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Plant Physiol, February 2000, Vol. 122, pp. 553-562 Nitrogenase Activity in Alnus incana Root Nodules. Responses to O2 and Short-Term N2 Deprivation1Department of Plant Biology, Swedish University of Agricultural Sciences, P.O. Box 7080, S-750 07 Uppsala, Sweden.
O2 and host-microsymbiont interactions are key factors affecting the physiology of N2-fixing symbioses. To determine the relationship among nitrogenase activity of Frankia-Alnus incana root nodules, O2 concentration, and short-term N2 deprivation, intact nodulated roots were exposed to various O2 pressures (pO2) and Ar:O2 in a continuous flow-through system. Nitrogenase activity (H2 production) occurred at a maximal rate at 20% O2. Exposure to short-term N2 deprivation in Ar:O2 carried out at either 17%, 21%, or 25% O2 caused a decline in the nitrogenase activity at 21% and 25% O2 by 12% and 25%, respectively. At 21% O2, nitrogenase activity recovered to initial activity within 60 min. The decline rate was correlated with the degree of inhibition of N2 fixation. Respiration (net CO2 evolution) decreased in response to the N2 deprivation at all pO2 values and did not recover during the time in Ar:O2. Increasing the pO2 from 21% to 25% and decreasing the pO2 from 21% to 17% during the decline further decreased rather than stimulated nitrogenase activity, showing that the decline was not due to O2 limitation. The decline was possibly due to a temporary disturbance in the supply of reductant to nitrogenase with a partial O2 inhibition of nitrogenase at 25% O2. These results are consistent with a fixed O2 diffusion barrier in A. incana root nodules, and show that A. incana nodules differ from legume nodules in the response of the nitrogenase activity to O2 and N2 deprivation.
Biological N2 fixation is inhibited by
atmospheric levels of O2 because of the
O2 sensitivity of nitrogenase.
N2-fixing organisms living in an aerobic
environment therefore use various physiological and biochemical
mechanisms to provide an acceptable O2
concentration for nitrogenase and, simultaneously, to allow the use of
O2 in oxidative phosphorylation. Root nodules of
many legumes such as soybean, alfalfa, lupin, and clover have an
apparently variable barrier to gas diffusion that may function to
regulate internal O2 concentration and protect
nitrogenase activity in the rhizobial microsymbiont (Hunt and Layzell,
1993 In several actinorhizal N2-fixing symbioses,
symbioses between the actinomycete bacterium Frankia and a
range of plant species including Alnus incana, nitrogenase
activity also declined shortly after exposure to acetylene in
continuous flow assay systems (Rosendahl and Huss-Danell, 1988 Among actinorhizal root nodules there is a diversity in structural
organization in the host part as well as in the microsymbiont, and
their physiologies relative to O2 are apparently
also diverse (Silvester et al., 1990 A. incana nodules are considered well ventilated, having gas
diffusion pathways into the zone of infected cells (Tjepkema, 1979 Metabolic interactions between the plant host and the microsymbiont
clearly occur, since the host supplies Frankia with
reductant in some form. Also, the assimilation of
NH4+ in A. incana root nodules is likely to be carried out by the infected
plant cell rather than in Frankia, since neither of
Frankia's two forms of Gln synthetases are expressed in the
symbiotic stage (Lundquist and Huss-Danell, 1992 The goals of this study were: (a) to characterize the optimum pO2 of nitrogenase activity for this Frankia-A. incana symbiosis, and (b) to address the questions of whether Ar:O2 treatment causes a decline in nitrogenase activity and respiration, and if such a decline affected by external pO2?
Plant Material and Growth Conditions Cuttings of a clone of gray alder (Alnus incana [L.]
Moench) were rooted and inoculated with "the local source of
Frankia" lacking uptake hydrogenase activity (Sellstedt et
al., 1986 Gas Exchange Measurements Nitrogenase activity was measured as H2
evolution and respiration as net CO2 evolution in
a gas exchange system generally adapted from a system described
previously (Layzell et al., 1989 The H2 sensors, O2 sensors,
IRGA, and mass flow controllers were connected to a Macintosh IIfx
computer via an analog to digital interface board. Outputs and data
collection were operated through the program Workbench (Strawberry
Tree, Sunnyvale, CA), which was programmed so that any desired partial
pressure of N2, Ar, and O2
could be achieved without affecting the total flow. The signals were
averaged over 10 s and recorded every 20 s. Changes in
outputs to flow controllers were programmed in Workbench when possible
to improve reproducibility in gas composition and timing between
experiments. Two plants were measured simultaneously. The total flows
were controlled by the mass flow controllers and the flows to each
cuvette were manually adjusted to 0.8 L min During the experiments the plants were kept in a chamber covered with
plastic and containing a temperature-controlled water bath to provide a
stable cuvette temperature. The chamber was kept humid by spraying
water on the white absorbent paper covering the inside walls. A second
water bath acted as a heat trap below the metal halogen lamp. The PPFD
at 40 cm above the cuvette was approximately 450 µmol
m The H2 analyzers were calibrated using a standard
gas of H2 (1,787 µL L Responses to Changes in pO2 Two experiments were performed to characterize the responses of H2 evolution and CO2 evolution to pO2. In experiment 1, in which the response in N2:O2 was investigated, the nodulated root systems were first kept with a flow of N2:O2 at 21% O2 for 25 to 30 min. The rates had then been stable for 10 to 15 min and a short shift from N2:O2 to Ar:O2 and back was done for 3 min to measure the total nitrogenase activity. The pO2 was then kept at 21% for 15 min and then changed in steps of 1% every 5 min for different plants ending either at 17% or 25% O2, which was kept for the remaining part of the experiment. The gas composition was either kept as N2:O2 up to 75 min, or changed from N2:O2 to Ar:O2 15 min after reaching the final pO2, as described for studies of responses to N2 deprivation. In the second experiment, the nodulated root systems were kept in the
gas exchange system for 50 min at 21% O2. The
pO2 was then altered in steps of 2%
O2 every 10 min, first down to 15% O2, then up to 25% O2, and
finally back to 21% O2. During the 10-min period
at each pO2, the gas composition was first
N2:O2 for 4 min followed by
Ar:O2 for 2.5 min and then
N2:O2 for 3.5 min. The
plotted values are from the end of the 4-min
N2:O2 period and from the
end of the Ar:O2 period. The switch to
Ar:O2 was necessary to measure the total electron
flux through nitrogenase, since no N2 is reduced
in Ar:O2 and all electrons are used for H2 production. The electron allocation
coefficient (EAC) of nitrogenase was calculated according to the method
of Edie and Phillips (1983) Responses to N2 Deprivation To test the effect of a short period of N2 deprivation on H2 evolution and on the remaining H2 evolution in N2:O2 afterward, nodulated root systems were pretreated by keeping them in N2:O2 at 21% O2 for 50 min. The gas composition was then changed from N2:O2 to Ar:O2, kept for 15 min, and then changed back to N2:O2. The effect of a longer period of N2 deprivation on H2 evolution and CO2 evolution was tested by exposing nodulated root systems to Ar:O2 for 60 min at different pO2 values. This was done with the plants used in experiment 1 in the study of responses to changes in pO2 described above. The exposure to Ar:O2 was carried out at either 17% or 25% O2 starting 15 min after reaching that pO2, or at 21% after the same total time had elapsed. After the Ar:O2 period, the gas composition was changed back to N2:O2 for an additional 15 min. To determine whether O2 could stimulate or further inhibit H2 evolution during the Ar-induced decline, the pO2 was either increased to 25% O2 or decreased to 17% starting 15 min after the change from N2:O2 to Ar:O2. This pO2 was kept for 15 min and then returned to 21%. Finally, the gas composition was changed from Ar:O2 to N2:O2. The pretreatment for these plants was the same as for the experiments on N2 deprivation described above. To determine whether a complete elimination of N2
fixation was necessary until a decline occurred, the nodulated root
systems were exposed to various external N2
pressures (pN2) balanced by Ar at 21%
O2. Root systems were initially kept in
N2:O2 at 21% O2 for 30 min. The gas composition was then
changed to 20%/59%/21% (Ar:N2:O2) for 10 min,
followed by 20 min in
N2:O2. Using this protocol,
changes in gas composition followed, which sequentially exposed the
root systems to 39.5%, 20%, 10%, and 0% N2.
The H2 and CO2 evolution
rates at the various pN2s were normalized as percentages of
the maximum H2 evolution at 0%
N2 (Ar:O2). The rates at
which the rates of H2 evolution and
CO2 evolution declined were calculated (linear
regression) as the percent change per minute on the data obtained
during 5 min starting 5 min after the change from
N2:O2 to
Ar:N2:O2. A coefficient of
inhibition of N2 fixation was calculated as:
(H2 evolution in
N2:Ar:O2
Responses to Changes in pO2 The maximum H2 evolution rate occurred at about 20% O2 (Figs. 1 and 2A) when pO2 was either reduced or increased from 21% O2. During the time at low pO2, the H2 evolution adapted and increased to be about 8% higher the second time at 19% O2 compared with the first time (Fig. 2A). At 25% pO2, the H2 evolution rate decreased by 36% but increased slightly upon return to 21% O2 (Fig. 2A). The H2 evolution recovered by a few percent during 10 min at 21% O2.
CO2 evolution also responded to changes in pO2. In N2:O2 (Fig. 1), the CO2 evolution rate decreased by 5% when pO2 was reduced from 21% to 17% O2, and increased by 3% when pO2 was increased from 21% to 25% O2. Below 20% O2, the CO2 evolution was linearly correlated to the H2 evolution. A smaller but similar adaptation at low pO2 as described above for H2 evolution also occurred (Figs. 1 and 2B). Responses to N2 Deprivation Changing from N2 to Ar as balancing gas gave an immediate increase in H2 evolution (Figs. 3, 4A, and 5A), as expected for nitrogenase activity since removal of N2 allows allocation of all reducing equivalents to proton reduction. The H2 evolution increased on average to 252% (Figs. 3, 4A, and 5A). The average EAC of several plants used in different experiments and calculated from the first short shift to Ar:O2 in the pretreatment was 0.60 ± 0.01 (mean ± SE, n = 28). In the experiment where pO2 was varied (Fig. 2A), EAC was 0.60% at 21% O2 and showed a statistically significant lower value only at 25% O2. This was apparently because the value in Ar:O2 was recorded a few minutes after the value in N2:O2, when inactivation of nitrogenase at high pO2 had proceeded further.
During the exposure to N2 deprivation in Ar:O2 (79:21), the total nitrogenase activity declined, as seen by the decline in H2 evolution (Fig. 3). The declined H2 evolution remained when the gas composition was changed back to N2:O2 after 15 min in Ar:O2 (Fig. 3), which is about when the activity was at its lowest point in Ar:O2 (Fig. 4A). The remaining activity in N2:O2 was 87% of the initial activity in N2:O2 and therefore had decreased as much as the activity in Ar:O2 had decreased compared with the initial peak activity in Ar:O2 (Fig. 3). The decline in H2 evolution was related to the pO2 at which the N2 deprivation treatment was carried out. The change from N2:O2 to Ar:O2 caused a decline in H2 evolution within a few minutes when the change was carried out at 21% or 25% O2 (Fig. 4A). The activity declined to a minimum at 88% of the peak activity after 17 min at 21% O2 and to 75% of the peak activity after 24 min at 25% O2. Following the decline, the activity increased and after 60 min in Ar:O2, it had recovered almost completely at 21% O2, but only partially at 25% O2. In contrast, at 17% O2 there was no decline in H2 evolution following the change to Ar:O2, and over the 60-min period in Ar:O2 the activity increased to 113% of initial activity in Ar:O2 (Fig. 4A). After the change back to N2:O2 the differences in H2 evolution between the treatments remained. H2 evolution of plants kept as controls at 17% or 25% O2 for 75 min without exposing them to Ar, increased gradually by 12% and 8%, respectively, over the 60-min period that corresponded to the Ar treatment (data not shown). To determine whether the decline in activity during N2 deprivation was an effect of the increased gas flow through the root system during the assay, two plants were grown for 2 and 3 weeks in the regular growth conditions with an air flow through the pot at the same rate as during the gas exchange measurements. These plants showed a similar decline and recovery at 21% O2 as in Figure 4A (data not shown). A large part of the CO2 evolution came from the nodules because only 50% of the nodulated root CO2 evolution remained after removing the nodules from the nodulated roots. CO2 evolution from the nodulated root systems decreased during N2 deprivation (Figs. 4B and 5B). During the 60-min period in Ar:O2 at 25% O2, the CO2 evolution rate decreased by about 10% of the initial CO2 evolution and was still 5% lower after the change back to N2:O2, thus showing the same pattern as for H2 evolution. In contrast, the CO2 evolution rate of the root systems kept at 17% and 21% O2 in Ar:O2 only decreased by 5% and returned to the initial rate upon the change back to N2. For nodulated root systems not exposed to Ar:O2 and kept at either 17% or 25% O2, the CO2 evolution rate increased by 3% and 7%, respectively, during the corresponding 60-min period (data not shown). No significant effects on respiration of changing from N2:O2 to Ar:O2 were found on root systems in which the nodules had been removed (data not shown). To further investigate the relationship between O2 and the Ar-induced decline in H2 evolution, the pO2 was changed 15 min after the change to Ar:O2 (Fig. 5). The decline could not be reversed by increasing the pO2. The pO2 was either increased to 25% or decreased to 17% and gave in both cases a further decrease in activity from 227% to 107% and from 214% to 189% of initial in N2:O2, respectively. In both cases H2 evolution recovered gradually at the new pO2. After 15 min at either 17% or 25% O2, the pO2 was changed back to 21%. A small drop in activity followed by a recovery occurred after the change from 17% to 21% O2. After an additional 15-min period, when Ar was replaced by N2, the activity was close to the initial rate for the plants temporarily exposed to 17% O2, but only 72% of the initial rate for the plants temporarily exposed to 25% O2. These latter plants recovered their activity to 88% of initial values within 15 min. The rate of the Ar-induced decline at different pN2s was not linearly correlated to pN2 (Fig. 6A). The decline occurred at pN2 lower than 20%, where the inhibition coefficient of N2 fixation indicated that N2 fixation was significantly reduced (Fig. 6B). The decline rate showed a linear correlation to the inhibition coefficient of N2 fixation (P < 0.05).
The experiments demonstrated the following major characteristics of nitrogenase activity in Frankia-A. incana root nodules. Nitrogenase activity has a sharp pO2 optimum. In response to N2 deprivation at pO2s above optimum, the nitrogenase activity declined followed by some recovery. In addition, the decline was not reversed by increased or decreased pO2. Responses to pO2 The pO2 giving maximum nitrogenase activity was 20% (Figs. 1 and 2A). Below 20% O2, nitrogenase activity seemed limited by O2 supply, since the activity decreased with decreasing pO2 and was stimulated by a return to higher pO2. Above 20% O2, nitrogenase activity became inactivated (Figs. 1 and 2A), which was partly irreversible since the activity remained lower when the pO2 was returned to 21%. The optimum at 20% O2, close to the pO2 of the growth
conditions, is consistent with earlier results for A. incana
nodules (Winship and Tjepkema, 1985 Respiration, measured as CO2 evolution, increased with increasing pO2 (Fig. 1). Below 21% O2 the change in respiration closely followed nitrogenase activity (Figs. 1 and 2). Some adaptation of nitrogenase activity and respiration occurred during the short period at pO2s lower than optimum (Fig. 2) and also during periods of 75 min at 17% and 25% O2 in N2:O2. However, the linear relationship between respiration and nitrogenase activity below optimum pO2 and the inhibition of nitrogenase activity above optimum are consistent with the presence of a fixed diffusion barrier to create a suitable internal O2 concentration for nitrogenase. Responses to N2 Deprivation The immediate rise in H2 evolution (Fig. 3)
showed that N2 fixation was eliminated by the
change from N2:O2 to
Ar:O2 due to removal of the substrate
N2. The EAC of approximately 0.6 found in the
present study is close to the results from intact root systems of
legumes (Hunt et al., 1987 The occurrence of a decline in nitrogenase activity following the
change from N2:O2 to
Ar:O2 (Fig. 3 and 4) resembles the acetylene-induced decline in nitrogenase activity of Alnus
spp. nodules during the acetylene reduction assay (Rosendahl and
Huss-Danell, 1988 First, the decline in nitrogenase activity could be caused by an
increased diffusion resistance for O2 in the
nodule. In some legume plants the nitrogenase activity declines
following exposure to Ar:O2 (e.g. Hunt et al.,
1987 Second, the decline in nitrogenase activity could be caused by a limitation in a supply of reductant to nitrogenase. Cessation of NH4+ production and assimilation in Ar:O2 could cause a disturbance in a supply of metabolites from the plant to Frankia that supports nitrogenase activity (compare with Fig. 7). The metabolism yielding reductant for nitrogenase in Frankia has not been elucidated. However, the overall high nitrogenase activity after 1 h with N2 deprivation at 17% and 21% O2 (Fig. 4A) suggests that there is not a simple short metabolic link between the production of NH4+ through N2 fixation and sustenance of nitrogenase activity as H2 evolution. Nevertheless, the decline in Ar:O2 at 21% O2, where nitrogenase activity operated more or less at its optimum pO2, since neither an increase or a decrease in pO2 stimulated nitrogenase activity (Fig. 5A), supports that nitrogenase activity declined due to decreasing amounts of reductant, ATP, or a specific metabolite. The decrease in CO2 evolution during the decline in Ar:O2 (Figs. 4B and 5B) could be interpreted as a decrease in an NH4+ assimilation-linked respiration. Also, the fact that a complete cessation of N2 fixation was not necessary for an Ar-induced decline to occur, but rather that the rate of the Ar-induced decline correlated to the degree of inhibition of NH4+ production (Fig. 6) supports the hypothesis that disturbances in a process linked to NH4+ assimilation such as plant carbon metabolism could be important and involved in the response of nitrogenase activity.
The effect of O2 on the occurrence of the Ar-induced decline (Fig. 4) in this context could be explained as follows. At the higher pO2 the total respiratory demand increased (Figs. 1 and 2). As a result, the respiratory system would have fewer metabolite reserves and therefore less ability to immediately compensate for the disturbance caused by Ar on a link between amino acid synthesis and carbon flow to Frankia. Also, at the lower pO2, nitrogenase activity becomes more O2 limited, so any changes in reductant supply may have less effect on nitrogenase activity. Nitrogenase activity and vesicle respiration could possibly be competing for reductant. If respiration is more successful at the higher pO2, it is possible that reductant limitation could cause the decline in nitrogenase activity. However, simply decreasing the pO2 from 21% to 17% after 15 min in Ar:O2 did not relieve any competition from respiration but, rather, decreased nitrogenase activity further, which is why this explanation does not seem likely. Third, the decline of nitrogenase activity in
Ar:O2 could be caused by inactivation of
nitrogenase by O2. The bigger decline at 25%
O2 compared with at 21% (Fig. 4A), and the high
sensitivity to increasing pO2 during the decline
in Ar:O2 (Fig. 5A) suggest that the decline is
due to O2 inactivation. The incomplete recovery of nitrogenase activity during the hour in Ar:O2
(Fig. 4A) and the remaining inhibition of nitrogenase after the change
back to N2:O2 (Figs. 3 and
4A) are further support for the idea that a partial inactivation of
nitrogenase causes the Ar-induced decline of nitrogenase activity, in
particular at 25% O2. A simple calculation of
the internal O2 concentration according to
Fick's first law of diffusion and assuming fixed diffusion resistances
(Sheehy et al., 1983 An increase in vesicle O2 concentration in response to short-term N2 deprivation at 25% O2, where Frankia vesicle respiration may be operating closer to saturation, would therefore take a longer time for the respiration to consume and consequently cause greater damage to nitrogenase. At 17% O2, it would be easier for the vesicle respiration to respond and consume any extra O2. The change from 21% to 17% O2 (Fig. 5A) would be expected to give an increase rather then a further decrease in nitrogenase activity if changing from 21% to 17% O2 relieved any O2 inhibition, and this result argues against a substantial O2 inhibition during the decline at 21% O2. The observed response to decreasing the pO2 from 21% to 17% O2 is consistent with nitrogenase becoming O2 limited, because of a limitation in factors such as ATP supply. Any possible relief of O2 inhibition may therefore be obscured by these factors. Some degree of O2 inhibition of nitrogenase at 25% O2 is possible, since there is very strong inhibition even before the Ar:O2 treatment begins. There are two possibilities through which the O2 concentration at the site of nitrogenase could increase. One explanation could be that the eliminated NH4+ production in Ar:O2 could disturb metabolite supply from the plant to Frankia in a similar way as discussed for the second hypothesis and lead to a decrease in the O2 consumption by the Frankia vesicle respiration due to substrate limitation. An alternative is that an increase in vesicle O2 concentration could be due to an increase in O2 concentration originating external to Frankia. Since NH4+ is assimilated in the plant host cells, exposure to Ar:O2 deprives the NH4+ assimilation metabolism in the host cell surrounding Frankia of its substrate. The elimination of NH4+ production and subsequent assimilation could therefore inhibit plant metabolism and mitochondrial respiratory O2 consumption through reduced turnover of ATP and NADH. This could lead to a temporary increase in O2 concentration in the plant cytoplasm and subsequently in the vesicle, which would inactivate nitrogenase or its electron donors. The decrease in CO2 evolution during the decline in Ar:O2 (Figs. 4B and 5B), which could be due to a decrease in NH4+ assimilation-linked respiration, and the correlation between the rate of the Ar-induced decline and the degree of inhibition of NH4+ production (Fig. 6) both suggest that plant metabolism is affected by Ar:O2. A decline and recovery in nitrogenase activity in response to
Ar:O2 has also been demonstrated for detached
nodules of the Frankia-M. gale symbiosis
(Tjepkema and Schwintzer, 1992
I thank Dr. L.J. Winship for stimulating discussions and technical advice, Dr. K. Huss-Danell for valuable comments on an earlier version of the manuscript, anonymous reviewers for constructive criticism, Dr. A. Sellstedt for generous lending of gas exchange equipment, Annika Höglund for assistance with plant cultivation, and the Department of Plant Physiology, Umeå University, Sweden, for providing general facilities.
Received May 6, 1999; accepted October 20, 1999. 1 This work was supported by the Swedish Natural Science Research Council (grant to K.H.-D.).
* E-mail Per-Olof.Lundquist{at}vbiol.slu.se; fax 46-18-673279.
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ABSTRACT
INTRODUCTION
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78°C in a dry ice-ethanol mix, and finally to the H2 and O2 sensors. One of
the two parallel measuring systems lacked the IRGA and the outlet
O2 sensor, but made it possible to measure H2 evolution
from two plants at the same time. Similar results were obtained in the
two systems.
) and CO2 evolution (
) rates in intact
nodulated A. incana root systems. The gas composition
was N2:O2 throughout the experiment. The
pO2 was first 21% O2 and then changed for
different plants to either 17% or 25% in steps of 1% O2
every 5 min. Values are means ± SE. Error bars are
smaller than the symbol when not displayed. Average 100% activities
were 31.4 ± 4.4, n = 10 (17% O2)
and 31.0 ± 3.2, n = 8 (25% O2)
µmol H2 plant
, EAC. In B, 
, in N2:O2; 
, in
Ar:O2.
