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Published January, 1991
Water Status Effect on Dinitrogen Fixation and Photosynthesis in Soybean
A. Djekoun and C. Planchon"
ABSTRACT
Water deficiency brings about a marked limitation in soybean
(Gfycine max. L. Merr.) yield by impairing photosynthesis and symbiotic N2 fixation. The objective of this study was to determine the
photosynthetic and N2-fixation response to short- and long-duration
water status variations in leaves and nodules. Plants were grown in
pots in a greenhouse. Carbon dioxide exchange rate was measured
by gas analysis and N2 fixation by the acetylene reduction method.
Leaf water status was determined with a pressure bomb and nodule
water potential with a psychrometer. Dinitrogen fixation decreased
steadily throughout the water deficiency period whereas photosynthesis first decreased only slightly, and then dropped dramatically.
After a severe water stress, partial recovery was slower for N2 fixation than for photosynthesis. The N2-fixation response appeared
to be directly related to a reduction in nodule mass, which affected
nodule structural constituents after a severe stress. During the water
deficiency period, the water status of the nodules responded only
partially to water status variations within the plant during the day.
Dinitrogen fixation depended more on the water status of the nodules
than on the changes in nodule mass. There was a higher dependence
of N2 fixation on nodule water content than on nodule water potential, particularly for values <-1.2 MPa. The N2-fixation activity
maintained during the severe phase of water stress, when photosynthesis was zero, resulted from the relative independence of the nodules on the daily water variations within the plant and from the
strong binding of the nodule water.
Laboratoire d'Amelioration des Plantes, Institut National Polytechnique (E.N.S.A.T.), 145 avenue de Muret, 31076 Toulouse Cedex,
France. Received 5 Feb. 1990. Corresponding author.
Published in Agron. J. 83:316-322 (1991).
HE SENSITIVITY of symbiotic N fixation to water
deficiency can be a major limiting factor of spyT
bean yield (Sinclair et al., 1987). Dinitrogen fixation
2
is more impaired than photosynthesis during soil dehydration (Kuo and Boersma, 1971) as its decrease
precedes the drop in photosynthesis (Durand et al.,
1987). However, there is an interdependence between
photosynthesis and N2 fixation in legumes (Bethlenfalvay et al., 1978a,b). Dinitrogen fixation is reduced
by short-duration changes in photosynthate availability and is also influenced by the O2 flux rates into the
nodules (Denison and Sinclair, 1985; Sinclair et al.,
1985). Nodules are more sensitive than the root system to limitations in photosynthate supply (Sprent,
1972). Soil rehydration brings about the fast and nearly total resumption of photosynthesis (Silvius et al.,
1977) whereas the recovery of N2 fixation is slower
(Sprent, 1981; Keba, 1987). A water stress can reduce
N2 fixation by a direct action on the nodules and its
effects can also be aggravated by the inability of the
stressed leaves to supply photosynthates to the nodules (Finn and Brun, 1980). Dinitrogen fixation has
been shown to decrease with leaf and nodule water
potentials and stomatal conductance (Pankhurst and
Sprent, 1975; Patterson et al., 1979; Finn and Brun,
1980).
Nitrogen metabolism as well as photosynthesis depend directly on the plant water status and the water
fluxes between the various organs. The relationships
between the water status of the nodules and that of
DJEKOUN & PLANCHON: WATER STATUS-DINITROGEN FIXATION AND PHOTOSYNTHESIS
317
the various plant parts or of the soil are difficult to
ascertain as the vascular connections between nodules
and roots are very small (Ismalli et al., 1983). Thus,
Huang et al. (1975a,b) reported nodule water potential
values that were very close to soil values, whereas
Durand et al. (1987) mentioned that water potential
values always remained higher in the nodules than in
the leaves, particularly at the predawn leaf water potential which can be considered as close to the soil
water potential. The decrease observed in nodule gas
permeability during a drought stress (Weiz et al.,
1985) seems to indicate that O2diffusion into nodules
might be an important factor determining N,-fixation
rates. However, the mechanism which controls N2fixation rates does not involve changes in the nodule
respiration rate (Weiz and Sinclair, 1987).
The aim of this study was to analyze the variations
of the nodule water status in relation to soil moisture
and to daily variations within the plant. Dinitrogen
fixation was determined in parallel to photosynthesis.
Limitations in photosynthesis availability to the nodule can be, with nodule water status, major limiting
factors of N2-fixation rates.
MATERIAL AND METHODS
The investigations were carried out in a glasshouse under
controlled conditions with day/night temperatures of 20 to
25 "C/l5 to 18 "C, 60%relative humidity, 700 pmol rn-, s-'
photosynthetic photon flux density (PPFD)' and 10 to 14 h
photoperiod. The soybean cultivar Hodgson was grown in
pots (14 cm in diam and 18 cm deep; 1 plant per pot) on a
1: 1: 1 sand-soil-peat mixture (Typic udifluvent loamy sand
soil). The plants were regularly watered to field capacity until
the flowering stage (stage R2according to Fehr and Caviness,
1977). The duration of the experiments, which included
three series of plants, was 18 d. The first series, which constituted the control plants (6 replications), was normally
watered and analyzed every 2 d (Fig. lA), and comprised a
set of 54 pots. The second series of plants was submitted to
daily measurement during the 10-d water stress period
(wateringwas discontinued). Within it, three sub-series, with
4 replications each, were used for the analyses at the beginning of the light period, and after 4 and 8 h of irradiance.
The whole set included 120 pots. The third series was used
for analyzing plant recovery ability after a water deficiency
period of variable duration followed by rewatering (Fig. 1A).
Three water deficiency levels were defined by assessing plant
condition 6, 8, and 10 d after watering had been stopped.
The plant recovery ability was determined after 4 and 8 d
for each level of water deficiency (6 replications; set of 54
pots). The experimental design was a randomized complete
block.
Whole plant C0,-exchange rate was measured in a translucent chamber with a Defor-Maihak CO, infrared gas analyzer (Defor-Maihak, Hamburg, GDR) at a temperature of
25 "C under three HPLR 400 W Philips (Eindhoven, The
Netherlands) lamps delivering an average irradiance of 600
rmol rn-, s-' PPFD at the top of the plant. A perspex lid
and Terosta (Terostat 90 10, Teroson, Asnikres, France) sealant ensured the separationbetween the aerial plant parts and
the root system. Dinitrogen fixation was measured in situ
using the acetylene reduction assay (Koch and Evans, 1966).
Acetylene was injected into the root system after the pot was
tightly sealed (the acetylene volume amounted to 10%of the
I
Abbreviation: PPFD, photosynthetic photon flux density.
0
4
-
C
z O
1
1
DAYS OF WATER TREATMENT
Fig. 1. Net photosynthesis (A), N2 fixation (acetylene reduction) (B)
and nodule dry weight (C) during the three water deficiency periods and 4 or 8 d after rewatering (the bars represent 2 SE of
the mean).
318
AGRONOMY JOURNAL, VOL. 83, MARCH-APRIL 1991
total porosity of the sand-soil-peat mixture in the pot). After
60 min of incubation (Balandreau and Dommergues, 1971),
samples were removed to determine ethylene concentration
by gas chromatography (Delsi model Di. 200; Suresnes,
France). Minchin et al. (1983, 1986) reported a dramatic
decrease in nitrogenase activity when legume nodules were
exposed to saturatingconcentrationsof acetylene. However,
in soybean, the decrease was limited, since 95%of the maximum rate was maintained after 14 min of acetylene exposure (Weiz and Sinclair, 1987).Ten individual assays showed
that, after 60 min of acetylene exposure, the acetylene reduction activity was 80% of the maximum value. The new
assay technique (Weiz and Sinclair, 1988)based on the time
required by nodules to reach steady-state ethylene production after being exposed to acetylene was not used in the
present investigations, which had been undertaken prior to
its appearance in print. Photosynthesis and N,-fixation
measurements were camed out in the morning after 4 h of
irradiance (series 1, sub-series 2, and series 3). The various
parameters characterizing the water status of the plant organs
were measured on all plants. The leaf water potential was
determined on the first and fifth trifoliate leaves using the
pressure bomb method of Scholander et al. (1965). The predawn leaf water potential was measured after a dark period
to provide an estimate ofthe soil water potential. The nodule
water potential was determined psychrometrically with a
Wescor (Logan, UT) HR-33T micro-voltmeterand a C-52
sample chamber. Ten to 15 split nodules sampled from the
whole root system were placed in the sample holder. Equilibration required 15 to 30 min depending on the severity
of the water stress. The unit had previously been calibrated
with NaCl solutions. The water content of the nodules
(Sprent, 1976), expressed as a percentage with respect to
nodules of control plants, and soil moisture were obtained
from weights before and after oven-drying at 70 "C for 48
h. The number of nodules and the nodule dry weight were
determined after unpotting for each level of water deficiency.
The data were analyzed using variance analysis procedures
and the means were compared using the LSD at P < 0.05.
. o
2
4
6
8
DAYS OF WATER TREATMENT
10
Fig. 2. Plant N,-fixation rates (as measured by the acetylene reduction activity) in percent of the well watered controls during water
deficit period (closed circles) and effect due to the decrease in
nodule mass (open circles) (calculated from Fig. lC), during the
water stress (the bars represent -t SE of the mean).
and B). Thus, on Day 6 of the water stress period,
photosynthesis and N2 fixation had decreased by 20
and SO%, respectively, with respect to the well watered
control plants. On Day 10, the CO, exchange rate was
zero while there was still significant N2-fixation activity. The nodule mass was also rapidly affected by water
deficiency: The 65% decrease observed after 10 d of
water deficiency may partly result from the impaired
photosynthesis (Fig. 1C).
After a water deficiency period of 8 d, photosynthesis and N, fixation had recovered almost completely 8 d after rewatering. However, N2-fixation recovery
was slower. After 10 d of water stress, recovery of
photosynthesis and N2fixation was less complete than
for the 8-d stress period and, again, recovery was slower for N2 fixation (Fig. 1A and B). In this latter case,
the reduction in N2 fixation appeared to be more related to a decrease in nodule mass (Fig. IC) than to a
drop in nodule activity. The change in nodule weight
per plant was due to a decrease in weight per nodule.
In control plants, during the corresponding period, a
slight increase in the number of nodules and in the
average nodule weight was observed. (Table 1).
The N2-fixation activity per plant, per nodule, and
per unit of nodule dry weight, 8 d after rewatering for
the different water deficiency levels (6 and 8 d), was
RESULTS AND DISCUSSION
Photosynthesis and dinitrogen fixation during water
dejciency
At the beginning of the water deficiency period, N2
fixation was more sensitive to water stress than was
photosynthesis, but as the water stress became more
severe, photosynthesis dropped dramatically (Fig. 1A
Table 1. Nitrogen fixation, number of nodules, and nodule dry weight during the water deficiency and the rewatering period.
Days of
experiment
Days of
water stress
Days with
water supply
T
nodule?
Nodule
number
dry weight*
N, fixation (moles C2H3
(-MPa)
0
4
8
12
18
6
10
14
8
12
16
10
14
18
LSD (0.05)
0
0
4
8
12
18
6
0
4
8
0
4
8
0
4
8
8
10
t r nodule : nodule water potential.
$ Nodule dry weight is reported value X
0.21
0.20
0.18
0.18
0.20
0.17
0.18
0.21
1.20
0.17
0.21
1.76
0.22
0.22
0.03
~~
0
24.0
24.8
25.3
26.0
26.0
25.3
25.7
26.1
25.1
25.7
26.1
25.6
26.2
26.0
1.9
0.220
0.226
0.230
0.232
0.240
0.170
0.200
0.224
0.160
0.195
0.221
0.140
0.182
0.196
0.08
9.2
9.1
9.1
8.9
9.2
6.7
7.8
8.6
6.1
7.6
8.5
5.4
6.9
7.5
0.2
3.14
3.16
3.18
3.31
3.37
1.59
2.57
3.30
0.83
1.59
2.97
0.43
1.30
2.28
0.04
0.130
0.127
0.127
0.127
0.126
0.060
0.110
0.127
0.033
0.062
0.110
0.016
0.050
0.087
0.003
14.3
14.0
13.8
14.3
14.0
9.9
13.2
14.7
5.2
8.1
13.9
3.1
7.1
11.5
1.5
319
Fig. 3. Relationship between relative nodule water content (A), nodule water potential (B) and gravimetric soil moisture (the bars represent
r SE of the mean).
close to that of the control plants (Table 1). This result,
which is in agreement with those of Bennett and Albrecht (1984), most likely reflects a preservation of the
cellular structures of the nodules at the nodule water
potential values reached ( - 1.2 MPa).
However, after a longer (10 d) water stress, the level
of N2-fixation activity, whether expressed per plant,
per nodule, or per unit of nodule dry weight, remained
below that of the control plants 8 d after rewatering,
in spite of the satisfactory water status of the nodules.
The decrease in nodule dry weight might then be due
to the degradation of the structural components of the
nodule. The variation of N2 fixation during the water
stress period was due to the decrease in nodule mass
and to the effects of other parameters, particularly the
nodule water status. Figure 1 can be used to calculate
the variation in N2 fixation and nodule mass, which
are expressed as a percentage of the well watered control plants during the water deficit period. The contribution of the effects related to the nodule weight
loss can then be distinguished from that due to nodule
dehydration (Fig. 2). Thus, after 10 d of water stress,
the decrease in nodule mass was 34% and N2 fixation
was decreased by 87% The effect of nodule dehydration corresponded to the difference 87 - 34 = 53%.
The decrease in N2-fixationactivity after 10 d of water
stress can thus be assigned to nodule dehydration for
the largest part and, to a lesser extent, to nodule mass
reduction.
Water status of the nodules and relationship
with soil moisture and leaf water status
During the water deficiency period, the water status
of the nodules, as defined by relative water content or
by water potential, changed with soil moisture (Fig.
3). For soil moisture values below 7%, water was more
strongly retained by the nodules, as shown by the
marked increase in nodule water potential and the
nearly stable relative humidity of the nodules (Fig. 3A
and B). Besides, the water status of the nodules also
depends on the water fluxes within the plant.
Three different sources contribute to the water supply of the nodules: (i) water absorption through direct
contact with soil, (ii) root water flux via the xylem,
and (iii) water circulation via the phloem (Pate, 1976;
Sprent, 1976). Nodules were relatively independent of
rapid daily fluctuations of plant water content as
shown by the relationships between leaf (1 and 5 )
water potentials during the light period and nodule
water potentials (Fig. 4). The relative stability of the
nodule water potential, as compared to the aerial sys-
Fig. 4. Relationships between: (A) leaf 5 water potential at the beginning of light period (closed circles), after 4 h (closed rectangle) and 8 h
(closed triangle), of light and nodule water potential during the water deficiency period. (B) leaf5 (closed squares) and leaf 1 (closed triangle)
water potentials after 4 h of light and nodule water potential during the water deficiency period (the bars represent r SE of the mean).
320
AGRONOMY JOURNAL,
VOL. 83, MARCH-APRIL
tem, during the day, is illustrated by the lower value
of the nodule water potential relative to the water potentials of Leaf 5 after the dark period (Fig. 4A) and
of Leaf 1 after 4 h of light (Fig. 4B), and the higher
value of the nodule water potential relative to the leaf
water potentials observed after a period of light (Fig.
4A). These differences were corroborated by analyzing
the changes in the nodule and leaf water potentials
during the light period (Table 2). The nodule water
potential always remained lower than the soil water
potential (estimated by the predawn leaf water potential), but the change in the nodule water potential during the day was smaller than the variation in the leaf
water potential, in particular during the severe water
stress period. Thus, the rapid daily variations of the
leaf water status, which were determined by the water
1991
flux between the plant and the atmosphere, affected
the nodule water status only to a limited extent.
The limited water exchanges between the nodules
and the plant during these daily cycles were likely the
result of the very fine connections between the nodules
and the roots (Ismaili et al., 1983). Calculations (Raven et al., 1989) suggest, however, that more water
transporting organic N from the nodules must migrate
to the xylem than enters the phloem transporting photosynthate. The shortfall in water entry via the phloem
can be made up for by parenchymatic water flux from
the root to the nodule. The decrease in the average
midday leaf water potentials parallels that of the nodule water potential during the water stress period (Durand et al., 1987). However, for field-grown plants in
sandy soils, desiccating nodules can reach water po-
Table 2. Nodule water potential and leaf water potential (Leaf 5) during the water deprivation period, at the beginning of the light period and
after 8 h of light.
Days
3
0
Parameters
Nodule water potential (-MPa)
Leaf water potential (-MPa)
Gravimetric soil moisture (%)
Nodule water content (%)t
6
8
10
light
dark
light
dark
light
dark
light
dark
light
dark
0.27
0.15
19.5
365
0.36
0.40
19.3
365
0.38
0.23
16.6
306
0.51
0.63
15.9
294
0.62
0.52
9.4
207
0.73
0.84
9.4
202
1.10
0.83
7.0
169
1.19
1.41
7.2
170
1.38
1.18
5.2
140
1.43
1.95
5.2
140
LSD 0.05
0.20
0.10
1.1
26
t Nodule water content expressed on a dry weight basis.
A
I
,
-n
-
Y= - a 4 + 0.034~
+ 0.0076X
r2=0.93**
Y=-0.39
4-
-
r%0.93**
I
c
5
3-
-s
2-
0
E
3
I-
2U
I
I
I
I
0
20 40 60 80 100 0
1.o
2D
(%I
RelativeNoduleWaterContenW) Nodule Water Potential(-Mpa)
Fig. 5. Relationship between N2fixation (as measured by acetylene reduction activity) and water content (percent of dry weight) (A), relative
water content (percent of control plants) (B) and water potential of the nodules (C).
100
0
300
Nodulewater Content
s
B
A
n
Y= 0.13 t 0.031X
rZ om**
&'
U
3
Y= 011 t 0.14 X
Y=1667-11.41X
&0.88**
rZ=0.88**
C
+ 1.96X2
0
u)
8-
u)
I
I
NoduleWer Content
I .
500
300
(%I
I
I
I
1
I
20
40
60
80
1000
RelatiiNodubWaterC&ent
I
I
11)
I
I
21)
NocluleWater Potential (-ha
Fig. 6. Relationship between N2 fixation (as measured by acetylene reduction activity)per unit of nodule dry weight and water content (percent
of dry weight) (A), relative water content (percent of control plants) (B), and water potential of the nodules (C).
DJEKOUN & PLANCHON: WATER STATUS-DINITROGEN FIXATION AND PHOTOSYNTHESIS
tentials that are lower than those observed for leaf
tissue (Bennett and Albrecht, 1984).
Nitrogen fixation and nodule water status
Nitrogen fixation rates appeared to be highly correlated with the nodule water content whether the latter was expressed as percent of the dry weight (r =
0.97) or as percent of the well-watered control (r =
0.96). The relationship with the nodule water potential
was less clear, particularly at low potential values (Fig.
5 A,B> and C). The variation of the nodule water content paralleled that of the nodule water potential when
soil moisture was above 6.5% (Fig. 3) but, below that
value, the nodule water became more and more tightly
bound and the nodule water potential increased rapidly whereas the nodule water content changed only
slightly. Thus, below nodule water potential values of
—1.2 MPa, the nodule water content decreased slowly
and the N2-fixation rate appeared to be less dependent
on the nodule water potential, whereas it remained
closely associated with the nodule water content. The
results obtained for the N2 fixation per mass unit (Fig.
6A,B, and C) show that the relationships observed
were not directly related to the decrease in nodule
mass. The data obtained reflect a higher dependence
of the N2 fixation upon the nodule water content than
upon water retention forces, particularly for nodule
water potential values below —1.2 MPa. The data obtained for nodule water potential values above —1.2
MPa are in agreement with those of Bennett and Albrecht (1984).
CONCLUSION
The data presented above have shown that, for a
10-d water stress resulting in a nodule water potential
of — 2.0 MPa, two effects can reduce N2 fixation. The
more important direct effect is related to the nodule
water status whereas the lesser effect is associated with
the decrease in nodule mass during the water stress as
a likely result of the decrease in photosynthesis. However, at such a level of nodule water status, some degradation of the structural components together with a
possible onset of nodule senescence occurred. Although the acetylene reduction activity technique appeared to be reliable for the comparison of well
watered plants, high acetylene concentrations can interact with the other parameters that are responsible
for the decrease in N2 fixation. Dinitrogen fixation was
shown to be more sensitive than photosynthesis to a
moderate drought stress. However, a severe water
stress appeared to allow the maintenance of some N2fixation activity, whereas photosynthesis disappeared
completely. The N2-fixation activity resulted from the
relative independence of the nodules from the daily
water fluxes within the plant and from its relationship
with the nodule water content. Dinitrogen fixation actually seems to be more closely correlated with the
nodule water content than with the nodule water potential, particularly for water stresses resulting in nodule water potential values below —1.2 MPa.
321
322
AGRONOMY JOURNAL, VOL. 83, MARCH-APRIL 1991
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