Journal of Experimental Botany, Vol. 49, No. 323, pp. 1015–1020, June 1998
Hydrogen measurements provide direct evidence
for a variable physical barrier to gas diffusion
in legume nodules
John F. Witty1 and Frank R. Minchin
Institute for Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, Ceredigion SY23 3EB, UK
Received 9 December 1997; Accepted 9 January 1998
Abstract
A considerable weight of evidence has accumulated
to show that O diffusion into legume nodules is con2
trolled by a variable physical barrier which balances
the influx of this gas against its respiratory consumption. Recently, however, the existence of such a barrier
has been challenged on the grounds of lack of evidence for structural changes and on the possibility that
there are metabolic and/or biochemical alternatives
which might substitute for the barrier. Such speculation may be justified for the apparent diffusion resistance to O and CO where a range of potential
2
2
metabolic reactions make it difficult to separate physical and chemical processes. However, this ambiguity
does not apply to H within nodules formed by rhizob2
ium strains lacking an uptake hydrogenase (Hup−ve).
Within such nodules H generated as a by-product of
2
N fixation cannot be further metabolized. Thus the
2
steady-state relationship between internal concentration of H and its rate of efflux from the nodule can
2
only be determined by the resistance of a physical
barrier to diffusion. Data are presented here on H
2
concentrations and rates of efflux from nodules of
soyabean (Clarke/USDA16 symbiosis) subjected to
detopping and stepped increases in pO , which pro2
vides incontrovertible evidence for the existence of
such a variable physical barrier.
Key words: Root nodules, diffusion resistance, hydrogen
production, oxygen uptake, respiratory quotient.
Introduction
Excess inputs of O to infected cells of legume nodules
2
will lead to loss of nitrogenase activity, whilst insufficient
O initially curtails N fixation and eventually causes
2
2
oxidant-induced nodule senescence ( Escuredo et al.,
1996). It is now generally accepted that O supply to N 2
2
fixing bacteroids is limited by a barrier to diffusion in the
inner cortex formed by cell layers with reduced intercellular air spaces. The initial controversy over the exact
location of this barrier (see review by Witty et al., 1986)
was largely resolved by micro-electrode studies of O
2
gradients across the nodule ( Tjepkema and Yocum, 1974;
Witty et al., 1987) and by micrographic evidence for
layers which lacked intercellular spaces (Parsons and
Day, 1990). There is now an accumulation of evidence
which suggests that the resistance of this physical barrier
can be rapidly varied to balance O influx with respiratory
2
consumption. However, some workers have questioned
this conclusion.
Evidence relating to the location and function of the
nodule diffusion barrier has been extensively reviewed by
Witty et al. (1986) and by Hunt and Layzell (1993). The
major points of evidence which suggest that the resistance
of this barrier can vary relate to (a) apparent changes in
nodule permeability following stress or exposure to Ar,
acetylene or increased external pO (b) microelectrode
2,
studies which show only a transitory increase in pO
2
within the inner cortex when external O concentrations
2
are increased, (c) the observation that nitrogenase is not
damaged by large increases in external pO even though
2
these were not balanced by increases in nodule O con2
sumption, (d ) oximetry studies on the extent of oxygenation of leghaemoglobin within nodules exposed to Ar,
elevated pO and stress, (e) microscopic evidence for
2
increases in the occlusion of intercellular spaces in the
cortex under stress, and (f ) studies of O diffusion in He
2
atmospheres which suggest that gas-filled pores traversing
the cortex close under stress ( Witty and Minchin, 1994).
1 To whom correspondence should be addressed. Fax: +44 1970 828357. E-mail: [email protected]
© Oxford University Press 1998
1016
Witty and Minchin
Arguments against a physical barrier are largely based
upon a lack of evidence for structural changes within the
nodule cortex ( Van Cauwenberghe et al., 1994; Hartwig
et al., 1997), but also include speculation about changes
in carbohydrate biochemistry within infected cells
(Streeter, 1995).
Such speculations may be justified for the diffusion of
O or CO where metabolic reactions make it difficult to
2
2
separate physical and biochemical processes. However,
the only known mechanism involving the metabolism of
H which is produced as a by-product of nitrogenase
2,
activity, is an uptake hydrogenase (Hup) which occurs
within the bacteroids of some rhizobial strains. Thus,
within nodules formed by Hup−ve strains, H produced
2
from nitrogenase cannot be further metabolized. In this
circumstance the steady-state relationship between the
internal concentration of H and its rate of efflux from
2
the nodule can only be determined by the resistance of a
physical barrier to diffusion. This paper presents data on
induced changes in H concentrations and efflux from
2
soybean nodules which provide incontrovertible evidence
for such a variable physical barrier. Parts of this work
have been reviewed during a NATO Advanced Research
Workshop ( Witty and Minchin, 1997).
Materials and methods
Seeds of soyabean (Glycine max L. cv. Clarke) were sown into
12 cm pots of vermiculite and inoculated with Bradyrhizobium
japonicum strain USDA 16 at sowing and at germination. Plants
were watered with a nitrogen-free nutrient solution and grown
in Saxcil controlled-environment cabinets with a 16 h photoperiod and a day/night temperature of 25/20 °C. After 7 weeks
the root systems were sealed into the pots and measurements
were made using an open flow-though gas system (Minchin
et al., 1983).
Hydrogen production and O uptake were estimated in the
2
effluent gas stream using the detector system described by Witty
and Minchin (1998). Carbon dioxide production was estimated
by infrared gas analysis. Plants were exposed to two treatment
regimes: (1) removal of the plant top about 2 cm above the
root and (2) exposure of the nodulated roots of intact plants
to stepped increases in gas stream O concentration.
2
Following each experiment, nodules were removed, counted
and weighed so that total nodule surface area pot−1 could be
calculated. Surface area per nodule was estimated from average
wet weight per nodule on the assumptions that specific gravity
was unity and that nodules were spherical.
Separate plants were used for the determination of H
2
concentrations inside root nodules. For treatment (2) nodules
were exposed to increasing gas phase pO using the cuvette
2
system shown in Fig. 1. Vermiculite at the top of a pot was
removed and a small circular magnet covered by a disc of
wetted capillary matting (Fig. 1) was pushed under a suitably
positioned nodulated root. A short length of plastic tubing
(0.75 cm length cut from the barrel of a 5 ml plastic syringe
barrel ) was placed around the nodule and covered with Saran
wrap. This thin transparent sheet was tensioned and held down
on the magnetic base by a thick steel washer surrounding the
plastic tube. To prevent the roots from being crushed appro-
Fig. 1. Sectional view of cuvette used to hold undisturbed attached
root sections so that H concentrations within the nodule could be
2
measured at different gas phase O concentrations. The small circular
2
magnet (28 mm diameter) was pushed beneath an appropriate attached
root at the surface of the pot. A section of plastic tube was placed
around a root nodule and a small sealed chamber was formed by
covering this with Saran wrap which was held onto the magnetic base
with a thick steel washer. Wetted capillary matting beneath the root
and a humidified gas stream prevented desiccation of the nodule during
experiments. At the beginning of experiments an H -selective microelec2
trode was inserted through the Saran wrap into the nodule.
priate grooves were made in the lower edge of the plastic tube
and in the capillary matting beneath the washer. The gas seal
achieved in this process does not need to be perfect. With a gas
flow of 300 ml min−1 as used in these experiments, inward
diffusion of air through any minor leaks was prevented by gas
outflow. The inlet gas stream was humidified by passage
through water and, at the start of each experiment, a H 2
specific microelectrode ( Witty, 1991) was pushed through the
Saran wrap and inserted to a position near to the centre of
the nodule.
The cuvette system described above provides a convenient
way of holding nodules in position for electrode insertion and
preventing desiccation. For this reason the cuvette was also
used to examine the effects of detopping (treatment 1) on H
2
concentrations in the nodule, even though controlled gas phase
composition was not required in these studies.
Results
Effects of detopping
Nodule activity remained more or less constant for 20 min
following the removal of plant tops and then H evolution
2
and CO production declined progressively to about one2
half and to about one-third of their initial value, respectively ( Fig. 2). Oxygen consumption also dropped over
this period (data not shown) in proportion to the decrease
in CO production so that the RQ value (Fig. 2) remained
2
nearly constant.
Oxygen consumption and H production were linearly
2
related over the decline period with a correlation coefficient of r>0.99 ( Fig. 3). The intercept value shown
in this figure, where H production reaches zero
2
(2.78 mmol min−1), represents background O consump2
Evidence for a physical barrier to diffusion
Fig. 2. Effects of detopping on respiratory CO production, H
2
2
evolution and RQ of the Soyabean (Clarke/USDA 16 symbiosis). The
plant top was excised after 20 min as indicated by the vertical arrow.
Values are means of three replicates. Standard errors were less than
10% of the mean.
1017
a 4 min period at 30 min intervals ( Table 1). Hydrogen
concentrations within nodules increased only slightly over
this period (item 2) although H production decreased
2
from 1.21 to 0.33 mmol min−1 (item 1). Thus nodule
diffusion resistance calculated according to Fick’s law
from these values and total nodule surface area ( Table 1,
footnote ‘a’) increased by a factor of 4.4 from 0.19 to
0.83 s m−1×10−6 (item 3).
From the data generated in this experiment an estimate
of the apparent change in the resistance of nodules to O
2
diffusion (R[O ]) can also be calculated. This has been
2
done previously on the assumption that RQ values remain
at unity (Minchin and Witty, 1990) so that O flux into
2
the nodule is equal to measured efflux of CO . Measured
2
values for RQ increased slightly following detopping from
1.05±0.01 to 1.12±0.01 (mean values for 0–20 and 110–
120 min, Fig. 2) so that the assumption that RQ=1 would
produce some error. The values for R[O ] presented in
2
Table 1 have been calculated from values for O uptake
2
by the nodules (item 5) derived from measured values
for total root uptake (item 4) less background respiration
determined from the regression intercept presented in
Fig. 3. The increase in R[O ] following detopping, from
2
0.57 to 2 s m−1×10−6 (item 6), represents a proportional
increase of 3.5 which is somewhat smaller than the
proportional increase in R[H ].
2
Effect of stepped increases in oxygen concentration
Fig. 3. Regression of O production upon H evolution obtained from
2
2
data in the detopping experiment shown if Fig. 2. The line fits to the
equation; O =7.41H +2.78, with a linear regression coefficient of 0.998.
2
2
tion of roots and nodules which was not associated with
nitrogenase activity. The mean initial value for total O
2
uptake was 11.44±0.024 mmol min−1 so that about 75%
of total root respiration in the undisturbed plants was
linked with nitrogenase activity. This value is similar to
the values obtained for lucerne and field bean from the
regression of CO upon ethylene ( Witty et al., 1983).
2
Small errors in both of these estimates may be associated
with bacterial respiration in the rhizosphere and, in the
case of the O /H regression some error could be caused
2 2
by Hup+ve bacteria in the rhizosphere. This latter error
is probably negligible in flow through systems because
H is rapidly swept away in the gas stream.
2
In order to examine changes in resistance of nodules
to H diffusion (R[ H ]) following detopping, values for
2
2
H production and H concentration were averaged over
2
2
The effect of stepped increases in gas phase pO on H
2
2
evolution and respiratory CO production by the
2
soyabean/USDA 16 symbiosis are shown in Fig. 4. The
change from atmospheric concentration to 30% O in N
2
2
resulted in a rapid decrease in both measured parameters,
but these recovered to more or less the starting value
after 60 min. A similar, but smaller, decrease and recovery
occurred when O was increased to 40%, but this effect
2
was not evident at the higher O steps. Steady-state rate
2
of CO production obtained after each O step were
2
2
similar to the initial value although a transient 50%
decrease occurred at the end of the experiment when 60%
O /N was replaced with air. In contrast, steady-state H
2 2
2
production increased progressively at O concentrations
2
above 30%, but showed a similar 90% decline when
nodules were returned to air.
It is was not possible to obtain reliable estimates of O
2
uptake during changes in gas phase O concentrations
2
with the system used here, because of minor differences
in gas mixing times in control and sample pots ( Witty
and Minchin, 1998). However, good estimates were
obtained during the steady-state interval following each
O step and these are presented in Table 2, together with
2
steady-state values for H production and concentration.
2
Hydrogen concentration within nodules (item 2) increased
with pO more rapidly that the rate of H production
2
2
1018
Witty and Minchin
Table 1. Effects on nodule function and derived parameters of detopping soyabean (Clarke/USDA 16 symbiosis)
Measurements were made in an open flow-through gas system as described in the text. Except for H concentrations within the nodules the values
2
shown are the means of three replicate plants ±SE. The values for H concentrations are means of microelectrode measurements on eight nodules
2
taken from each of three plants ±SE.
Parameter
(1)
(2)
(3)
(4)
(5)
(6)
(7)
H evolution ( mmol min−1)
2
H concentration in nodule (mol m−3)
2
R[H ]a (s m−1×10−6)
2
O uptake by nodulated root ( mmol min−1)
2
O uptake by nodulesb ( mmol min−1)
2
R[O ]c (s m−1×10−6)
2
R[O ]/R[ H ]
2
2
Before detopping
1.21±0.03
0.41±0.023
0.19±0.005
11.38±0.12
8.60±0.11
0.57±0.007
3.00±0.08
Time after detopping (min)
10
40
70
100
1.14±0.10
0.41±0.033
0.20±0.005
11.26±0.14
8.47±0.14
0.58±0.009
2.90±0.06
0.74±0.12
0.45±0.024
0.34±0.066
8.08±0.31
5.29±0.31
0.94±0.054
2.73±0.08
0.46±0.05
0.47±0.037
0.59±0.142
6.25±0.25
3.46±0.25
1.45±0.099
2.46±0.09
0.33±0.04
0.47±0.035
0.83±0.215
5.32±0.21
2.54±0.21
1.99±0.146
2.43± 0.11
aResistance to H diffusion out of the nodule. Values calculated according to Fick’s law assuming that the H concentration outside nodules in
2
2
the flowing gas stream is zero, so that the concentration gradient driving outward diffusion is equal to the internal concentration of this gas. Thus
R[H ]=nodule surface area×(internal H concentration/H flux s−1). Measured nodule surface area=9.34×10−3 m2 pot−1.
2
2
2
bCalculated as total root consumption minus O uptake not coupled to nitrogenase activity, determined from the regression intercept of O
2
2
uptake upon H production ( Fig. 2).
2
cResistance to O diffusion into the nodule. Values calculated according to Fick’s Law on the assumption that all O entering the nodule is
2
2
metabolized and internal concentrations are very close to zero. The concentration gradient driving inward O diffusion is thus equal to the external
2
concentration (8.694 mol m−3 in ambient atmosphere at 20 °C ). Thus R[O ]=nodule surface area×(8.694/O flux into nodule s−1). Nodule surface
2
2
area as in ‘a’ above.
total respiration used in fixation in this experiment is the
same as in the detopping experiments. Based upon this
assumption R[O ] increased with pO by a factor of 2.59
2
2
from 0.52 to 1.35 s m−1×10−6. This increase is again
considerably smaller than the proportional increase in
R[ H ].
2
Discussion
Fig. 4. Effect of stepped increases in gas phase O concentration on
2
respiratory CO production and H evolution by undisturbed nodulated
2
2
roots of soyabean (Clarke/USDA 16 symbiosis). After 10 min in air O
2
concentrations were increases in a series of 10% steps as indicated by
dotted vertical lines. The system was returned to air after 260 min. The
RQ decreased progressively from 1.07±0.04 at atmospheric pO to
2
0.97±0.04 at 60% O (data not shown). Values are the means of three
2
replicates. Standard errors were less than 10% of the mean.
(item 1) so that R[ H ] increased by a factor of 4.37 from
2
0.19 to 0.83 s m−1×10−6 (item 3).
Values for R[O ] cannot be calculated directly from
2
this data set as they were for the detopping experiment
( Table 1) because a regression of O upon H cannot be
2
2
obtained (O consumption did not increase with H
2
2
production). Thus the intercept value representing background O consumption not coupled to nitrogenase activ2
ity (as in Fig. 2 for the detopping experiment) is not
available. An estimate of this parameter (item 5) can,
however, be obtained by assuming that the proportion of
Within nodules formed by rhizobial strains such as USDA
16, which lack an uptake hydrogenase system, H pro2
duced by nitrogenase cannot be further metabolized. For
nodules in a flowing gas stream containing no H the
2
external concentration of this gas is effectively zero. At
steady-state the rate of H efflux from the nodule is equal
2
to its rate of production and under this circumstance the
only factor which relates internal H concentration with
2
rate of production is the resistance of a physical barrier
to diffusion out of the nodule. If this resistance remained
constant then changes in H production would be accom2
panied by proportional changes in internal concentration.
This was not the case; when rates of H production were
2
altered by either detopping the plant or exposing the root
systems to increased pO the relationship between H
2
2
production and internal concentration changed considerably. The application of Fick’s law to these values shows
a greater than 4-fold increase in R[ H ] for both treatments
2
( Tables 1, 2, item 3) which can only be explained by
changes in a physical barrier to diffusion.
Changes in the apparent R[O ] in these experiments
2
are not in exact proportion to changes in R[ H ]; the ratio
2
R[O ]/R[ H ] decreases as resistance increases ( Tables 1,
2
2
2, item 7). These changes may be due to the use of O in
2
Evidence for a physical barrier to diffusion
1019
Table 2. Effects on nodule function and derived parameters of stepped increases in O concentration followed by a return to air, as
2
shown in Fig. 2
Measurements were made using undisturbed soyabean (Clarke/USDA 16 symbiosis) in an open flow- through system as described in text. Except
for H concentrations within nodules values are the means of three replicates ±SE. The values for H concentrations are means of microelectrode
2
2
measurements on eight nodules taken from each of three plants ±SE.
Parameter
(1)
(2)
(3)
(4)
(5)
(6)
(7)
H evolution ( mmol min−1)
2
H concentration in nodule (mol m−3)
2
R [ H ]a (s m−1×10−6)
2
O uptake by nodulated root ( mmol min−1)
2
O uptake by nodulesb ( mmol min−1)
2
R[O ]c (s m−1×10−6)
2
R[O ]/R[H ]
2
2
Gas phase O concentration (mol m−3)
2
8.69
12.5
16.64
20.80
24.96
8.69
0.80±0.083
0.41±0.023
0.19±0.009
7.74±0.188
6.09±0.188
0.52±0.024
2.73±0.052
0.73±0.059
0.57±0.019
0.26±0.004
7.81±0.226
6.16±0.226
0.74±0.037
2.84±0.196
0.81±0.031
1.22±0.143
0.50±0.005
7.37±0.24
5.72±0.24
1.09 ±0.044
2.18±0.114
1.08±0.027
1.64±0.092
0.55±0.006
7.95±0.22
6.30±0.22
1.20±0.040
2.07±0.116
1.21±0.016
2.78±0.240
0.83±0.005
8.37±0.29
6.72±0.29
1.35±0.055
1.62±0.057
0.83±0.076
0.38±0.063
0.17±0.006
7.02±0.26
5.37±0.26
0.59±0.28
3.47±0.232
aResistance to H diffusion out of the nodule. Values calculated according to Fick’s assuming that the H concentration outside nodules in the
2
2
flowing gas stream is zero and the concentration gradient driving outward diffusion is equal to the internal concentration of this gas. Thus R[ H ]=
2
nodule surface area×(internal H concentration/H flux s−1). Nodule surface area=6.05×10−3 m2.
2
2
bCalculated as total root consumption minus O uptake not coupled to nitrogenase activity. See text for details.
2
cResistance to O diffusion into the nodule. Values calculated according to Fick’s Law on the assumption that all O entering the nodule is
2
2
metabolized and the internal concentrations can be taken as zero. The concentration gradient driving inward O diffusion is then equal to the
2
external concentration indicated at the top of the table). Thus R[O ]=nodule surface area×(ambient O concentration/O flux into nodule s−1).
2
2
2
Nodule surface area as in ‘a’ above.
metabolic processes within the nodule which invalidate
the application of Fick’s law (Streeter, 1995). However,
an alternative explanation relates to differences in the
diffusion coefficients of H and O when resistance is
2
2
provided by a gas-filled or a liquid-filled barrier. A
comparison of published values for the diffusion ratios
of O and H in air and in water suggests that it would
2
2
be relatively more difficult for H to diffuse out of the
2
nodule if the barrier were liquid filled. One consequence
of this is that H concentrations would be higher within
2
nodules bounded by a liquid filled barrier than in nodules
where the barrier was formed by small air-filled pores
(Denison et al., 1992). This observation has been confirmed in comparisons of soyabean and lupin; based upon
changes in rate of O diffusion into the nodule when
2
background mixing gases were altered from Ar to He,
Witty and Minchin (1994) concluded that in unstressed
lupin nodules (internal H concentration about 2%) the
2
barrier was liquid filled whereas in unstressed soyabean
nodules (internal H concentration about 1%) about half
2
the O entering the nodule did so via air-filled pores. This
2
study also showed that, as the barrier in soyabean nodules
closed in response to stress, air-filled pores were lost and
the barrier became liquid filled. The operation of this
mechanism following detopping and O stepping would
2
offer one explanation for changes in the relative resistance
to O and H obtained in experiments described here.
2
2
Based on published values2 the ratio R[O ]/R[ H ] would
2
be about 3.6 if all gas exchange passed via open pores,
but would decrease to about 1.9 as the barrier filled with
liquid. If half of the gas exchange to the infected zone
passed through air filled pores ( Witty and Minchin, 1994)
then these values would be 2.75 and 1.9, which approximate to those obtained experimentally following detopping and O stepping ( Tables 1, 2, item 7). The possible
2
physiological and biochemical mechanisms underlying
these changes have been recently reviewed (Minchin,
1997).
The data presented here on the relationship between
H concentrations inside the nodule and production rate
2
of this gas represents incontrovertible evidence for
changes in the resistance of a physical barrier to gas
diffusion in response to detopping and increased pO .
2
This observation does not preclude the operation of other,
more subtle, mechanisms within infected cells which
modify O supply to the bacteroids (Bergersen, 1994;
2
Thumfort et al., 1994).
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2
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2 At 20 °C diffusion coefficients in air for H and O are 6.34×10−9 m2 s−1 and 1.78×10−9 m2 s−1 respectively. Diffusion coefficients in water for H
2
2
2
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2
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