Journal of Experimental Botany, Vol. 47, No. 301, pp. 1067-1073, August 1996
Journal of
Experimental
Botany
Inhibition of nitrogenase activity and nodule oxygen
permeability by water deficit1
Rachid Serraj2 and Thomas R. Sinclair3
USDA-ARS, Agronomy Department, Agronomy Physiology Laboratory, IFAS Building No. 164, University of
Florida, PO Box 110840, Gainesville, FL 32611-0840, USA
Received 12 December 1995; Accepted 18 April 1996
Abstract
Introduction
Short-term effects of water deficit on nitrogenase
activity were investigated with hydroponically grown
soybean plants (Glycine max L. Merr. cv. Biloxi) by
adding polyethylene glycol (PEG) to the hydroponic
solution and measuring nitrogenase activity, nodule
respiration, and permeability to oxygen diffusion (PJ.
These experiments showed a rapid decrease in acetylene reduction activity {ARA) and nodule respiration. A
consequence of the decreased respiration rate was
that Po calculated by Fick's Law also decreased.
However, these results following PEG treatment were
in direct conflict with a previous report of stability in
Po determined by using an alternative technique. To
resolve this conflict, an hypothesis describing a
sequence of responses to the initial PEG treatment is
presented. An important finding of this study was that
the response to water deficit induced by PEG occurred
in two stages. The first stage of decreased nodule
activity was O2-limited and could be reversed by exposing the nodules to elevated pO2. The second stage
which developed after 24 h of exposure to PEG
resulted in substantial loss in nodule activity and this
activity could not be recovered with increased pO2.
Severe water deficit treatments disrupt nodule activity
to such a degree that 0 2 is no longer the major
limitation.
Several studies have shown that symbiotic nitrogen fixation in legumes is more sensitive to drought than other
physiological parameters such as photosynthesis (Durand
et al., 1987), transpiration (Sail and Sinclair, 1991), or
nitrate assimilation (Obaton et al., 1982). However, the
mechanisms by which water deficit inhibits nitrogen fixation by legume nodules are still poorly understood. In
field conditions, this inhibition results from complex
events affecting both nodule development and functioning
(Sprent, 1976).
Early experiments (Sprent, 1972) have shown that the
optimum soil water content for symbiotic N2 fixation in
soybean was close tofieldcapacity. Decreasing soil water
resulted in a decrease of N2 fixation rates associated with
decreased nodule permeability to O2 diffusion (Po)
(Pankhurst and Sprent, 1975). The relationship between
nitrogen fixation and the regulation of nitrogenase activity
by oxygen under drought conditions was further confirmed by several studies in both field and pot experiments
(Weisz et al., 1985; Durand et al, 1987; Guerin et al.,
1990) indicating an effect of drought stress on a barrier
to gas diffusion, located in the nodule cortex (Weisz and
Sinclair, 1987; Hunt et al., 1987). It is also well established
that inhibition of nitrogenase activity is related to a
decrease of Po under a variety of other stress conditions
including nitrate application (Vessey et al., 1988), phloem
sap deprivation (Minchin et al., 1986; Hartwig et al.,
1987), and exposure to salt (Serraj et al., 1994). Several
hypotheses of Po regulation in the nodule cortex by
environmental factors have recently been proposed, invol-
Key words: Glycine max, N2 fixation, soybean, oxygen
permeability, water deficit.
1
Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the US Department of Agriculture and
does not imply approval or the exclusion of other products that may also be suitable.
2
Permanent address: Laboratoire de Physiologic Vegetale, Departement de Biologie, Faculte des Sciences-Semlalia, BP S 15 Marrakech, Morocco.
3
To whom correspondence should be addressed. Fax: +1 352 374 5852. E-mail: aksch©gnv.ifas.ufl.edu
Abbreviations: ARA, acetylene reduction activity; PEG, polyethylene glycol; P,, permeability to oxygen diffusion.
© Oxford University Press 1996
1068
Serraj and Sinclair
ving glycoproteins (Iannetta et cil., 1995), osmoelectric
regulation (Denison and Kinraide, 1995) or osmocontractile cells in the inner nodule cortex (Serraj et al., 1995).
However, the mechanism involving Po alteration in the
drought stress inhibition of nodule activity has been
disputed in recent reports. Diaz del Castillo et al. (1994)
and Diaz del Castillo and Layzell (1995) measured nodule
response to drought-stress after several days of soil drying.
They concluded that inhibition by drought stress was due
mainly to a decrease in respiratory capacity rather than
a decrease in O2 availability inside the nodule. Purcell
and Sinclair (1995) imposed a rapid stress on soybean
plants by adding polyethylene glycol (PEG) in the solution around the roots. They found that decreases in
nitrogenase activity and respiration preceded a decline in
nodule permeability.
In view of the contradictory reports on the role of
permeability in the drought response of nitrogen fixation,
this study was undertaken to provide more comprehensive
data on the response of nodules to the rapid imposition
of water deficit treatments using polyethylene glycol. The
first objective was to measure respiration rates of nodules
in response to water-deficit treatments by in situ O2
consumption. The second objective was to examine the
induced changes in nodule respiration rates relative to
nitrogenase activity and nodule permeability.
Materials and methods
Plant material and growth conditions
Soybean plants (cv. Biloxi) were grown in a greenhouse in a
hydroponic system (Serraj et al., 1992). Pregerminated seeds
were inoculated with a commercial preparation of
Bradyrhizobium japonicum (Nitragin, Milwaukee, WI) and
transferred for growth into a 45 1 container (8 plants/container).
About 301 of minus N nutrient solution was kept in the
container so that there was a 15 cm air-space above the solution.
The nutrient solution was renewed twice a week, the pH was
maintained close to 7.0 by adding 0.2 g I" 1 CaCO3 and air was
continuously bubbled through the solution at a flow rate of 2 1
min"1. The nutrient solution was supplemented with 2mM
urea during the first 10 d, i.e. before nodule emergence. Nodules
were visible within 7-10 d of transfer to the hydroponic system,
and nodules developed above the level of the nutrient solution
on the top 15 cm of the root. Natural light was supplemented
with incandescent lamps during a 16 h photoperiod. Day/night
temperatures were 28/20 °C.
Plants were transferred from the greenhouse to the laboratory
24 h before nodule gas exchange experiments began. The
nodulated roots of the intact plant were sealed in a 0.4 1 flowthrough assay chamber with humidified airflowpassing through
the chamber at 0.5 1 min"1. The volume of nutrient solution
was maintained at 20% of the assay chamber volume, so that
the nodules were above the nutrient solution. Water-deficit
stress was induced by adding PEG (molecular weight = 6000)
to the hydroponic solution that was in the bottom of the assay
chamber (Purcell and Sinclair, 1995). The PEG-treated plants
and the controls were monitored simultaneously. All experi-
ments were conducted with 4-week-old plants and repeated at
least, three times.
Nodule gas exchange measurement
The acetylene reduction activity (ARA) was assayed in an open
flow system, using a steady-flow gas mixture of 21 kPa O2,
69 kPa N 2 and 10 kPa C2H2, generated with mass flow meters
(MKS Instruments Inc., Andover, MA). Fifteen minutes were
allowed for steady-state conditions to be achieved. Outflow
ethylene concentration was determined with a gas chromatograph equipped with a flame ionization detector. After ARA
measurements, the nodulated roots were flushed with acetylenefree gas for 15 min before respiration measurements were made.
Nodulated root respiration was measured by both O2
consumption and CO2 evolution measurements, in the absence
of C2H2. The CO2 content of the outflow was determined with
an infrared gas analyser (Beckman Instruments Inc., IL 60173).
Measurement of nodulated root O2 consumption was achieved
using a closed system in which the chamber gas was pumped
through an oxygen sensor (Figaro Inc., IL 60091). O2
consumption was computed as the rate of O2 disappearance
during 20 min periods (Serraj et al., 1994).
At the end of the experiments, nodules were removed from
the root, the root system respiration was determined, and
nodule O2 consumption and CO2 evolution were calculated as
the difference between nodulated root respiration and root
respiration. After the measurements, plants were harvested for
determination of nodule surface area (Serraj et al., 1994) and
dry weight.
In a separate experiment, ARA response to oxygen enrichment
was measured using the same system, by varying the O2 and
N2 partial pressures (Drevon et al., 1988). The mass flow
meters were used to vary the composition of the gas mixture
and maintain a 0.5 1 min"1 constant flow during the assay. To
prevent inhibition of nitrogenase activity by oxygen excess, the
increase in pO2 was imposed gradually at a rate of 0.6 kPa
min"1 rather than by a step change. A concomitant increase in
diffusion barrier resistance during pO2 alteration may prevent
the inhibition of nitrogenase activity by oxygen excess (Hunt
et al., 1989). ARA was measured at 20, 30, 40, and 50 kPa O2;
for every pO2, the outflow C2H4 concentration was determined
at least three times during the steady state. Approximately 2.5 h
were required for the complete measurement of pO2 response.
Calculation of nodule Po
Nodule permeability to O2 diffusion was calculated as described
previously (Serraj et al., 1994), using Fick's law, as the ratio
between nodule O2 consumption and external concentration of
O2 (0,), assuming that the internal O2 concentration (O,) of the
nodules was negligible compared to Oc.
Statistical analysis
Student r-tests were used to establish significant differences
(P<0.05) between treatments with SigmaStat software.
Results
After exposure of the nodulated roots to a gas stream
containing lOkPa C2H2 for ARA determination, the
concentration of C2H4 in the outflow rose rapidly during
the first 5 min and then reached a steady state (Fig. 1),
without showing any induced decline in nodule ARA over
Nodule responses to water deficit
1069
Similarly, nodule respiration (O2 uptake and CO2
evolution) showed an inhibition which paralleled ARA
inhibition (Fig. 3A) when the nodulated roots were
exposed to PEG (-0.5 MPa) for up to 30 h. Nodule
respiration showed a higher inhibition percentage than
root respiration (Table 1). After 7 h treatment with PEG
(—0.5 MPa), respiration values of the roots with nodules
removed declined to about 85% of its control values,
whereas nodule O2 consumption and CO2 evolution represented only 45% and 60% of the control, respectively.
Consequently, the nodule respiratory quotient (RQ), the
ratio between the rate of CO2 evolution and the rate of
O2 consumption, was calculated to have changed as a
result of PEG stress (Table 1). RQ was significantly
(/><0.05) higher in the PEG-stressed nodules than in the
120
5
10
15
20
25
Time (min)
Fig. 1. Development of steady-state ethylene production during acetylene reduction activity assay. The different symbols correspond to four
different soybean plants. Values are expressed as a percentage of the
maximum activity. The average value of ARA was 248.8 ±25.0 fimol
K"1 DW nodule h" 1 .
20 min. The average initial value of ARA in control plants
was 248.8±25.0 ^mol g" 1 DW nodule h" 1 .
The exposure of the intact nodulated roots to
— 1.0 MPa PEG resulted in a rapid and dramatic decrease
of ARA (Fig. 2). After 7 h of treatment, ARA of the
stressed plants represented only about 50% of its initial
value. This short-term inhibition varied with PEG concentration in the medium (data not shown) and disappeared
20 h after the PEG was removed (Fig. 2).
—r~
1
A
100
i
- a
1
1
•
O
ARA
0 2 Consumption'
•
CO2 Evolution
»
60
40
20
1
I
10
15 20
Tlme(h)
25
30
25
30
0.0
10
15
20
25
30
Time(h)
Fig. 2. Effect of PEG (-1.0 MPa) application to the root system on
acetylene reduction activity. The arrow indicates the time when root
exposure to PEG was removed by changing the nutrient solution.
Values are expressed as a percentage of the initial activities. The average
initial value of ARA was 248.8±25.0 junol g~' DW nodule h~ l . Each
value is the mean ( ± S E ) of four replicates. The dark bar indicates the
night period.
5
10
15
20
Time(h)
Fig. 3. (A) Relative acetylene reduction activity, nodule O 2 consumption
and CO 2 evolution versus time following exposure of intact nodulated
roots of soybean (Glycine max L. cv. Biloxi) to PEG ( - 0 . 5 MPa).
Values are expressed as a percentage of the control (Table 1). (B)
Nodule permeability for control and PEG-stressed plants calculated
from O 2 consumption data. Each value is the mean ( ± S E ) of four
replicates. The dark bar indicates the night period.
1070
Serraj and Sinclair
Table 1. Effects of 7 h treatment with PEG (—0.5 MPa) on acetylene reduction activity (ARAJ, nodule and root respiration and
respiratory quotient
Average values of root and nodule dry weights were 0.30 g and 1.18 g, respectively. Each value is the mean±SE (n = 4).
O2 consumption"
(,imol g'1 DWh" 1 )
CO2 evolution
(/xmolg-'DWh- 1 )
RQ>
(^mol h" 1 plant" 1 )
109.2 ±8.1
72.2 ±8.5
458.6±43.8
206.7±27.7
450.7 ±55.2
270 3 ±52.7
0.98 ±0 06
1.31 ±0.11
_
-
128.5± 13.0
108.3 ±2.07
129 3±6.4
110.9±2.3
1.01+0.05
1.02 ±0.02
ARA
Nodule
Control
PEG
Root
Control
PEG
" Root respiration was determined after nodule removal; nodule respiration was calculated as the difference between nodulated root respiration
and root respiration.
* RQ was calculated as the ratio between the rate of CO2 evolution and the rate of O2 consumption.
Discussion
Nodule O2 uptake measurements were used to estimate
nodule Po and to test the hypothesis of oxygen Limitation
of nodule activity under water deficit conditions. In order
to avoid eventual artefacts due to the presence of C2H2
(Minchin et al., 1986), nodule respiration measurements
were conducted in the absence of C2H2. It was verified
that in the experimental conditions used here, there was
no C2H2-induced decline within the 15 min exposure to
600
A
500
nol plant
controls. For both treatments, the RQ of the roots was
not significantly different from 1.0 (Table 1).
Nodule Po was calculated using Fick's law, as the ratio
between the flux of O2 entering the nodules and the
external O2 concentration. Consequently, the observed
decrease in O2 consumption of PEG-stressed nodules
resulted in a parallel decrease in calculated Po with time
following the PEG treatment (Fig. 3B). After 7 h, Po
decreased to less than 50% of the controls, and after 30 h
it was less than 20% of the controls.
Linear regressions between ARA and nodulated root
respiration as measured by O2 consumption (Fig. 4A)
and by CO2 evolution (Fig. 4B) for the control plants
were 2.51 and 2.81 /xmol /xmol"1 C2H4, respectively
(Fig. 4). The slope values increased significantly (P<0.05)
after PEG application to 3.06 and 3.51 fimol ^mol" 1
C2H4 for O2 consumption and CO2 evolution,
respectively.
In a separate experiment, the responses of nodule ARA
to the increase of external oxygen pressure (pO2) were
initiated 4, 7 and 24 h after treatment with PEG
(—1.0 MPa). After 4 h and 7 h treatments, an increase in
pO2 induced a stimulation of nodule ARA which was
greater in the stressed plants than in the well-watered
ones (Fig. 5). This stimulation resulted in a complete
recovery of nodule ARA in the plants stressed during 4 h
and a partial recovery in the plants stressed during 7 h;
no stimulation by pO2 was observed 24 h after PEG
application (Fig. 5).
%
c
Q
s
a.
400 300
200
(
100
- .•J&'
§
8
O
y£
O .<
O .C<^
cP
-
#
Control -
o
PEG
I
o
. B
-~ 500 -
.
.•'o
_
3a. 400 CD
300 200 -
!
1
d
o
(
Control
100 -
o
I
0
i
I
t
1
PEG
i
i
20 40 60 80 100 120 140 160
Acetylene reduction activity (umol CjH 4 plant'1 h'1)
Fig. 4. Plot of (A) nodulated root O2 consumption and (B) nodulated
root CO2 evolution against acetylene reduction activity for control
plants (unstressed) and plants exposed to —0.5 MPa PEG. The linear
regressions were for A: y = 36.5 + 2.5\x (control), y = 47.2 + 3.06.x
(PEG) and for B: y = 41.1 +2 81.v (control), ;> = 50.5 + 3.51.v (PEG).
C2H2 of nodule ARA (Fig. 1) nor root nodule respiration
(data not shown).
The RQ value for control nodules of about 1.0 was
consistent with previous reports (Sprent and Gallacher,
1976; Serraj et al., 1994). The nodules on PEG-stressed
plants had RQ values of about 1.3 (Table 1), which
supported the previous conclusion that drought stress
Nodule responses to water deficit 1071
120
110
30
40
50
pO2 (kPa)
Fig. 5. Effect of external pO2 on acetylene reduction activity of soybean
nodules exposed to - l . O M P a PEG during 4, 7 and 24h. Values are
expressed in percent of initial activity. Each value is the mean (±SE)
of three replicates.
stimulates the fermentative pathways (Sprent and
Gallacher, 1976; Irigoyen et al, 1992) and restricts oxygen
diffusion into nodules, resulting in O2-limitation of nodule
activity (Pankhurst and Sprent, 1975; Weisz et al, 1985;
Durand et al, 1987).
The regression of respiration against ARA for wellwatered plants (Fig. 4) resulted in slope values close to
those reported previously for soybean and other legume
species (Witty et al, 1983). The increase in slope under
PEG stress is consistent with an increase observed in
dehydrating subterranean clover {Trifolium subterraneum
L.) nodules (Davey and Simpson, 1990). A similar
increase in the slope of respiration versus ARA was found
with salt stress in alfalfa (Medicago sativa) (Ikeda et al,
1992).
The results of this study showed that PEG treatment
resulted in a continual decrease in respiration and nitrogenase activity over 30 h (Fig. 3A). Consistent with the
results of Purcell and Sinclair (1995), the response to
PEG was rapid with decreases in respiration and nitro-
genase activity occurring within 4 h of treatment.
Importantly, the PEG-induced decline in the first 4-7 h
after treatment was completely or partially reversible by
increasingpO2 around the nodules (Fig. 5). These results,
therefore, indicated that a pO2 limitation within the
nodules inhibited respiration and nitrogenase activity
within the first hour following the PEG treatment.
Since Po was calculated from respiration rates, the
decrease in respiration that followed the PEG treatment
resulted in a decrease in calculated Po (Fig. 3B). This
decrease in Po contrasts with the results of Diaz del
Castillo et al (1994), Diaz del Castillo and Layzell
(1995), and Purcell and Sinclair (1995).
Comparisons of the results reported here with those of
Diaz del Castillo et al. (1994) and Diaz del Castillo and
Layzell (1995) may be complicated by the nature of the
stress treatments. In these previous reports, droughtstresses were induced by several days of dehydration of
the rooting media. As a result, the level of stresses induced
on the nodules were fairly severe. Even in the 'mild'
treatment, hydrogen evolution was decreased to 52% of
the control. Therefore, the stresses in Diaz del Castillo
et al. (1994) are probably compared appropriately only
to the stress level induced after a 30 h exposure to PEG
in the experiments.
In addition, there is uncertainty in interpreting the
results obtained with nodule oximetry used by Diaz del
Castillo and Layzell (1995). The nodule oximetry technique itself has been reported to decrease nodule permeability, resulting in a greater O2-limitation of nodule
metabolism, in the absence of any stress (Kuzma et al,
1993; Diaz del Castillo and Layzell, 1995). Therefore, it
is likely that the effect of drought stress on nodule Po
would be underestimated in such conditions. The Po
values reported by Diaz del Castillo and Layzell (1995)
even for the control nodules (0.34 and 0.66 urn s'1) were
several-fold less than those measured in the present work
i the
h
s'1)1 and those reportedd in
(approximately 1.9
s" 1 ) (Sheehy et al, 1983; Minchin
literature (1.0-3.0
et al, 1986; Weisz and Sinclair, 1987).
The results of Purcell and Sinclair (1995) showed a
decrease in respiration within 4 h after PEG treatment
consistent with that found here. Consequently, determinations of Po by the lag-phase technique and from respiration data appear to give conflicting estimates of changes
in Po. The lag-phase technique estimates a physical barrier
to nodule gas permeability based on the evolution of a
relatively inert gas (i.e. ethylene) from the interior of the
nodule. This technique is dependent on several assumptions concerning gas diffusion, and its evaluation is vulnerable to several sources of error (Weisz and Sinclair, 1989).
A possibility exists that the PEG treatment resulted in a
decrease in O 2 diffusion, but not a decrease in the physical
barrier to ethylene diffusion. However, it seems unlikely
1072 Serraj and Sinclair
that two gaseous molecules of similar size could have
substantially different diffusion characteristics.
An alternative explanation for the apparent difference
in the determination of Po from the lag-phase technique
and respiration measurements might result from an erroneous assumption in the calculation of Po from total
nodule respiration data. The calculation of Po by Fick's
Law from respiration data assumes that the total respiratory flux is a result of activity inside the diffusion barrier.
In fact, it is likely that a reasonable portion of the nodule
gas flux may originate in the cortex. A cortical region
that is 300 /^m thick in a 2 mm diameter nodule accounts
for half the volume of the nodule. The cortical cells are
metabolically active and include vascular bundles where
compounds are loaded and unloaded in association with
transport processes. Certainly, calculation of nodule Po
from total nodule oxygen flux assuming Fick's Law will
be in error depending on the level of cortical respiration
(Weisz and Sinclair, 1987; Minchin et al., 1992).
The problem of using oxygen flux to estimate Po may
be especially important in these dynamic studies of the
temporal response of nodules to PEG treatment, where
there may be a temporal separation in changes in respiratory rates of the cortex and the inner nodule. An hypothesis
that the PEG treatment has a more immediate effect on
the cortical respiration as a result of changes in the
metabolic activity of the vascular bundles, allows a possible resolution in understanding the observed changes in
nodule respiration while obtaining stability in Po using
the lag-phase technique (Purcell and Sinclair, 1995). An
immediate effect of the PEG treatment on cortical respiration would result in an early, small decrease in O2
consumption and CO2 evolution as observed. Changes in
cortical respiration can occur without necessarily being
accompanied by any changes in Po.
A likely consequence of decreased cortical respiration
would be a feedback into the interior of the nodule that
would inhibit nitrogenase activity. In fact, a decrease in
nitrogenase activity is readily observed in response to the
PEG treatment, and the decrease in ARA lagged behind
the decrease in O2 consumption in response to the PEG
treatment (Fig. 3A). The final response in the sequence
following the PEG treatment might be a decrease in the
physical barrier to gas diffusion as measured by the lagphase technique.
The hypothetical sequence of responses to PEG treatment might also provide an explanation of nodule
response to elevated pO2 in the early stages of the PEG
treatment. In the early stages of treatment, increased pO2
would help to overcome both some of the inhibitory
effects of the PEG treatment on cortical respiration, and
also the usual limitation of the physical diffusion barrier
on respiration inside the nodule as evidenced by the
response of the control nodules (Fig. 5).
Exposure to the PEG treatment for 24 h or more
resulted in very low respiration and nitrogenase activity
by the nodules. Increasing pO2 failed to result in recovery
of nodule activity (Fig. 5) indicating that serious disruptions in nodule functioning had taken place. The lack of
response to pO2 after exposing the roots to PEG for 24 h
is similar to the results of Diaz del Castillo et al. (1994)
on plants that had low activity after being subjected to
prolonged drought. Consistent with their conclusion,
oxygen limitations seem to be less important in the more
severe stages of water deficit stresses in limiting nodule
activity.
Therefore, the important finding of this study is that
water deficit induced by PEG results in a two-stage
inhibition of nodule activity. The first stage clearly
involves a rapid decrease in respiration and nitrogenase
activity. The hypothesis presented here is that the initial
decrease in nodule respiration may result from changes
in cortical respiration. Based on stability in Po after the
initial treatment with PEG reported by Purcell and
Sinclair (1995), it appears that Po changes occur after
the initial changes in respiration and nitrogenase activity.
The second stage of inhibition occurs after prolonged
stress that developed in these experiments after 24 h of
exposure of roots to the PEG treatment when nodule
activity was less than half of the initial rates. Nitrogenase
in this second stage is constrained by factors other than
Po as previously concluded by Diaz del Castillo et al.
(1994).
Acknowledgements
Partial support for this research was provided by United
Soybean Board project No. 4008. We thank Drs JJ Drevon,
FR Minchin, LC Purcell, JK Vessey, and KB Walsh for prereviewing the manuscript.
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