Plant and Soil 191: 157–161, 1997. 157c 1997 Kluwer Academic Publishers. Printed in the Netherlands.

The acetylene-induced decline in nitrogenase activity in root nodules ofElaeagnus angustifolia

Gordon V. Johnson1, Christa R. Schwintzer2 and John D. Tjepkema2;3

1Department of Biology, University of New Mexico, Albuquerque, NM 87131-1091, USA and 2Department ofPlant Biology and Pathology, University of Maine, Orono, ME 04469-5722, USA. 3Corresponding author�

Received 9 October 1996. Accepted in revised form 20 March 1997

Key words: acetylene reduction, acetylene-induced decline, argon-induced decline, actinorhizal plants, Elaeagnus,Frankia, nitrogen fixation

Abstract

The rate of C2H2 reduction by nodulated seedlings of Elaeagnus angustifolia (Russian olive) was followed as afunction of time. Our goals were to: 1) determine whether there is an C2H2-induced decline in nitrogenase activity;and 2) investigate the mechanism of any decline. We found a peak rate of C2H2 reduction at 1.5 min after theintroduction of C2H2 that was followed by a rapid decline in activity to 56% of the peak value. After the declinethere was a partial recovery to 67% of the peak value at 60 min. When the pO2 was decreased during the declinethere was no significant effect (p�0.05) on nitrogenase activity. When the C2H2 reduction assay was preceded by anincubation in a gas mixture (20 kPa O2) with Ar substituted for N2, there was little decline in nitrogenase activity asa function of time, but the rate of C2H2 reduction per gram nodule was reduced by approximately 50%. From theseresults we conclude that Elaeagnus angustifolia exhibits a pronounced C2H2-induced decline and consequently theinitial peak rate C2H2 reduction must be determined to obtain a valid measure of nitrogenase activity. We furthersuggest that cessation of NH3 formation initiates the decline and that the decline is not caused by a change in nodulepermeability to gases.

Introduction

The reduction of C2H2 to C2H4 is a widely used mea-sure of nitrogenase activity because of its high sensitiv-ity, speed, low cost, and nondestructive nature ( Vessey,1994; Winship and Tjepkema, 1990). However, ques-tions have been raised about the proper use of this assay(Minchin et al., 1983, 1986, 1994). When legume rootnodules are exposed to C2H2, an initial peak rate ofC2H4 production is observed which is often followedin a few minutes by an irreversible decline in the rateof C2H4 formation. The initial peak rate is consideredto be the only reliable measure of nitrogenase activityin legume nodules that have a decline (Minchin et al.,1983, 1994). When legume root nodules are exposedto gas mixtures in which Ar has been substituted forN2, there is a similar decline in nitrogenase activi-ty termed the argon induced decline (Minchin et al.,

� FAX No: +12075812969. E-mail: tjepkema@maine.maine.edu

1983; Witty et al., 1984). Acetylene and Ar, as wellas He (Minchin et al., 1983), affect nodule activityvia cessation of ammonia production (Minchin et al.,1983; Witty et al., 1984). This in turn causes eventsleading to an increase in the resistance of the nodule togaseous diffusion thus causing the decline in nitroge-nase activity (Witty et al., 1984).

The situation in actinorhizal plants is not clearbecause only a few species have been examined todate. The nodules of actinorhizal plants differ fromthose of legumes in being induced by Frankia ratherthan rhizobia and in being modified roots instead ofbeing tumor-like (Newcomb and Wood, 1987). As inlegumes, actinorhizal nodules have an initial peak rateof C2H4 formation followed by a decline. But in con-trast to legumes, this is followed by a recovery ofactivity to stable rates in Alnus, Myrica and Casuarina(Silvester and Winship, 1990; Tjepkema et al., 1988).The extent of the decline and the extent of the recov-

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ery depend on the plant species and the conditionsunder which the plants are grown and assayed (Tjep-kema et al., 1988; Tjepkema and Schwintzer, 1992;Schwintzer and Tjepkema, 1994). In contrast, there islittle or no recovery from the decline in Coriaria andDatisca (Silvester and Harris, 1989; Tjepkema et al.,1988). Because these two genera share a unique nod-ule morphology (Newcomb and Wood, 1987) it is notclear whether there are other genera that have little orno recovery.

The mechanism of the C2H2-induced decline isnot well understood in actinorhizal nodules. Most ofthe work on this question has been done with Myri-ca gale (Schwintzer and Tjepkema, 1994; Silvesterand Winship, 1990; Tjepkema and Schwintzer, 1992).However actinorhizal plants belong to eight differ-ent plant families, including the Betulaceae, Elaeag-naceae, and Myricaceae, and are very diverse (Bakerand Schwintzer, 1990). Major physiological as well asstructural differences have been found between nod-ules formed by plants belonging to different plant fami-lies (Newcomb and Wood, 1987; Silvester et al., 1990).

In the present work we investigated the time courseof C2H2 reduction by nodules of Elaeagnus angustifo-lia (Russian olive), a member of the Elaeagnaceae. Thetime course of C2H2 reduction has not been previouslyexamined in this family. Our goals were to determinethe extent of the C2H2-induced decline, whether or notthere is a recovery, and to investigate the mechanismof the decline.

Materials and methods

To determine the extent of the decline and recovery, wemeasured the time course of C2H2 reduction in an open,flow-through system (Tjepkema et al., 1988; Winshipand Tjepkema, 1990) in a gas mixture consisting ofair with C2H2 and O2 added to obtain 10 kPa C2H2

and 20 kPa O2 (standard experiment). To investigatethe mechanism of the decline, we examined the effectof preincubation in argon to determine whether thedecline is initiated by the cessation of NH3 production(argon experiment) and examined the effect of decreas-ing pO2 during the decline to determine whether thedecline is caused by reduced O2 permeability of thenodules (oxygen experiment).

Plant growth

Elaeagnus angustifolia seeds were collected nearAlbuquerque, NM. Seeds were scarified with concen-trated H2SO4, rinsed with water, prechilled for sixweeks at 2 �C, and then germinated in vermiculite andgrown in a greenhouse in Albuquerque.Six weeks aftergermination began, the seedlings were transferred indi-vidually to 1-L containers of aerated hydroponic medi-um. The nutrient solution contained (�mol L�1): 2,000MgSO4, 500 CaCl2, 910 KH2PO4, 92 K2HPO4, 1,960K2SO4, 5,980 CaSO4, 9.0 FeEDDHA, 46 H3BO3, 9.1MnCl2, 0.81 ZnSO4, 0.29 CuSO4, 0.10 Na2MoO4,0.10 NiSO4, 0.085 CoCl2. Plants were maintained ina growth chamber with 14 h days at 25 �C and 10 hnights at 20 �C. Light was supplied at an intensity of225 to 250�mol m�2 s�1 photosynthetic photon flux atplant height by cool white fluorescent lamps with sup-plemental incandescent lamps. Two days after trans-fer to hydroponic medium, the seedlings were inocu-lated with a suspension of crushed nodules preparedfrom field-collected nodules of Elaeagnus angustifo-lia. Nodules were observed on most plants within 19days.

Eight weeks after inoculation the seedlings weresent by overnight express to Orono, Maine, where theywere maintained in a growth chamber under similarconditions as above with a light intensity of approxi-mately 225 �mol m�2 s�1 and were allowed to recov-er for at least one week prior to the beginning of theexperiments. In Orono, the seedlings were grown in250-mL, opaque, wide-mouth, plastic bottles contain-ing 125 mL of the nutrient solution described aboveat one-fourth strength. The solutions were replacedevery two or three days and distilled water was addedto maintain the solution level between replacements.Almost all nodules were in the upper one fourth of theroot system and the nutrient solution was kept belowthis level to acclimate the nodules to a gaseous envi-ronment. A few plants had single nodules lower onthe root system which were removed when the plantsarrived in Orono. At the time of the experiments theplants had a mean dry mass of 1.32 g plant�1 and themean nodule dry mass constituted 5.30% of total drymass. There were no signifcant differencess (p�0.05)in plant 10 mass or % nodules between experiments.

C2H2 reduction assays

All C2H2 reduction measurements were made usingan open, flow-through system (Tjepkema et al., 1988;

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Winship and Tjepkema, 1990) consisting of a gas sup-ply, peristaltic pump, a cuvette containing an intactroot system of a plant, and tubing venting waste gas tothe outdoors. Just before each C2H2, reduction assay, aplant was transferred to a cuvette consisting of a rigidplastic tube (34 mm inner diameter, 151 mm long).The bottorn was sealed with a neoprene stopper and 65mL of nutrient solution was placed in the bottom of thecuvette. The plant remained in the same neoprene stop-per which held it during growth in the growth chamberand the stopper was inserted into the top of the cuvettewith the lower portion of the roots submerged in thenutrient solution. The space between the stem of theplant and the edge of the hole in the center of thestopper was sealed with Kromopan (LASCOD, SestoFiorentino, Italy), an alginate-based compound usedin making dental impressions. The nodules were onthe upper section of the root system above the nutrientsolution in a gas volume of approximately 47 mL. Agas flow rate of 150 mL per minute was used, via tub-ing sealed into holes on opposite sides of the cuvette atpositions above the nutrient solution. The cuvette wasimmersed in a water bath (26.0 �C) in the laboratory.

Gas mixtures were made in saran bags (ANSPEC,Ann Arbor, MI, USA) and contained air supplementedwith O2, C2H2, and N2 as needed. Small amounts ofwater were kept in the bags to maintain 100% relativehumidity. All mixtures contained 20 kPa O2 and 10kPa C2H2 unless otherwise specified. Using an excessof water, C2H2 was generated by adding CaC2 to thewater. Gas was pumped through the cuvette with a peri-staltic pump, and connections were made with PVC(Tygon) tubing of 2.4 mm inner diameter and 0.8 mmwall thickness. Gas samples exiting the cuvette weretaken in 3-mL disposable plastic syringes, and werestored in the syringes for 1 to 15 min before analy-sis. The syringes were tested for leakage and lost only2% of their C2H4 in 60 min. Analysis for C2H2 andC2H4 employed a gas chromatograph equipped witha sampling loop and flame ionization detector. Beforethe introduction of C2H2, humidified air was pumpedthrough the cuvette (150 mL min�1) for 20 to 30 minin the standard and oxygen experiments. In the argonexperiment, a mixture of 20 kPa O2 and 80 kPa Arwas pumped through the cuvette for 60 min before theintroduction of C2H2. After C2H2 was introduced, theinitial gas samples were taken at 0. 5, 1, 1.5, 2, 2.5,3, 4, 6, 8, and 10 min. In the oxygen experiment, thepO2, of the gas mixture was shifted to 16 kPa at 18min after the introduction of C2H2 and back to 20 kPaat 33 min.

Figure 1. Rates of C2H2 reduction in Elaeagnus angustifoliaseedlings as a function of time after C2H2 addition. The plants in thestandard experiment received no pretreatment and the plants giventhe argon pretreatment were exposed to a gas mixture (20 kPa O2)in which Ar was substituted for N2 6 for 60 min prior to exposure toC2H2. Nodule mass is dry mass. Values are mean �SE, n = 6.

Means were compared by ANOVA followed by aTukey HSD multiple comparisons test where appro-priate. Rates of nitrogenase activity before and aftera shift from 20 to 16 kPa O2 were compared with apaired t-test.

Results and discussion

The nature of the C2H2-induced decline

Following the introduction of C2H2, the peak rate ofC2H4 formation occurred at 1.5 min and was followedby a rapid decline (Figure 1, standard experiment).This is similar to results obtained for other actinorhizalnodules assayed by the same method (Silvester andHarris, 1989; Silvester and Winship, 1990; Tjepkemaet al., 1988). There was only a small recovery fromthe decline. At 60 min after C2H2 addition the rate hadrecovered to only 67% of the peak rate, after being at57% of the peak rate during the minimum of the decline(Figure 1). This recovery was much smaller than thattypical of Alnus, Myrica, and Casuarina (Schwintzerand Tjepkema, 1994; Silvester and Winship, 1990;Tjepkema and Murry, 1989; Tjepkema et al., 1988).This small recovery also differs from Coriaria andDatisca, where there is essentially no recovery fromthe decline (Silvester and Harris, 1989; Tjepkema etal., 1988).

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Figure 2. Effect of reducing pO2 during the C2H2-induced declinein Elaeagnus angustifolia seedlings. The specifc activity at the peakrate of acetylene reduction was 1.93�0.23 �mol min�1 g�1 noduledry mass (mean�SE, n = 6).

From detailed investigation of the nodules of Myri-ca gale, it has been concluded that the peak rate ofC2H2 reduction represents the most accurate measure-ment of nitrogenase activity (Schwintzer and Tjepke-ma, 1994; Tjepkema and Schwintzer, 1992). Likewisethe peak rate is the best measure of nitrogenase activityin legume nodules (Minchin et al., 1983, 1986). Fromthese results and the presence of a distinct initial peakrate followed by a substantial decline, we conclude thatfor Elaeagnus it is necessary to measure the initial peakrate of C2H2 reduction in order to obtain an accuratemeasure of nitrogenase activity. A flow-through sys-tem is the only practical method for measuring such apeak rate (Minchin et al., 1994).

Mechanism of the decline preincubation with argon

When 10% C2H2 is introduced into the root zone,essentially all N2 fixation stops, with nitrogenase activ-ity being diverted to the reduction of C2H2 to C2H4

(Hunt and Layzell, 1993). Thus NH3 is no longer beingformed. This cessation of NH3 formation is thought toinitiate the C2H2-induced decline in legume nodules(Minchin et al., 1983; Witty et al., 1984). The evidencefor this comes from following nitrogenase activity viathe evolution of H2. When the O2 concentration is keptconstant but N2 is replaced with Ar or He there is adecline in nitrogenase activity that is very similar tothe C2H2-induced decline (Minchin et al., 1983). Sim-ilar results have been found in Myrica gale (Tjepkemaand Schwintzer, 1992).

After preincubating the root system of Elaeagnusin a gas mixture in which N2 was replaced by Ar, therewas essentially no C2H2-induced decline and nitroge-nase activity was nearly constant as a function of time(Figure 1). However the specific nodule activity wasonly half that in the standard (Figure 1) and oxygen(Figure 2) experiments both of which lacked the Arpretreatment. This is consistent with a partial loss ofnitrogenase activity during the Ar pretreatment. It islikely that the cessation of NH3 formation caused byC2H2 and Ar both reduce nitrogenase activity by thesame mechanism. Thus after the Ar pretreatment therewas no further loss of activity when C2H2 was intro-duced.

Mechanism of the decline - O2 shift

In legume nodules, the C2H2-induced decline isthought to be initiated by cessation of ammonia for-mation (Minchin et al., 1983; Witty et al., 1984) whichleads to a decrease in permeability of the nodules tooxygen (Witty et al., 1984). This in turn decreasesnitrogenase activity via a reduction in the diffusionof O2 to the sites of respiration that generate the ATPrequired for nitrogenase activity. Thus O2 supply limitsnitrogenase activity during the C2H2-induced declineand any decrease in pO2 toward the end of the C2H2-induced decline should decrease nitrogenase activity.This is indeed what happens in garden peas (Pisumsativum) where reducing the pO2 from 20 to 16 kPatoward the end of the C2H2-induced decline caus-es nitrogenase activity to decrease by approximately20% (Schwintzer and Tjepkema, unpublished). Acti-norhizal plants, however, may be different. In Myricagale decreasing the pO2 toward the end of the C2H2-induced decline actually increases nitrogenase activ-ity. This may be due to relief of oxygen inhibitionof nitrogenase or there may be competition betweennitrogenase and respiration for reductant (Tjepkemaand Schwintzer, 1992).

In Elaeagnus we found no significant effect(p�0.05) when we shifted the pO2 between 20 and 16kPa toward the end of the C2H2-induced decline (Fig-ure 2). The data in Figure 2 are presented as relativerates of nitrogenase activity with the peak rate for eachplant set at 100%. We did this because normalizingthe rates substantially reduces between plant variabil-ity. Lack of a significant effect when shifting the pO2

between 20 and 16 kPa toward the end of the acetylene-induced decline is evidence against the possibility thatthe decline in Elaeagnus is due to a decrease in perme-

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ability to oxygen. A decrease in permeability shouldresult in a strong dependence of nitrogenase activityon external pO2. Thus for a shift from 20 to 16 kPa onewould expect a decrease in C2H4 formation approach-ing 20%. There was an apparent mean decrease of4.9% between the last measurement at 20 kPa and thefirst measurement at 16 kPa O2 (Figure 2) but this wasnot significant (p�0.05).

The lack of a significant O2 effect during the C2H2-induced decline in Elaeagnus suggests that the declineis due to some limiting factor for nitrogenase other thanpO2 or ATP supply. One possibility is that cessationof NH3 formation in some way reduces the supply ofreductant needed by nitrogenase. If a malate-aspartateshuttle is involved in the transport of carbon and reduc-tant from the host cytoplasm to the microsymbiont(Huss-Danell, 1990), this might be disrupted by thecessation of NH3 formation.

Conclusions

The occurrence of a large C2H2-induced decline inElaeagnus emphasizes the need for measuring the ini-tial peak rate of nitrogenase activity when doing C2H2

reduction assays on actinorhizal plants. Use of closedsystems and assumptions of constant nitrogenase activ-ity following the introduction of C2H2 are not justified.The elimination of the decline in Elaeagnus by prein-cubation in Ar supports the hypothesis that in acti-norhizal nodules, as in legumes (Minchin, 1983; Wittyet al., 1984), the cessation of NH3 formation initi-ates the C2H2-induced decline. In contrast to resultsfor legume nodules, it appears that the C2H2-induceddecline in Elaeagnus nodules is not associated with adecrease in permeability of the nodules to O2.

Acknowledgements

This work was supported by the US Department ofAgriculture, NRICGP Grant No 94-37106-1060 toGVJ and Grant No 92-37305-7758 to JDT.

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