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

Nitrogenase activity and root nodule metabolism in response to O2 and

short-term N2 deprivation in dark-treated Frankia-Alnus incana plants

Per-Olof Lundquista*, Torgny Nasholmb and Kerstin Huss-Danellc

aDepartment of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences, PO Box 7080, SE-750 07 Uppsala,SwedenbUmea Plant Science Center, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, SE-90183 Umea, SwedencDepartment of Agricultural Research for Northern Sweden, Crop Science Section, Swedish University of Agricultural Sciences, PO Box4097, SE-904 03 Umea, Sweden*Corresponding author, e-mail: [email protected]

Received 21 November 2002; revised 6 April 2003

Inhibition of nitrogenase (EC 1.18.6.1) activity by O2 hasbeen suggested to be an early response to disturbance in

carbon supply to root nodules in the Frankia-Alnus incanasymbiosis. 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 recordedas CO2 evolution. Dark-treated plants had lower initial nitro-

genase activity in N2:O2 (68% of controls), which declined

further during a 1-h period in the assay system in N2:O2 at 21and 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 20min,

compared with control plants continuously kept in their nor-mal light regime. In contrast, there was no decline in dark-

treated plants at 13% O2, and only a smaller and temporarydecline in control plants at 21% O2. When dark-treated

plants were kept at 21% O2 during 45min prior to N2 depri-

vation 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 depriva-

tion, suggesting decreased assimilation of NH41. 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 concludethat 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 ofNH4

1 production.

Introduction

Several kinds of altered environmental conditions forN2-fixing Alnus, such as the addition of ammonium toplants, drought stress and prolonged darkness affectAlnus root nodule physiology and decrease nitrogenaseactivity (Huss-Danell 1990, 1997). During a prolongeddark period that induced carbon starvation it was shownthat the decrease in nitrogenase activity of intact plantswas due to inactivation and loss of nitrogenase proteins(Lundquist and Huss-Danell 1991a, b). Disappearanceof starch granules in uninfected cells and a later degrada-

tion of plant cells and Frankia also occurred in the rootnodules (Vikman et al. 1990, Lundquist and Huss-Danell1991a, b). Inactivation of nitrogenase and inhibition ofnitrogenase synthesis by O2 were proposed as earlyresponses following the disturbance in carbon supply tothe nodule (Lundquist and Huss-Danell 1991a, b).Avoiding O2 inactivation of nitrogenase is an essential

strategy for N2-fixing organisms because nitrogenase isvery O2 labile. Nitrogenase activity of intact Alnus incanaroot nodules has a clear optimum pO2 at 20%, which is

PHYSIOLOGIA PLANTARUM119: 244–252. 2003 Copyright# Physiologia Plantarum 2003

Printed in Denmark – all rights reserved ISSN 0031-9317

Abbreviations – pO2, oxygen partial pressure; Gln, glutamine; Cit, citrulline.

244 Physiol. Plant. 119, 2003

close to the pO2 of the growth condition (Lundquist2000). The protection of nitrogenase in Frankia as afree-living bacterium, and also during symbiosis inAlnus nodules, is believed to be carried out by a diffusionbarrier to O2 mainly in the Frankia vesicle envelope,together with O2 consuming respiration (Winship andSilvester 1989, Kleemann et al. 1994). In the symbioticstage, Frankia is dependent on host-plant metabolism inthe sense that the host provides carbon compounds insome form and at the same time assimilates the nitrogenproduced by Frankia.Effects of short-term N2 deprivation was previously

studied on unstressed A. incana plants (Lundquist2000). When intact nodulated root systems were exposedto N2 deprivation in Ar:O2, it was found that above thepO2 optimum (i.e. at 21 and 25% O2) a change fromN2-fixing conditions to a non-N2-fixing condition in Ar:O2

caused a decline in nitrogenase activity (Lundquist 2000).This decline was followed by a recovery phase. Theseresults were consistent with a fixed diffusion barrier toO2 in these root nodules (Lundquist 2000). The resultsalso suggested that the decline in nitrogenase activity ofthose unstressed plants was due to a temporary distur-bance in supply of reductant to nitrogenase in combina-tion with a partial O2 inhibition of nitrogenase duringthe non-N2-fixing condition in Ar:O2. Our hypothesis isthat reduced supply of NH4

1 to the plant cell surround-ing Frankia could lead to a disturbance in supply ofreductant to O2 consuming respiration in Frankia vesi-cles and/or plant mitochondria. Carbon starvation dueto a prolonged period of darkness is likely to induce analtered plant metabolism. In the present study wetherefore chose to investigate carbon-starved plants tobetter understand metabolic and physiological interac-tions in Alnus nodules. We were able to make use of aunique strain of Frankia lacking hydrogenase uptakeactivity that allowed measurements of nitrogenaseactivity as H2 evolution in N2:O2 of intact nodulatedroot systems, and combine this in a study of rootnodule metabolism.The ammonia formed in N2 fixation is likely to be

assimilated into amino acids in the plant cell surroundingN2-fixing Frankia (Blom et al. 1981, Lundquist andHuss-Danell 1992). When ammonia production bynitrogenase is inhibited in Ar:O2 the amino acid assim-ilating metabolism is expected to demand less ATP. Thenodule contents of amino acids and the adenylates ATPand ADP were therefore studied as indicators of hownodule metabolism is affected by short-term N2 depriva-tion.The questions we specifically addressed in this study

on N2-fixing Alnus incana nodules were: (1) is nitrogen-ase activity under N2-fixing conditions more sensitive toO2 in dark-treated plants than in control plants? (2) whatare the relationships between nitrogenase activity, short-term N2 deprivation and O2 in dark-treated plants?, and(3) is root nodule metabolism affected by short-term N2

deprivation?

Materials and methods

Plant material and growth conditions

Plant material and growth conditions were as describedearlier (Lundquist 2000). In brief, rooted cuttings of aclone of grey alder (Alnus incana (L.) Moench) wereinoculated with a crushed nodule inoculum lackinghydrogenase uptake activity (the local source of Frankia;Sellstedt et al. 1986, Huss-Danell 1991), and planted intopots with gravel of granite rock as support. The plantswere grown in a climate chamber with the conditions17 h light at 25�C and 7 h darkness at 15�C, RH about70% and a PPFD of about 250mmolm2 s�1. After4weeks the PPFD was raised to about 600 mmolm2 s�1.The plants were used at 10.8±0.3 (mean±SE, n¼ 60)weeks after inoculation and were 69± 3 (mean±SE) cmhigh.

Gas exchange measurements

Gas exchange measurements of H2 evolution and CO2

evolution were carried out on nodulated root systems ofintact plants where the pot was sealed to serve as acuvette as described earlier (Lundquist 2000). In brief,the gas exchange system was a computer-controlled sys-tem where mass flow controllers controlled the gas flows,and where the effluent gas was analysed by H2 sensors,O2 sensors and an infra-red gas analyser (IRGA). Out-puts and data collection were operated through the pro-gram Workbench (Strawberry Tree Inc., Sunnyvale, CA,USA). The gas volume of the cuvette was approx. 0.25Lwhen it contained roots and gravel. The total flow rate ofthe gas through the cuvette was 0.8 lmin�1. The systemhad two separate channels, although without an IRGAin one of the channels, which made it possible to measureH2 evolution from two plants at the same time.Some plants were kept in darkness for a total period of

22 h until the start of the assays. This was achieved byplacing the plants under a stand covered with black clothin the growth chamber (Lundquist and Huss-Danell1991a) starting at the end of the regular light period.To investigate the sensitivity of nitrogenase activity to

pO2 and to short-term N2 deprivation in Ar:O2 the con-trol plants and dark-treated plants were treated as fol-lows. The pots were mounted in the gas exchange systemand were left with a flow of N2:O2 at a pO2 of either 21,17 or 13%, which was kept throughout the experiment.After about 1 h and 15min the gas composition waschanged to Ar:O2, kept for 60min and then changedback to N2:O2 for an additional 15min.The H2 evolution in N2:O2 of dark-treated plants

reached a small plateau within 10–15min after connect-ing them to the gas exchange system and then began todecrease for some plants (plants kept at 21% O2). Thatfirst peak value was used to calculate the initial H2

evolution presented in Table 1. The H2 evolution ratewas expressed per plant height since there was a corr-elation between the peak value of H2 evolution in

Physiol. Plant. 119, 2003 245

N2:O2 and plant height (H2 evolution ¼0.53�plant height� 4.1 mmolH2m

�1 h�1, n¼ 25, r¼ 0.67,P, 0.0003), and also a correlation between plant heightand nodule weight (plant height¼ 4.0�noduleweight1 70, n¼ 8, r¼ 0.869, P, 0.005). To calculatethe effect of pO2 and time in the gas exchange systemon nitrogenase activity, the H2 evolution in N2:O2 60minprior to the shift to Ar:O2, and the H2 evolution justbefore the 60min Ar:O2 period were used.To test whether a high pO2 during the time before the

exposure to Ar:O2 would affect the presence of thedecline in Ar:O2, one set of plants was kept at 21% O2

as described above. After 45min the pO2 was lowered insteps of 1% O2 every 5min ending at 17% O2, which wasmaintained for 15min until the change from N2:O2 toAr:O2 occurred.To investigate if there was a relationship between the

rates of decline of H2 evolution and of CO2 evolution,the slopes of the declines following the change to Ar:O2

were calculated for the plants kept at 21% O2. Thedecline rates were calculated by using linear regressionwith the data that were obtained 7–12min after thechange to Ar:O2. The decline rate of CO2 evolution wasplotted as a function of the decline rate of H2 evolution.

Analyses of metabolites

To investigate if root nodule metabolism was affected byshort-term N2 deprivation the contents of amino acidsand adenylates were analysed. This was done in a sepa-rate experiment where the treatment of the plants includ-ing the time they were kept in the gas exchange systemwas the same as described above for the study of theeffect of short-term N2 deprivation in Ar:O2. As above,the pot was used as cuvette, but the pot was sealed tightlywith a plastic bag as a lid to be able to rapidly pick thenodules without disturbing the gas atmosphere. H2 evo-lution was also measured in this experiment, but the gaswas sampled from the pot instead of from the water-trapand gave similar results as before. Root nodules wereharvested 5min before the change to Ar:O2, and 10minand 45min after the change to Ar:O2. Several nodules(approximately 0.5–1.0 g per plant) were picked whilestill in the gas flow, immediately put into liquid N2 andstored there.The nodules from each plant were crushed separately

in a mortar with liquid N2. A fraction of crushed nodules(0.3–0.6 g) to be used for ATP measurements were

rapidly weighed at about the temperature of liquid N2

and stored in liquid N2. The remaining nodules werefurther ground to a powder and 0.15–0.2 g were weighedand stored at �20�C until amino acid extraction. Aminoacids were extracted by percolating with 80% ethanoland analysed as 9-fluorenylmethyl formate derivativesusing reversed-phase high-performance liquid chromato-graphy and fluorescence detection according to Nasholmet al. (1987). Some samples were analysed further toconfirm that the peak attributed to Cit did not containSer.The fraction of crushed nodules to be used for adeny-

late extraction were further ground in a mortar withliquid N2 with the addition of 600ml 10% perchloricacid and 0.04–0.06 g insoluble polyvinylpolypyrrolidone.After thawing, extracts were neutralized with buffer (5M

KOH, 1M triethanolamine), and ATP and ADP weremeasured using the firefly luciferase method essentiallyaccording to Gardestrom and Wigge (1988). Three tofour measurements were made on each extract fromeach plant.

Statistical analyses

Significant differences between mean values among gasexchange data and among amino acid data were testedby Student’s t-test. Significant differences between meanvalues of adenylate data were tested by two-way analysisof variance. Differences were considered significant ifP, 0.05.

Results

Responses of nitrogenase activity

Apparent nitrogenase activity (H2 evolution in N2:O2 perplant height) of dark-treated plants was 68% of thecontrol plants (Table 1). The dark-treated plants weresensitive to the initial period in N2:O2 in the gasexchange system, and this inhibition of nitrogenase activ-ity was related to the pO2 (Table 1). During the 60-minperiod in N2:O2 the dark-treated plants kept at 21 and17% O2 lost 23 and 13% of this activity, respectively. Incontrast, the dark-treated plants kept at 13% O2, as wellas the control plants kept at 13 and 17% O2, increasedtheir activity by about 10%.In response to short-term N2 deprivation in Ar:O2, the

H2 evolution increased as expected because of allocation

Table 1. Effects of prolonged darkness (22 h) and pO2 on H2 evolution in N2:O2 by Alnus incana root nodules. See Material and Methods fordescription of calculations. Data are shown as means±SE for the number of plants in parentheses. Significant differences (P, 0.05) betweendark-treated and controls are indicated as asterisks.

Activity Control plants Dark-treated plants

Initial H2 evolution at 21% O2 (mmol H2m�1 h�1) %

remaining H2 evolution after 1 h in N2:O2

47±3.2 (26) 32±3.3 (17)*

at 21% O2 104±1.8 (15) 77.1±4.0 (14)*at 17% O2 111±2.8 (6) 87.3±3.9 (6)*at 13% O2 111±4.2 (6) 110±2.0 (6)


of all reducing power to H2 production in the absence ofthe substrate N2 (Fig. 1). During the N2 deprivation inAr:O2, the nitrogenase activity of the dark-treated plantsrapidly declined and the decline was bigger at high pO2

(Fig. 1). Expressed as a percentage of the peak activitiesin Ar:O2, nitrogenase activity decreased to minimumvalues of 63, 74 and 90% after 22, 20 and 16min, respect-ively, for the dark-treated plants kept at 21, 17 and 13%O2

(Fig. 1A–C). The effect of N2 deprivation on dark-trea-ted plants at 21% O2 was, in fact, even greater sincethose plants did not reach a stable peak activity butrather immediately started to decline in activity inAr:O2 (Fig. 1A). No decline was observed for controlplants, except at 21% O2 where H2 evolution declinedto 88% of initial peak activity after 17min and wasfollowed by a recovery (Fig. 1A). The H2 evolution ofdark-treated plants initially kept at 21% O2 and then at17% O2 during the period in Ar:O2 showed only a smalldecline in response to short-term N2 deprivation(Fig. 1D).At the end of the time period in Ar:O2, the nitrogenase

activity of the plants exposed to 21 and 17% O2 haddeclined significantly to 60 and 71%, respectively, whenexpressed relative to the activity of the control plants(Fig. 1A and B). Nitrogenase activities of the dark-treated plants kept at 13% O2 (Fig. 1C) or initially keptat 21% O2 and later at 17% O2 (Fig. 1D) were notsignificantly different from control plants.When the gas phase was returned from Ar:O2 to

N2:O2, H2 evolution decreased rapidly (Fig. 1). After a15-min recovery period in N2:O2 the remaining nitrogen-ase activities were 75 and 71% of initial activity in N2:O2

for the dark-treated plants kept at 21 and 17% O2,respectively (Fig. 1A and B). Nitrogenase activity of thedark-treated plants kept at 13% O2 was as the initialactivity in N2:O2 (Fig. 1C), and the control plants had7–14% higher activities than initial (Fig. 1A–C). Thenitrogenase activity of dark-treated plants initially keptat 21% O2 and later at 17% O2 during the N2 depriva-tion, was 15% higher after the period in Ar:O2, but werenot different from the corresponding control plants(Fig. 1D).

Responses of CO2 evolution

CO2 evolution declined in Ar:O2 and was at the end ofthe 60-min period 83–88% of initial in N2:O2 for dark-treated plants and 88–95 of initial for control plants(Fig. 1). Fifteen minutes after the return from Ar:O2 toN2:O2, the remaining CO2 evolution was 88–91% ofinitial CO2 evolution in N2:O2 for the dark-treated plants(Fig. 1A–C), which corresponds to 91–95% of the CO2

evolution of control plants.The CO2 evolution of dark-treated plants initially kept

at 21% O2 and later at 17% O2 during the N2 depriva-tion, was 90% of initial at the end of the period in Ar:O2,but was not significantly different from the correspond-ing control plants (Fig. 1D). Fifteen minutes after the

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Fig. 1. Time course of changes in H2 evolution and CO2 evolutionrates in intact nodulated root systems of 22 h-dark-treated Alnusincana plants during short-term N2 deprivation in Ar:O2 at threedifferent pO2 values. The root systems were kept at either (A) 21 (B)17 or (C) 13% O2 throughout the experiment. One group of plants(D) was initially kept at 21% O2 for 45min and gradually broughtto 17% O2 prior to the Ar:O2 treatment. The gas composition waschanged from N2:O2 to Ar:O2 as indicated by the top panel. Theactivities in N2:O2 at the time just before the change to Ar:O2 wereset to 100%. Mean 100% activities ranged between 15.9 and38.8mmolH2 plant

�1 h�1 (n¼ 6–14) for dark-treated plants, 31.2–42.8 mmol H2 plant

�1 h�1 (n¼ 3–6) for control plants, 389–700mmolCO2 plant

�1 h�1 (n¼ 3–7) for dark-treated plants and418–622mmolCO2 plant

�1 h�1 (n¼ 2–3) for control plants. (——)H2 evolution of control plants (——) H2 evolution of dark-treatedplants (— -—) CO2 evolution of control plants and (- - - -) CO2

evolution of dark-treated plants. Significant differences (P, 0.05)between dark-treated and controls at selected time-points areindicated as asterisks.


return from Ar:O2 to N2:O2, the CO2 evolution was 98%of initial and similar to control plants.The decline rates of CO2 evolution and H2 evolution

were faster in dark-treated than in control plants(Figs 1A and 2). The rate at which the H2 evolutiondeclined during Ar:O2 at 17 and 21% O2 correlatedwith the decline rate in CO2 evolution (Fig. 2, DH2 evo-lution ¼ 13.1�DCO2 evolution� 0.185, n¼ 20, r¼ 0.69,P, 0.0008).

Contents of amino acids and adenylates

Cit and Glu were the most abundant free amino acids inthe root nodules representing 38 and 36%, respectively,of the total amino acids in the control plants (Table 2).Other common amino acids were Asp, Gln and Ala. Thetotal free amino acid content of nodules of dark-treatedplants was 84% of the control plants. The content of Citwas significantly lower in dark-treated plants and was67% of the control.In response to short-term N2 deprivation the amounts

of Gln (Fig. 3A) decreased rapidly in control plants aswell as in dark-treated plants to 42 and 38% of initial,respectively, after 45min in Ar:O2. The content of Cit(Fig. 3C) decreased in dark-treated plants to 55% after45min in Ar:O2. The contents of Glu, Asp and Ala didnot change significantly (Fig. 3B, D and E). A reductionin Asn content was observed in control plants after45min with N2 deprivation (not shown). Changes inthe remaining amino acids in response to N2 deprivationwere not statistically significant.The root nodules contained high amounts of ATP and

ADP (Table 3), but there were no significant differencesbetween the dark-treated plants and the control plants.The ATP/ADP ratio was 4.5 and 5.1 in control plantsand dark-treated plants, respectively. Short-term N2

deprivation did not cause any significant changes in thecontents of these adenylates.

Discussion

Responses of nitrogenase activity to prolonged darkness

and O2

Nitrogenase activity in A. incana root nodules decreasedin response to darkness for 22 h (Table 1). The decreasewas smaller than that found in earlier studies (Lundquistand Huss-Danell 1991a, b) where an acetylene reductionassay was used to measure nitrogenase activity.Decreased nitrogenase activity in response to prolongeddarkness may be attributed to carbon starvation of theplants and the root nodules. Alternatively, feedback inhi-bition of root nodule metabolism and nodule growththrough accumulation of one or several amino acidshas been suggested as an important regulatory mechan-ism (Parsons et al. 1993). Exposure to prolonged dark-ness could lead to closure of stomata, inhibition oftranspiration and therefore a reduction in the removalof amino acids from the root nodules in the xylem sap.However, we found that none of the detected aminoacids in the root nodules increased compared to thecontrol plants. Instead the most abundant amino acidCit decreased significantly (Table 2). This result does notsupport the idea that nitrogenase activity decreased dueto a feedback mechanism sensitive to an overall accumu-lation of the detected amino acids in root nodules in thisparticular treatment.In several respects the dark-treated plants were more

sensitive than control plants to altered conditions. In

∆ CO2 (% min–1)

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Fig. 2. Rate of decline of H2 evolution vs. the rate of decline of CO2

evolution in response to short-term N2 deprivation at 21% O2 inintact nodulated Alnus incana root systems of dark-treated (*) andcontrol plants (*). The H2 and CO2 evolution rates obtainedduring 5min starting 7min after the change from N2:O2 to Ar:O2

were normalized as percentage of themaximumevolution rates inAr:O2,and the rates at which the decline occurred were calculated. Linearregression analysis gave the relationship DH2 evolution¼ 13.1�DCO2

evolution� 0.185, n¼ 20, r¼ 0.69, P, 0.0008.

Table 2. Amino acid content of Alnus incana root nodules fromcontrol plants and plants kept in prolonged darkness of 22 h. Theroot systems were kept in the gas exchange system in N2:O2 for 1 hprior to harvesting the root nodules. Data are shown as means (fivecontrols and four dark-treated)±SE. Significant differences(P, 0.05) between treatments are indicated as asterisks. Otheramino acids (, 0.14mmol g�1 FW) include Asn, Tyr, GABA, Val,Lys, Pro, Met.

Amino acidControl plants(mmol g�1 FW)

Dark-treated plants(mmol g�1 FW)

Gln 0.53±0.02 0.40±0.05Asp 1.60±0.19 1.63±0.18Cit 6.83±0.66 4.56±0.17 *Glu 6.44±0.21 6.08±0.11Arg 0.23±0.09 0.18±0.12Gly 0.30±0.09 0.29±0.04Thr 0.34±0.03 0.26±0.01 *Ala 0.44±0.05 0.42±0.05Phe 0.23±0.07 0.17±0.02Ile1 leu 0.14±0.02 0.16±0.03Orn 0.18±0.06 0.16±0.06Others 0.65±0.1 0.68±0.28Total 17.9±1.12 15.0±0.43


addition to the loss of nitrogenase activity due to the 22 hin darkness, the dark-treated plants also lost a significantfraction of nitrogenase activity during the time period inthe N2:O2 gas composition at 21 and 17% O2 (Table 1).This suggests that they were more sensitive to O2. In viewof the lack of any evidence for a variable diffusion barrieragainst O2 in A. incana nodules (Lundquist 2000), ourresults suggest inactivation of nitrogenase by O2. Itappears that for nodules of dark-treated plants kept atan external pO2 of 21%, the respiratory O2 consumptionis not high enough to completely reduce the intracellularpO2 to an acceptable level for nitrogenase (Table 1). Alower respiration of dark-treated plants could be due to a

limitation in respiratory capacity of Frankia, and/oramounts of carbon substrate supporting respiratory O2

consumption. In an earlier study of effects of prolongeddarkness (Vikman et al. 1990), symbiotic Frankia vesicleclusters were rapidly purified from the same symbiosis asin the present study. The study showed that the capacityto respire various added substrates was lower alreadyafter a 24-h-period of prolonged darkness (Vikman et al.1990). Moreover, the conditions in the cuvette of the gasexchange analysis system should create higher ventilationof the root nodules compared to during normal growth,which the dark-treated plants appeared to perceive as anincreased pO2. Therefore, the results suggest that respiration

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Fig. 3. The effect of short-term N2 deprivation at 21%O2 on contents of Gln, Glu,Cit, Asp and Ala in rootnodules of Alnus incanacontrol plants (*) and 22 h-dark-treated plants (*). Dataare shown as means±SE forthree to five plants. Significantdifferences (P, 0.05) due toshort-term N2 deprivationcompared to initial contentsare indicated by asterisks.

Table 3. Contents of ATP and ADP, and ATP/ADP ratios, in Alnus incana root nodules from control plants and plants kept in prolongeddarkness of 22 h. Prior to harvesting the root nodules the root systems were kept in the gas exchange system in N2:O2 for 1 h, and for someplants also in Ar:O2 for 10 or 45min. Data are shown as means±SE for the number of plants in parentheses.

Control plants Dark-treated plants

TreatmentATP(nmol g�1 FW)

ADP(nmol g�1 FW)

ATP / ADPratio

ATP(nmol g�1 FW)

ADP(nmol g�1 FW)

ATP / ADPratio

N2:O2 184±12 41.3±1.8 4.5±0.21 (4) 182±15 36.5±4.0 5.1±0.13 (3)Ar:O2, 10min 192±21 39.6±4.2 5.0±0.32 (7) 171±15 32.9±1.5 5.2±0.40 (3)Ar:O2, 45min 174±11 34.1±3.0 5.3±0.50 (4) 176±16 36.2±4.6 5.0±0.37 (4)


of dark-treated plants is less able to adapt to a perceivedincreased pO2 making these nodules more sensitive toO2. At the lower pO2, respiration could be able to con-tribute to maintain an acceptable pO2 for nitrogenase(Table 1), because of the lower O2 stress, and alsobecause less amounts of carbon substrate is used at thelower respiration rate in the low pO2.Avoiding O2 inactivation of nitrogenase is an import-

ant role of the Frankia vesicle. Since the protection inFrankia is considered to largely depend on the fixeddiffusion barrier in the vesicle envelope together withO2 consumption inside the vesicle, a continuous reduc-tant supply is critical for maintaining a stable flux andconsumption of O2. In addition, a continuous electronflux through nitrogenase can also contribute in prevent-ing inactivation of nitrogenase. Nitrogenase of the aero-bic nitrogen-fixing bacterium Azotobacter sp. has beenshown to be able to account for O2 consumption throughreduction to H2O2, and under certain conditions theN2-fixing activity can be inactivated in a reversible way(Thorneley and Ashby 1989). To reduce intracellular O2

concentration the respiratory O2 consumption plays acrucial role in N2-fixing root nodules on legume plants(Layzell and Hunt 1990), as well as in root nodules onactinorhizal plants including Alnus (Silvester et al. 1990,Lundquist 2000). For unstressed A. incana plants grownat an ambient pO2 of 21% the nitrogenase activityoccurred at a maximal rate at 20% O2 (Lundquist2000). At a pO2 above 20% nitrogenase graduallybecame inactivated, and below 20% O2, nitrogenaseactivity was limited by diffusion barriers in the nodule.With this notion of a fixed diffusion barrier for O2, weconclude that stress situations, which limit carbon allo-cation to respiration, are intimately related to oxygenstress for nitrogenase. O2 inactivation of nitrogenasedue to an increasingly carbon-limited respiratory O2 con-sumption is likely to be an important factor during thegradual loss of nitrogenase activity during prolongeddarkness.

Responses of nitrogenase activity to short-term N2

deprivation and O2

Short-term N2 deprivation of dark-treated plants causeda decline in nitrogenase activity that was dependent onthe pO2 (Fig. 1A–C), in a similar way to what was earliershown for unstressed A. incana plants (Lundquist 2000).However, again the dark-treated plants were more sensi-tive to pO2 than control plants such that the declineoccurred at lower pO2 values in dark-treated plants com-pared to unstressed plants (Fig. 1A–C and Lundquist2000). Furthermore, the decreased nitrogenase activityof dark-treated plants remained significantly lower aftera 15-min-recovery period in N2:O2.As discussed previously (Lundquist 2000), there could

be a couple of mechanisms explaining the decline innitrogenase activity during N2 deprivation. An import-ant component is disturbance to plant and bacterial

metabolism during the cessation of NH41 production.

This could lead to a reduced reductant supply to Frankiathat would limit nitrogenase activity as such, limit pro-tective electron flux through nitrogenase and also limitprotective O2-consuming Frankia vesicle respiration.Disturbance to plant amino acid metabolism in the sur-rounding plant cell would also affect plant O2-consumingrespiration. In case the respiratory processes in thenodules become limited by substrate this would lead toan increase in the cellular concentration of O2 whichcould cause inactivation of nitrogenase by O2. In thesecarbon-starved plants, the metabolite reserves would besmaller and therefore less able to immediately compen-sate for disturbance caused by the cessation in NH4

1

production on a link between amino acid synthesis andcarbon flow. At 21 and 17% O2 these metabolite reserveswould be more rapidly used up compared to 13%because of the higher respiration rate. This in turnwould lead to inactivation of nitrogenase by O2.In support of this are the results obtained from the

dark-treated plants initially kept at 21% O2 for 45min,and later at 17% during N2 deprivation (Fig. 1D), whichdid not show a decline in nitrogenase activity during theN2 deprivation. Furthermore, these results are consistentwith the hypothesis that the same mechanism explainsthe decline during N2 deprivation as well as the effect ofO2 during the 1-h period in N2:O2 at 21% O2 (Table 1).The results suggest that inactivation occurred in a frac-tion of the nodules of each plant that is particularlysensitive, or in a sensitive fraction of each root nodule.The sensitive fraction appears to have been inactivatedduring the initial period at 21% O2 leading to abolish-ment of a decline during the following period of N2

deprivation at 17% O2 (Fig. 1D). In this context theresults shown in Fig. 1A and 1B could be interpreted asthat it was the most sensitive fraction that was affected byN2 deprivation at 17% O2. At 21% O2 a bigger fractionalso containing less sensitive nodules was affected.During short-term N2 deprivation, the rate of the

decline in nitrogenase activity was overall correlated tothe decline rate of CO2 evolution (Fig. 2). This couldreflect a reduction in the energy use of nitrogenase activ-ity. In addition, this result could also reflect a lowermetabolic rate and lower O2 consumption in the plantfraction of the nodule due to cessation of NH4

1 produc-tion and corresponding inhibition of amino acid meta-bolism. The decline rates of H2 evolution and CO2

evolution were higher in the dark-treated plants(Fig. 1A and B and Fig. 2). The decline rate of H2

evolution versus CO2 evolution also seemed to be com-paratively higher in the dark-treated plants (Fig. 2) whichfurther shows the higher sensitivity of nitrogenase activ-ity in dark-treated plants.

Responses of root nodule metabolism to prolonged

darkness and short-term N2 deprivation

Citrulline, the suggested transport form of nitrogen tothe shoot in Alnus (Gardner and Leaf 1960), was the


most abundant amino acid in the root nodules (Table 2).This is also consistent with a series of experiments onA. glutinosa (Baker et al. 1997) which showed that Citcould act as a store for nitrogen. The significant decreasein Cit due to the dark treatment (Table 2) clearly showedthat root nodule metabolism was affected by prolongeddarkness.Root nodule primary nitrogen metabolism responded

rapidly to the short-term N2 deprivation, as seen in thedecrease in the contents of the amino acids Gln and Cit(Fig. 3). Gln is commonly the first amino acid in the pathof NH4

1 assimilation in plants. Gln functions as anamino donor in the reductive transfer of the amide nitro-gen to a-ketoglutarate catalysed by glutamate synthase(GOGAT). Gln is also the amino donor in the synthesisof carbamoyl phosphate, a precursor in the synthesis ofCit (Schubert and Boland 1990). Decreased Gln concen-tration during N2 deprivation is explained by ceasedproduction of NH4

1 by N2 fixation, and a continuedmetabolism of Gln.The decrease in Cit concentration can, in consequence,

be explained by lack of Gln available for carbamoylphosphate synthesis together with transport from thenodules, degradation of Cit or further metabolism toArg. The remaining Cit in dark-treated nodules decreasedrelatively more rapidly in dark-treated plants than incontrols (Fig. 3), indicating that a more efficient trans-port, a more active degradation or a slower synthesis wasinduced during N2 deprivation in the dark-treated plants.Glutamate is suggested to serve as the amino donor in

several steps in the synthesis of Cit (Schubert and Boland1990). A high turnover rate, but not necessarily largepools, is therefore expected. The large amounts of gluta-mate found in the present study therefore suggest thatthere are at least two pools of glutamate present in theroot nodule, a small with high turnover rate and a largerstorage pool. The lack of change in the amount of glu-tamate in response to N2 deprivation (Fig. 3B) neitherfor dark-treated plants nor for control plants is consis-tent with this idea.The present report is the first on adenylate contents in

Alnus nodules. The amount of ATP plus ADP in theseA. incana nodules was about 200 nmol g�1 FW. This islower than, but in the same order of magnitude as, inmaize root tips (approximately 400 nmol g�1 FW, Xiaet al. 1995) and in soybean root nodules (approximately300 nmol g�1 FW, Oresnik and Layzell 1994). Interest-ingly, the fairly high ATP/ADP ratio of about 5 inthese A. incana nodules suggests an aerobic metabolismin the root nodules (Raymond et al. 1987), despite thehigh sensitivity of nitrogenase to inactivation by O2. As acomparison, maize roots pretreated for 6 h in 100% O2

or 3% O2 had ATP/ADP ratios of about 5 and 1, respec-tively (Xia et al. 1995). The present results thus supportearlier studies of Alnus root nodule anatomy, whichshowed that these nodules appear well aerated with acontinuity of air spaces into groups of infected cells(Tjepkema 1979). In contrast, the root nodules of legumeplants appear to have a microaerobic condition in the

major part of the root nodule, which is also reflected inan overall nodule ATP/ADP ratio of 1.5–2.7 (Oresnikand Layzell 1994, Kuzma et al. 1995). The present resultconfirms that Alnus nodules have a different O2 metabo-lism compared to legume nodules.No significant changes were detected in overall root

nodule contents of ATP and ADP in response to theprolonged darkness treatment (Table 3), suggesting thatthe plants were not severely stressed. The activity ofsome of the enzymes in glycolysis and in mitochondrialmetabolism can be inhibited by a high ATP/ADP ratio(Raymond et al. 1987). Also, addition of NH4

1 to nitro-gen-starved cells can reduce the ATP/ADP ratio(Raymond et al. 1987). The ATP/ADP ratio was thereforealso investigated during N2 deprivation as an overallmetabolic indicator, which could indicate inhibition ofmetabolism important for O2-consuming respiration.Our results did, however, not show any significant differ-ences in response to short-term N2 deprivation for eithercontrol plants or dark-treated plants after 10 or 45min inAr:O2 (Table 3). The ATP/ADP ratios that were deter-mined were the overall ratios of the entire root nodules,and further studies should include fractionation of thenodules. The two compartments that probably have thehighest turnover of adenylates are the Frankia vesicleand the surrounding infected plant cell in which synthesisof Cit is likely to occur. Nitrogenase use about 10–15ATP per ½N2 reduced in vivo (Burris 1991) while synth-esis of Cit can cost about 5 ATP per N (Schubert andBoland 1990). If the adenylate pool sizes of these twocompartments were proportional to what is needed forthese major metabolic reactions this would indicate ahigher amount in Frankia compared to the plant cell.The ATP/ADP ratio of the Frankia vesicles does notnecessarily need to differ much between the N2:O2 con-dition and the Ar:O2 condition since nitrogenase is stilloperating at a high activity producing H2 gas. In theplant cell the demand for ATP is likely to decrease dur-ing cessation of NH4

1 production in Ar:O2. If anincrease in the ATP/ADP ratio occurs in the infectedplant cell in response to the shift to non-N2-fixing andnon-assimilating condition, the increase may however, beconfounded in the ratio of overall nodule adenylates. It isalso likely that metabolic changes that would result in anincrease in the ATP/ADP ratio are adjusted fairly rapidly.In conclusion, we have shown that nitrogenase activity

of carbon-starved A. incana plants is more sensitive to O2

inactivation than control plants. Short-term N2 depriva-tion was found to rapidly lead to inhibition of nitrogen-ase activity of carbon-starved plants caused by O2 andlack of ability to adjust their metabolism. We alsoshowed that plant-related amino acid metabolism wasrapidly affected by short-term N2 deprivation. We con-clude that root nodule nitrogen metabolism is intimatelyconnected to O2 protection of nitrogenase in A. incananodules.

Acknowledgements – We thank Dr P. Gardestrom for advice onthe adenylate analysis, Dr A. Sellstedt for generous lending of


equipment, A. Sandstrom for helpful assistance with plant cultiva-tion, M. Zetterstrom for help with amino acid analyses and theDepartment of Plant Physiology, Umea University, Umea, Sweden,for general facilities. This work was supported by the SwedishNatural Science Research Council (K.H.-D.) and the SwedishResearch Council for Environment, Agricultural Sciences andSpatial Planning (T.N.).

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Edited by J. I. Sprent


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