02 mm3 s-' for the ambient andoxygen regulated treatments, respectively.

bclC

0

t8

I0

E

E.R 40

mo2°9 2 4 6 8 10 12 14

Time (minutes)

FIG. 4. Ethylene production rate as a percent of the maximum rateachieved versus time since the chamber was switched from 0.0 to 0.1mm3 mm-3 acetylene.

c

.a

O,

0)

z

-5

BE

0

0

_3

coX

Time (hours)

FIG. 5. Nodule respiration (@) and acetylene reduction ([) as a

percent of the initial value versus time since the growth chamber lightscame on for individual plants assayed under ambient conditions andthen switched to 0.1 mm3 mm-3 oxygen. Arrows indicate the time whenthe oxygen concentration was altered. See Table I for absolute values ofnodule respiration and acetylene reduction rates.

reduction rates did not differ over the 24-h period these plantswere assayed between those at ambient and those at 0.3 mm3mm-3 oxygen. On May 18, the oxygen concentration in thosechambers at 0.3 mm3 mm-3 was further increased to 0.35 mm3mm-3 and the plants were assayed for another 24 h. Althoughon the night of May 19, the mean Vm,, for the elevated oxygenplants was significantly lower (a = 0.05) than that of the controlplants, the mean Vmax did not differ significantly between treat-ments on any other date.Hydroponic Plant Response to Saturating Acetylene Concen-

trations. Nitrogenase activity as a percent of the maximum rateofethylene production was plotted against time since the additionof acetylene to the assay chamber for nine individual assays (Fig.4). The concentration of acetylene in the chamber reached 0.10mm3 mm-3 after 3 min and maximum ethylene productionoccurred about 1 min later or a total of 4 min after the additionof acetylene to the gas supply. Acetylene reduction rates werethen stable for approximately 5 more min and then began a slowdecline reaching 95% of the maximum rate at 14 min. Minchinet al. (16) reported a more dramatic decrease in nitrogenaseactivity when legume nodules are exposed to saturating concen-trations of acetylene than that shown in Figure 4.As the acetylene concentration in the assay chamber required

3 min to reach steady-state levels, it is possible that an inhibitionof nodule activity occurred before the 4 min peak in ethyleneproduction indicated in Figure 4. To test this, the flow rate tothe assay chamber was increased to 10 ml s- so that the 0.1mm3 mm-3 steady-state acetylene concentration in the chamber

903

D-1 NoAmbient OxygenaOxygen Regulated

1:ii.~~~~~~'r4 t -1

60 8

0

9

0

II

https://plantphysiol.orgDownloaded on January 8, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

Plant Physiol. Vol. 84, 1987

Table I. Steady-State Valuesfor Acetylene Reduction and Nodule Respiration at Ambient and 0.1 mm3mm-3 Oxygen

Acetylene Reduction Nodule RespirationExpt. Final Final

Initial ambient sub-ambient % Initial ambient sub-ambientmm3 g-' dwt s-' mm3g3- dwt s-'

1 0.803 0.797 99 2.35 2.51 1072 0.627 0.600 96 2.27 2.73 1203 0.939 4.484 0.920 0.893 97 3.47 3.60 1045 0.661 0.609 92 2.57 2.35 916 0.577 0.516 89 1.90 2.00 1057 0.558 0.515 92 1.62 1.81 1128 1.115 1.154 103 3.31 3.65 1109 1.077 1.006 93 3.12 3.41 10910 1.006 0.900 89 2.77 2.79 101

Mean 0.828 0.777 94.4 2.79 2.76 106.6SE 0.067 0.072 1.6 0.27 0.23 2.7

-- Fr- ag

:R .".......... And f~~~~~-

-

+-.i

p- -1C-1.;P..(1)1-11--..L

.71.2 -,') v1-1

4.

C', .- .

..,

1 2'h 2 3

T 1e n

FIG. 6. Nodule respiration (0) and acetylene reduction (0) as a

percent of the initial value versus time after the growth chamber lightscame on for individual plants assayed under ambient conditions andthen switched to 0.4 mm3 mm-' oxygen. Arrows indicate the time whenthe oxygen concentration was altered. See Table II for absolute values ofnodule respiration and acetylene reduction.

was reached in 5 s (27). Ethylene production was assayed every4 s for 120 s after the introduction of acetylene. Inhibition ofnitrogenase activity did not occur within this initial 120-s period.These data demonstrated the absence of an acetylene inhibitionin nitrogenase activity when the assay was completed within 10

min of the initiation of a change in acetylene concentration.Non-Steady-State Response to Altered Oxygen Concentra-

tions. To observe the transient responses to altered oxygen con-

centration, plants were assayed at ambient oxygen and thenswitched to 0.1 mm3 mm-3 oxygen and periodically assayed untilsteady-state was attained. Nodule respiration and maximumacetylene reduction rate as a percent of that observed underambient conditions are plotted for four plants in Figure 5. Nodulerespiration rate decreased dramatically when the oxygen concen-

tration was dropped to 0.1 mm' mm- and then recovered slowlyover the next few hours. Similarly, acetylene reduction ratedecreased when the oxygen concentration was lowered, but thisdecrease was also transitory as there was a recovery to ratesinitially observed under ambient oxygen. Initial and final steady-state nodule respiration and acetylene reduction rates for allplants switched from ambient to 0.1 mm3 mm-3 are given inTable I. Nodule activity always recovered to values similar totheir pretreatment values.Nodule respiration and acetylene reduction were assayed for

four plants under ambient oxygen and then switched to 0.4 mm3mm-3 oxygen. Nodule respiration initially decreased after in-creasing the oxygen concentration and then recovered to ratescomparable to those initially observed (Fig. 6). Non-steady-stateacetylene reduction rates were measured for two of these plantsand, like nodule respiration, initially decreased after increasingthe oxygen concentration. Final steady-state acetylene reductionrates for all four plants were similar to rates initially observed atambient oxygen (Table II).

DISCUSSIONIn the field experiments, no long-term effects (greater than 8

h) on acetylene reduction rate were observed from lowering the

Table II. SteadyState Valuesfor Acetylene Reduction and Nodule Respiration at Ambient and 0.4 mm3mm-3 Oxygen

Acetylene Reduction Nodule RespirationExpt. Final

Initial ambient Fisal % Initial ambient Final %sub-ambient sub-ambient

mm3 g9' dwt s-' mm3 g-' dwt s-'1 0.875 0.775 89 2.64 2.75 1042 0.893 0.825 92 2.39 2.43 1023 0.578 0.607 105 2.19 1.95 894 0.784 0.785 100 2.05 2.16 105

Mean 0.782 0.748 96.5 2.32 2.32 100.0SE 0.072 0.048 3.7 0.13 0.17 3.7

904 WEISZ AND SINCLAIR

for

..

O

https://plantphysiol.orgDownloaded on January 8, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

NODULE RESPIRATION AND NITROGEN FIXATION RESPONSE TO OXYGEN

rhizosphere oxygen concentration to either 0.06 or 0.1 mm3mm-3. Similarly, there were no consistent long-term effects ofoxygen concentration elevated to either 0.3 or 0.4 mm3 mm-3.These field results on intact plants confirm the lack of responseto long-term exposures to altered oxygen concentrations (5, 6,14, 23) and further demonstrate that at least after an 8 h exposureto altered oxygen concentration in the range of 0.06 to 0.4 mm3mm-3, there are no long-term effects of oxygen concentration onnitrogenase activity.As in the field experiments, the hydroponic plants exhibited

steady-state acetylene reduction rates which were independent ofthe external oxygen concentration from 0.1 to 0.4 mm3 mm-3.There were, however, short-term responses ofnitrogenase activityto altered oxygen concentrations. Decreasing the oxygen concen-tration to 0.1 mm3 mm-3 resulted in decreased ethylene produc-tion rates and a dramatic decrease in nodule respiration. Thesedecreased rates were transitory and full recovery ofboth acetylenereduction and nodule respiration rates was complete in 4 to 8 h.The mean value and standard error of nodule respiration atambient oxygen concentrations for all plants was 2.65 ± 0.20mm3 g dwt-' s-'. This is in close agreement with nodule respi-ration rates of 1.90 to 3.44 mm3 g dwt-' s-' previously reportedfor 20- to 60-d-old soybean plants (20). Data from these hydro-ponically grown soybean plants indicated that, given enoughtime, nodules can acclimate to decreased rhizosphere oxygenconcentrations. Further, the fact that steady-state nodule respi-ration rates at 0.1 mm3 mm-3 oxygen were similar to thoseobserved under ambient conditions is inconsistent with the hy-pothesis that the mechanism for maintaining a constant nitrogenfixation rate under varying oxygen concentrations involves analteration in nodule respiration rate.

Exposure to 0.4 mm3 mm-3 oxygen resulted in short-termdecreases in both nodule respiration and acetylene reductionrates. Such a response to increased oxygen concentrations mayhave been caused by partial oxygen inactivation of nitrogenase.The data from the plants assayed at 0.1 mm3 mm-3 oxygenindicated that the nodule response time to altered oxygen con-centrations was several hours. In these experiments, the oxygenconcentrations in the assay chamber were changed in 5 min.Such a rapid increase in the external oxygen concentration mayhave overwhelmed the regulatory mechanism. During the periodthat the oxygen concentration in the chamber was left at thesesupra-ambient levels, the nodule respiration and acetylene re-duction rates recovered and the final steady-state rates werecomparable to those initially observed. Thus, while the externaloxygen concentration had been doubled, the nodule respirationrates remained the same. These data are also inconsistent withthe hypothesis that the internal, low nodule oxygen concentrationwas maintained by an alteration in nodule respiration rate.

All the results from this study support the hypothesis thatintact soybean nodules have a mechanism for adapting to a widerange of altered rhizosphere oxygen concentrations. Changes innodule activity which followed alterations in the rhizosphereoxygen concentration were always regulated back to the originallevel. This adaptive mechanism requires several hours for com-plete adjustment of nitrogen fixation rates. Contrary to thehypothesis that the adaptive mechanism involves a change innodule respiration rate, nodule respiration responded to theadaption in oxygen concentration in a manner parallel to acety-lene reduction. Respiratory control of nitrogen fixation ratesdoes not appear to play a role in the regulation of nitrogenaseactivity in response to changes in the rhizosphere oxygen con-centration.

LITERATURE C I ED1. BERGERSEN FJ 1962 The effects of partial pressure of oxygen upon respiration

and nitrogen fixation by soybean root nodules. J Gen Microbiol 29: 113-

1252. BERGERSEN FJ 1971 Biochemistry of symbiotic nitrogen fixation in legumes.

Annu Rev Plant Physiol 22: 121-1403. BERGERSEN FJ, GL TURNER 1975 Leghaemoglobin and the supply of 02 to

nitrogen-fixing root nodule bacteroids: presence oftwo oxidase systems andATP production at low free 02 concentrations. J Gen Microbiol 92: 345-354

4. BERGERSEN FJ, GL TURNER 1980 Properties of terminal oxidase systems ofbacteroids from root nodules of soybean and cowpea and of N2-fixingbacteria grown in continuous culture. J Gen Microbiol 1 8: 235-252

5. CRISWELL JG, UD HAVELKA, B QUEBEDEAUX, RWF HARDY 1976 Adaptationof nitrogen fixation by intact soybean nodules to altered rhizosphere pO2-Plant Physiol 58: 622-625

6. CRISWELL JG, UD HAVELKA, B QUEBEDEAUX, RWF HARDY 1977 Effect ofrhizosphere P02 on nitrogen fixation by excised and intact nodulated soybeanroots. Crop Sci 17: 39-44

7. DELIEU T, DW WALKER 1981 Polaragraphic measurement of photosyntheticoxygen evolution by leaf discs. New Phytol 89: 165-178

8. DENISON RF, TR SINCLAIR 1985 Diurnal and seasonal variation in dinitrogenfixation (acetylene reduction) rates by field-grown soybeans. Agron J 77:679-684

9. DENISON RF, TR SINCLAIR, RW ZOBEL, MM JOHNSON, GM DRAKE 1983 Anon-destructive field assay for soybean nitrogen fixation by acetylene reduc-tion. Plant Soil 70: 173-182

10. DENISON RF, PR WEIsz, TR SINCLAIR 1983 Analysis of acetylene reductionrates of soybean nodules at low acetylene concentrations. Plant Physiol 73:648-651

11. DENISON RF, PR WEIsZ, TR SINCLAIR 1987 Oxygen supply to nodules as alimiting factor in symbiotic nitrogen fixation. In RJ Summerfield, ed, WorldCrops: Cool Season Food Legumes. Martinus Nijhof, Dordrecht, Nether-lands. In press

12. FEHR WR, CE CAVINESS, DT BURMOOD, JS PENNINGTON 1971 Stage ofdevelopment descriptions for soybean, Glycine max (L.) Merrill. Crop Sci11: 929-931

13. IMSANDE J, EJ RALSTON 1981 Hydroponic growth and the nondestructive assayfor dinitrogen fixation. Plant Physiol 68: 1380-1384

14. MINCHIN FR, JE SHEEHY, JF WrrrY 1985 Factors limiting N2 fixation by thelegume-Rhizobium symbiosis. In H J Evans, P J Bottomley, W E Newton,eds, Nitrogen Fixation Research Progress Martinus Nijhoff, Boston, pp 285-291

15. MINCHIN FR, JE SHEEHY, JF WITrY 1986 Further errors in the acetylenereduction assay: Effects of plant disturbance. J Exp Bot 37: 1521-1591

16. MINCHIN FR, JF WrTTY, JE SHEEHY, M MULLER 1983 A major error in theacetylene reduction assay: decreases in nodular nitrogenase activity underassay conditions. J Exp Bot 34: 641-649

17. PANKHURsT CE, JI SPRENT 1975 Effects of water stress on the respiratory andnitrogen-fixing activity of soybean root nodules. J Exp Bot 26: 287-304

18. PETERSON JB 1985 The interaction ofoxygen with nitrogen fixation in soybeannodules. In R Shibles, ed, World Soybean Research Conference III: Proceed-ings. Westview Press, Boulder, CO, pp 807-814

19. RALSrON EJ, J IMSANDE 1982 Entry ofoxygen and nitrogen into intact soybeannodules. J Exp Bot 33: 208-214

20. RYLE GJA, RA ANNOTT, CE POWELL, AJ GORDON 1984 N3 fixation andrespiration costs of nodules, nitrogenase activity and nodule growth andmaintenance in Fiskeby soybean. J Exp Bot 35: 1156-1165

21. SHEEHY JE, FR MINCHIN, JF Wrm 1983 Biological control of the resistanceto oxygen flux in nodules. Ann Bot 52: 565-571

22. SINCLAIR TR, PR WEISZ 1985 Response to soil temperature of dinitrogenfixation (acetylene reduction) rates by field-grown soybeans. Agron J 77:685-688

23. SINCLAIR TR, PR WEISZ, RF DENSON 1985 Oxygen limitation to nitrogenfixation in soybean nodules. In R Shibles, ed, World Soybean ResearchConference III: Proceedings. Westview Press, Boulder, CO, pp 797-806

24. TJEPKEMA JD, CS YOcUM 1973 Respiration and oxygen transport in soybeannodules. Planta 115: 59-72

25. WEIsz PR, RF DENISON, TR SINCLAIR 1985 Response to drought stress ofnitrogen fixation (acetylene reduction) rates by field-grown soybeans. PlantPhysiol 78: 525-530

26. WEIsz PR, TR SINCLAIR 1987 Regulation of soybean nitrogen fixation inresponse to rhizosphere oxygen. II. Quantification ofnodule gas permeability.Plant Physiol 84: 906-910

27. WEISZ PR AND TR SINCLAIR 1987 Soybean nodule gas permeability: Devel-opment of a rapid non-destructive assay. Plant Soil. In press

28. Wrrry JF, FR MINCHIN, JE SHEEHY 1983 Carbon costs of nitrogenase activityin legume root nodules determined using acetylene and oxygen. J Exp Bot34: 951-963

29. WirrT JF, FR MINCHIN, JE SHEEHY, MI MINGUEZ 1984 Acetylene-inducedchanges in oxygen diffusion resistance and nitrogenase activity of legumeroot nodules. Ann Bot 53: 13-20

30. WINSHIP LJ, JD TJEPKEMA 1985 Nitrogen fixation and respiration by rootnodules of Alnus rubra Bong: effects of temperature and oxygen concentra-tion. Plant Soil 87: 91-107

905

https://plantphysiol.orgDownloaded on January 8, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.