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Plant Physiol. (1999) 119: 289-296
Involvement of Ureides in Nitrogen Fixation
Inhibition in Soybean1
Rachid Serraj2,
Vincent Vadez,
R. Ford Denison, and
Thomas R. Sinclair*
United States Department of Agriculture-Agricultural Research
Service, Agronomy Physiology Laboratory, Institute of Food and
Agricultural Sciences, University of Florida, P.O. Box 110965, Gainesville, Florida 32611-0965 (R.S., V.V., T.R.S.); and Department of Agronomy and Range Science, University of California,
Davis, California 95616 (R.F.D.)
 |
ABSTRACT |
The
sensitivity of N2 fixation to drought stress in soybean
(Glycine max Merr.) has been shown to be associated with
high ureide accumulation in the shoots, which has led to the hypothesis that N2 fixation during drought is decreased by a feedback
mechanism. The ureide feedback hypothesis was tested directly by
measuring the effect of 10 mM ureide applied by stem
infusion or in the nutrient solution of hydroponically grown plants on
acetylene reduction activity (ARA). An almost complete inhibition of
ARA was observed within 4 to 7 d after treatment, accompanied by
an increase in ureide concentration in the shoot but not in the
nodules. The inhibition of ARA resulting from ureide treatments was
dependent on the concentration of applied ureide. Urea also inhibited
ARA but asparagine resulted in the greatest inhibition of nodule
activity. Because ureides did not accumulate in the nodule upon ureide
treatment, it was concluded that they were not directly inhibitory to
the nodules but that their influence mediated through a derivative compound, with asparagine being a potential candidate. Ureide treatment
resulted in a continual decrease in nodule permeability to
O2 simultaneous with the inhibition of nitrogenase activity during a 5-d treatment period, although it was not clear whether the
latter phenomenon was a consequence or a cause of the decrease in the
nodule permeability to O2.
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INTRODUCTION |
The physiological basis of N2 fixation
inhibition by water deficits in legume nodules is not clearly
understood. A potential physiological basis for this water-deficit
sensitivity may be that drought stress decreases the
Po (Weisz et al., 1985 ), as has been shown with
other stresses such as temperature, salinity, or nitrate (Hunt and
Layzell, 1993; Serraj et al., 1994; Denison and Harter, 1995). The role
of O2 limitation in the response of nitrogenase
activity to drought stress has been discussed extensively (Diaz del
Castillo and Layzell, 1995; Purcell and Sinclair, 1995; Serraj and
Sinclair, 1996b; Serraj et al., 1999). However, the mechanisms by which
drought affects Po have
not been elucidated. It is not clear whether drought stress has a
direct effect on Po, or whether the decrease in
Po is a consequence of a decrease in nodule
activity.
An alternative explanation for the decrease in nitrogenase activity
under drought could be a feedback mechanism involving the accumulation
of N compounds. Pate et al. (1969) proposed that lower rates of water
movement out of the nodule during drought stress may restrict export of
products of N2 fixation, and the accumulation of
these products would inhibit nitrogenase activity. Others have
suggested that N2 fixation in legumes might be
regulated by a feedback mechanism involving N metabolism and the pool
of reduced N in the plant (Silsbury et al., 1986; Parsons et al., 1993;
Hartwig et al., 1994). Oti-Boateng and Silsbury (1993) reported an
inhibition of nitrogenase activity in fava bean after plant uptake of
Asn or Gln.
Soybean (Glycine max Merr.) usually exports more
than 80% of the N compounds out of the nodules in the form of the
ureides Aln and Alac. They are transported in the xylem to the shoots, where they are catabolized (Winkler et al., 1987). High accumulation of
petiole ureides has been measured during the inhibition of N2 fixation by drought in both controlled (de
Silva et al., 1996; Serraj and Sinclair, 1996a) and field (Purcell et
al., 1998) environments. Furthermore, in a comparison of grain legume
species, Sinclair and Serraj (1995) reported that those species
exporting ureides from the nodules had N2
fixation that was drought sensitive. Those species that exported little
or no ureide had N2 fixation that was relatively
drought tolerant.
An important possibility is that the accumulation of ureides in soybean
nodules under soil-water deficits might trigger a feedback mechanism
that results in decreased N2 fixation activity (Sinclair and Serraj, 1995; Serraj et al., 1999). This paper reports a
series of experiments to investigate the hypothesis of a ureide feedback inhibition of N2 fixation in soybean.
First, ureide levels were measured in plant tissue (nodules, roots, and
shoots) upon the imposition of water deficits to confirm that ureide
levels increased in the nodules themselves, and not just in the shoot. Second, the influence of ureides on nodule activity was examined by
introducing ureides, along with other compounds, into soybean plants.
These experiments were designed to examine the time course of the
response and to determine the concentration response. Third, data were
collected to determine if Po and the response of
N2 fixation to pO2 were
also sensitive to introduced ureides.
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MATERIALS AND METHODS |
Accumulation of Ureides in Soybean Tissues under Drought
(Experiment 1)
Soybean (Glycine max Merr. cv Biloxi) plants were
inoculated with a commercial preparation of Bradyrhizobium
japonicum
(Nitragin3,
Milwaukee, WI) and grown in pots (2.5 L) filled with a loamy sand-soil
mixture in a greenhouse (Serraj and Sinclair, 1996a). One plant was
allowed to develop in each of 15 pots under well-watered conditions for
4 to 6 weeks. When the plants had produced four or five fully expanded
leaves, all pots were watered to saturation. Seven pots were maintained
as controls in well-watered conditions, whereas eight drought-treatment
pots were allowed to dehydrate during the next 12 d as a result
of plant transpiration. The mean nodule dry weight of these
plants was 0.55 g, reflecting a good level of nodulation.
Four drought-treatment plants were harvested on d 7 and 10 of the
dry-down experiment. Leaf water potential was measured at midday on the
uppermost, fully expanded leaf using a pressure chamber. Ureides were
extracted from freshly harvested nodule, root, and leaf-blade tissues.
Samples of about 0.8 g fresh weight were extracted with 1.0 mL of
0.2 M NaOH in a boiling water bath for 30 min. Ureide
concentration in the extracts was determined colorimetrically according
to the method of Young and Conway (1942) and is expressed on a fresh
weight basis.
Techniques of Ureide Application (Experiment 2)
To measure the effects of ureides on nodule activity in soybean,
we compared two different techniques of ureide application: stem
infusion and ureide addition to hydroponic solutions. For stem
infusion, the plants were grown as described above for 4 weeks, and
then prepared for infusion by inserting three 26-gauge hypodermic
needles into the stem of each plant at the three top internodes. The
needles were connected with plastic tubing (Fisher Scientific) to 10-mL
syringes filled with the infusion solution, which was continuously
injected into the stem using an infusion apparatus (Sage Instruments,
Boston, MA) at a constant flow rate of 0.5 mL
h 1. This stem-infusion procedure was similar to
the technique described previously by Grabau et al. (1986), in which
metabolites were found to be distributed throughout the soybean shoots.
Two solutions were infused: 10 mM ureides (5 mM
Alac plus 5 mM Aln) and deionized water (control). The
infusion solution was renewed every day during the 7-d infusion period
by filling the syringes with the appropriate solution. The effects of
stem infusion on N2 fixation were measured by
daily assays of ARA using the procedure described below.
For the hydroponic method, the plants were germinated, inoculated with
commercial inoculant (Nitragin), and grown in 1-L Erlenmeyer flasks in
a greenhouse on a nutrient solution containing the following concentrations of macro- and microelements: CaCl2
(3.3 mM), MgSO4 (2.05 mM), K2SO4
(1.25 mM),
KH2PO4 (0.25 mM), H3BO3 (4 µM), MnSO4 (6.6 µM),
ZnSO4 (1.55 µM),
CuSO4 (1.55 µM),
NaMoO4 (0.12 µM), and FeEDTA (40 µM). The nutrient solution was changed twice weekly, the
pH of the solution was maintained close to 7.0 by adding 0.2 g
L1 CaCO3, and air was
continuously bubbled through the solution at a flow rate of 2 L
min1 (Serraj and Sinclair, 1996b). The volume
of nutrient solution was initially sufficient to immerse the roots, and
was then adjusted with root growth down to 20% of the flask volume, so
that the nodules developed above the nutrient solution. The nodule dry weight per plant when subjected to treatment was 0.25 to 0.50 g.
The intact, nodulated root systems of 4-week-old plants were exposed to
ureide treatment by replacing the nutrient solution in the flask with a
nutrient solution containing 10 mM ureides (5 mM Alac plus 5 mM Aln). ARA was measured daily
using the procedure described below.
Effect of Different N Compounds and Ureide Concentrations
(Experiments 3, 4, and 5)
Because the stem-infusion technique was more tedious than the
hydroponic method, all of the following experiments were done with
plants grown hydroponically in a greenhouse. The intact, nodulated root
systems of 4-week-old plants were exposed to different treatments by
replacing the nutrient solution in the flasks with a new solution
containing various N compounds. In experiment 3, the effects on nodule
activity of 10 mM urea, Aln, Alac (K salt), and Asn were
compared with nodule activity in control plants and with 10 mM KCl and malate treatments. Experiment 4 examined the effect of Aln and Alac concentrations (5, 10, and 20 mM) on
nodule activity. Finally, the effects of 2.5 and 5.0 mM
Alac, and the recovery of ARA after ureide removal, were investigated
in experiment 5. In the latter experiment, plants were harvested and
oven dried for 2 d, and the shoots and nodules were individually
ground. Ureide content was determined on each plant part using the
colorimetric method of Young and Conway (1942) and is expressed on a
dry weight basis.
Nodule Oximetry Measurements (Experiments 6 and 7)
There are several possible changes in the nodule in response to
the ureide treatment. To investigate whether a potential N feedback
mechanism could be linked to some changes in Po,
plants were grown hydroponically and exposed to solutions containing ureide or Asn. Po was measured by oximetry
(Denison and Layzell, 1991). This method is based on computer-monitored
measurement of the in vivo changes in leghemoglobin oxygenation
(measured spectrophotometrically) of individual nodules after exposure
of nodules to gas mixtures containing either 20 or 100 kPa
O2.
For experiment 6, the plants were grown in Erlenmeyer flasks for 3 weeks in a greenhouse as described previously. Approximately 4 d
before beginning the oximetry measurements, the plants were transferred
to 2-L clear pouches (Ziploc, Dow Chemical, Indianapolis, IN) with 200 mL of nutrient solution and moved to the laboratory under artificial
light (600 µmol m2 s1
for 20 h). Permeability was initially measured on three or four nodules (2-4 mm external diameter) per plant, three plants per treatment, using the oximeter. After the initial permeability measurement, each nodule was tagged with a droplet of methylene blue.
The nutrient solution was then replaced with one of four treatment
solutions: control, ureide (Alac), malate, or nitrate. The added
compounds were all at a concentration of 2.5 mM. Four days
after the plants were exposed to the various treatment solutions, Po was again measured with oximetry for each
tagged nodule. After measurement, tagged nodules were individually
harvested and fresh weight was determined to estimate the diameter of
the nodule, assuming a spherical nodule and a bulk density of 1.
For experiment 7, 4-week-old plants were exposed to ureides by
replacing the nutrient solution in the Erlenmeyer flasks with nutrient
solution only, nutrient solution containing 10 mM Alac, or
nutrient solution containing 10 mM Asn. Nodule ARA was
measured daily, as described below, during a 5-d period. Permeability
was also measured daily on three or four nodules (2-3 mm external diameter) per plant, three plants per treatment. Nodules used for the
permeability measurements were harvested immediately after the oximeter
measurement for determination of nodule diameter and fresh weight.
Acetylene Reduction Assays and Response to pO2
For all experiments described above except experiment 6, ARA was
assayed on undisturbed, intact plants sealed in the Erlenmeyer flasks
(Serraj and Sinclair, 1996b). An open-flow system with a steady flow of
1 L min1 was used to subject the nodules to a
gas mixture of 21 kPa O2, 69 kPa
N2, and 10 kPa acetylene, which was generated
with mass-flow meters (MKS Instruments, Andover, MA). Ten 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. Previous tests (Serraj and Sinclair, 1996b)
showed that this assay system using greenhouse-grown plants resulted in
no acetylene inhibition. After gas samples were obtained for the ARA
measurements, the nodulated roots were continuously flushed with
acetylene-free atmospheric gas. The values of ARA were expressed on a
per plant basis, and then commonly normalized against either control
plants or initial rates.
The response of nodule ARA to O2 enrichment was
measured on the plants from experiment 7 treated or not with 10 mM Alac for 5 d. The purpose of this experiment was to
determine if ARA in Alac-treated plants could be recovered to levels
achieved by nontreated plants when subjected to elevated
pO2 to overcome the potential limitation of
nodule permeability changes. ARA response to pO2 was measured by varying the O2 and
N2 partial pressures (Serraj and Sinclair,
1996b). The mass flow meters were used to vary the composition of the
gas mixture and to maintain a 0.5 L min1
constant flow during the assay. To prevent inhibition of nitrogenase activity by a sudden O2 increase, the increase in
pO2 was imposed gradually at a rate of 0.6 kPa
min1 rather than by a step change. ARA was
measured at 10, 20, 30, 40, 55, and 70 kPa O2 for
every pO2. After reaching the desired pO2, 10 kPa acetylene was introduced for only 10 min and then the outflow ethylene concentration was measured.
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RESULTS |
Accumulation of Ureides under Drought
Drought stress resulted in a significant increase in ureide
concentration in soybean tissues. Under well-watered conditions and
high leaf water potentials, ureide concentration was very low in
nodule, root, and leaf-blade tissues (Fig.
1). During soil dehydration leaf water
potential decreased substantially, indicating a fairly severe level of
drought stress, which was associated with an accumulation of ureides in
the leaf blades and nodules. Ureide concentration in the leaf blades
and nodules exceeded 4 µmol g1 fresh weight
when the plants were severely stressed (Fig. 1). The concentration
of ureides in the roots was extremely low and remained unchanged during
the drought-stress treatment.
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| Figure 1.
Leaf-blade (A), root (B), and nodule (C) ureide
concentrations of soybean plants as a function of leaf water potential
in a soil dehydration experiment (experiment 1). Each data point is for
individual plants harvested from the control and after 7 and 10 d
of stress. FW, Fresh weight.
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Stem Infusion and Ureide Application in Hydroponic Solution
Two techniques of plant exposure to ureides were compared: stem
infusion and addition of 10 mM ureide to the hydroponic
solution. Both treatments resulted in a dramatic decrease of ARA (Fig.
2). ARA was unaffected during the first
3 d after the initiation of the stem-infusion treatment and
started to decrease almost linearly after this time (Fig. 2A). On d 7, ARA of treated plants was less than 15% of that for plants infused
with deionized water. Ureide application in the root nutrient solution,
however, resulted in a faster inhibition of ARA compared with the
stem-infusion treatment. Exposure of nodulated soybean roots to
hydroponic solution with 10 mM ureide resulted in a decline
of ARA within 48 h after treatment (Fig. 2B). After 4 d of
treatment, ARA was almost nonexistent for ureide-treated plants,
whereas ARA was still increasing in the control plants.
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| Figure 2.
Effect of stem infusion (A) and addition to
hydroponic solution of 10 mM ureides (5 mM Aln
plus 5 mM Alac) (B) on ARA (experiment 2). Values are
means ± SE of four replicates.
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Although both stem infusion and ureide addition to hydroponic solution
resulted in an inhibition of nodule ARA, the first technique was more
tedious and less reproducible. Therefore, only the hydroponic solution
method was used in all subsequent experiments.
Effects of Different N Compounds on Nodule Activity
To examine further the effects of ureides on nitrogenase activity,
we compared the effects of 10 mM of various N compounds (Alac, Aln, Asn, and urea) and other compounds (KCl and malate) on ARA
after 3 d of treatment. All N compounds showed an inhibitory effect on ARA, whereas nonnitrogenous compounds showed no significant effect on ARA (Fig. 3A). Among the N
compounds, urea prompted the smallest decrease in ARA, i.e. only
25%. By contrast, there was a 90% inhibition of ARA by Asn. Ureides
induced a 70% inhibition of ARA compared with the control, with no
difference detected between Aln and Alac.
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| Figure 3.
Effect of 10 mM of various compounds
3 d after treatment (A, experiment 3) and concentrations of Aln
and Alac 4 d after treatment (B, experiment 4) on ARA of
4-week-old cv Biloxi soybean plants grown hydroponically. Values were
normalized against daily values of ARA for control plants. Values are
means ± SE of four replicates.
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The effects of Aln and Alac on nodule ARA were compared further at
different concentrations. Both ureide compounds resulted in large
declines in ARA (Fig. 3B), with no significant difference between the
effects of Aln and Alac at any concentration. The ARA decline was less
in the 5 mM treatment (an average 56% decrease after
96 h) than in the 10 and 20 mM treatments (an
average 88% decrease after 96 h).
Recovery of Nodule ARA from Ureide Inhibition
To investigate the reversibility of ureide effects, ARA recovery
was studied after treatment with 2.5 mM (Fig.
4A) and 5 mM (Fig. 4B) Alac.
The 2.5 mM ureide treatment prompted only a 40% decrease
of ARA, which was significant only after 4 d of treatment and
stabilized to this value on the 5th and 6th d (Fig. 4A). After 6 d, one-half of the plants were changed to a Alac-free nutrient solution, whereas the other plants were changed to a new 2.5 mM Alac nutrient solution. Thereafter, ARA decreased
another 30% in the Alac-treated plants, whereas there was an ARA
recovery in the other plants, reaching 80% of the control. The 5 mM treatment induced a 60% decrease in ARA within 3 d
after treatment (Fig. 4B). Switching the plants to an Alac-free
nutrient solution resulted in an ARA recovery to only 53% of the
control.
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| Figure 4.
Effect of 2.5 mM (A) and 5.0 mM (B) Alac on ARA of 4-week-old cv Biloxi soybean plants
grown hydroponically (experiment 5). ARA recovery was measured after
Alac removal from the nutrient solution. Data were double normalized
for daily values of ARA compared with the control plants and compared
with the mean value for 0 and 3 h. Values are means ± SE of four replicates.
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Ureides were measured in shoots and nodules at the end of this
experiment. Both Alac concentration treatments resulted in ureide
accumulation in shoots; there was a 5-fold increase with 5 mM Alac and a 4-fold increase with 2.5 mM Alac
(Table I). Plants recovered from the 5.0 mM Alac treatment showed a 3-fold increase in shoot ureide
compared with the controls, whereas the 2.5 mM treatment
showed a decreased shoot ureide concentration compared with the
control. By contrast, nodules did not show any treatment difference for
ureide concentration regardless of Alac concentration (Table I).
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Table I.
Ureide concentration in shoots and nodules of
4-week-old soybean plants after treatment with 2.5 or 5 mM
Alac, or after treatment with Alac followed by recovery after ureide
removal, compared with control plants (experiment 5)
Plants correspond to data reported in Figure 5 harvested after the last
ARA measurement. Data are means ± SE of four
replicates.
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Effects of Ureide on Po
The response of Po to 2.5 mM
Alac treatment was compared with the response to nitrate and malate
treatments at the same concentration. The treatments with the N
compounds resulted in a change in Po. After
4 d of treatment both nitrate and ureide applications resulted in
a significant 30% decrease in Po, whereas
Po after treatment with malate was statistically
equivalent to the control (Table II).
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Table II.
Po after 4 d of
exposure to 2.5 mM Alac, nitrate, or malate (experiment 6)
Data are means ± SE of 8 to 12 nodules.
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Nodule ARA and Po were measured simultaneously
during a 5-d period after the addition of either 10 mM Asn
or 10 mM Alac to the nutrient solution. After 2 d
there was a significant decrease in both ARA (Fig.
5A) and Po (Fig.
5B) in response to the Asn treatment. The measured variables seemed to
be nearly stable after 3 d, with ARA at 0.3 to 0.4 of the control
and with Po at 0.5 to 0.6 of the control,
respectively. The response of ARA and Po to the
ureide treatment was somewhat different from the response to the Asn
treatment. There was not a statistically significant decrease in either
ARA or Po until d 3 in response to the ureide treatment. The severity of the decrease in the Alac treatment was also
not as great as in the Asn treatment, with ARA at about 0.6 and
Po at about 0.7 of the control.
Vmax, as calculated from the oximeter data,
also decreased in the same pattern as ARA and Po
in response to the Asn and Alac treatments (data not shown).
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| Figure 5.
Effect of 10 mM Alac and 10 mM Asn on ARA (top) and Po (bottom)
in 4-week-old cv Biloxi soybean plants grown hydroponically (experiment
7). Po was measured on three or four nodules on
each plant with the nodule oximetry method (Denison and Layzell, 1991).
Data were double normalized for daily values compared with the control
plants and compared with the mean value for  24 and 0 h. Values
are means ± SE of three plants.
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To investigate the extent of O2 limitation in
nodules in response to ureide treatment, we examined the ARA response
to changes in external pO2 at 5 d after
ureide treatment (Fig. 6). An increase in
pO2 induced a stimulation of nodule ARA in the
control plants, whereas increasing pO2 did not
result in a statistically significant ARA recovery in ARA for the
ureide-treated plants.
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| Figure 6.
Effect of external pO2 on ARA of
soybean nodules exposed or not to 10 mM Alac during 5 d in experiment 7. Each value is the mean ± SE of
three replicates.
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DISCUSSION |
Previous research demonstrated that ureides accumulated in soybean
petioles in response to developing water deficits (de Silva et al.,
1996; Serraj and Sinclair, 1996a; Purcell et al., 1998). This study of
the ureide concentrations in plants exposed to drought confirmed not
only that leaf ureide levels increased but also that there was a large
increase in nodule ureide concentration (Fig. 1). This result is
consistent with the increases in nodule ureide levels observed by
Purcell and Sinclair (1995) after exposure to PEG and by Walsh et al.
(1989) in response to the imposition of stresses other than water
deficits on soybean plants.
The observed increase in nodule ureide concentration is consistent with
the hypothesis that ureides will accumulate in the nodule as a result
of the action of any factor that decreases phloem flow to the nodules
(Walsh et al., 1989; Streeter and Salminen, 1993; Serraj et al., 1999).
This hypothesis results from the apparent dependence of xylem water
flow from the nodule on the inflow of water in the phloem. Therefore,
factors that decrease phloem flow to the nodules decrease the export
rate from the nodules, and consequently, N2
fixation products such as ureides will accumulate. Certainly, the
development of soil-water deficits is likely to be an important factor
contributing to decreased phloem flow to the nodules, which then
results in the inhibition of nitrogenase activity (Serraj et al.,
1999).
Oti-Boateng and Silsbury (1993) found an inhibition of nitrogenase
activity after nodule exposure to Asn or Gln. Neo and Layzell (1997)
increased phloem N2 levels by exposing soybean
plants to an atmosphere enriched in ammonia, which resulted in
decreased N2-fixation activity. Whereas the
concentration of most nitrogenous compounds increased, Gln had the
greatest increase. Neo and Layzell (1997) suggested that Gln was the
most likely candidate for the feedback signal compound. However,
nitrate inhibition studies with soybean implicated changes in Asn
concentration in the regulation of N2-fixation
activity (Mizukoshi et al., 1995; Bacanamwo and Harper, 1997).
The main objective of this research was to determine if the
accumulation of ureides in soybean plants with developing water deficits is involved in a feedback inhibition of
N2-fixation activity. Both stem infusion with
ureides and exposure of hydroponically grown plants to ureides resulted
in decreases in ARA (Fig. 2). The inhibitory effect on ARA was
dependent on the concentration of the ureide treatment (Fig. 3B).
However, other N compounds, including Asn and urea, also had inhibitory
effects on nodule activity (Fig. 3A).
However, the results of our experiments indicated that ureides may not
be the direct signal compounds. Whereas ureide application resulted in
large increases of ureides in the shoot, ureide levels did not increase
in the nodules (Table I). Furthermore, exposure of the plants to Asn
resulted in a greater (Fig. 3A) and faster (Fig. 5A) decrease in ARA
than was observed with Alac. Therefore, it seems more likely that a
catabolic product of ureide resulting from the high concentrations of
ureides in the shoot (Table I) might be more closely associated with
the triggering mechanism for the observed feedback response. Such a
conclusion is consistent with the suggestion of Mizukoshi et al. (1995)
and Bacanamwo and Harper (1997) that Asn might be closely linked to a
feedback inhibition of nitrogenase activity.
The possibility of a shoot-derived signal on nitrogenase activity has
also been indicated in other experiments. Research using both grafting
and split-root experiments indicated feedback regulation of
N2 fixation that may not originate in the roots
but in the shoots (Silsbury et al., 1986; Rafin and Roumet, 1994). We
found in grafting experiments with soybean (Serraj and Sinclair, 1996a) that the drought tolerance of N2 fixation was
associated with both the root and the shoot. Reciprocal grafts were
done between the drought-tolerant cv Jackson and the drought-sensitive
cv Biloxi. Those plants that had cv Jackson rootstock were as drought
tolerant as cv Jackson itself. Those plants with cv Jackson as the
scion and cv Biloxi as the rootstock also showed a high level of
drought tolerance, although it was not as great as that of cv Jackson. Therefore, the shoots of cv Jackson contained a trait, which we now
speculate may be associated with ureide catabolism, that contributed to
drought tolerance for N2 fixation. In experiment
5 the recovery from 2.5 mM Alac treatment (Fig. 4A) showed
a decrease in shoot ureide, and the poor recovery from the 5.0 mM Alac treatment (Fig. 4B) was associated with the
maintenance of a high concentration of ureide in the shoot (Table I).
A hypothesis to explain the feedback response involving ureides might
be based on the regulation of Po (Parsons et
al., 1993; Streeter, 1993; Purcell and Sinclair, 1994; Denison and
Harter, 1995). Our results showed a decrease in
Po that paralleled ARA inhibition associated
with the Asn and ureide treatments (Fig. 5), indicating that the
inhibition of nitrogenase activity could be driven by a
pO2 limitation within the nodules. It was not
possible to determine from our data whether the decrease in
Po was directly responsible for decreasing
nodule activity, or whether a decrease in nodule respiration led to an
increase in nodule pO2, which then triggered a
decrease in Po.
If decreased Po was the main consequence of the
ureide treatment, then increased pO2 would help
to overcome this inhibitory effect on nodule activity. However,
increasing pO2 5 d after imposition of the
ureide treatment failed to induce a recovery of nodule activity (Fig.
6). This indicates that mechanisms other than O2 diffusion may be involved in the long-term response to the ureide inhibition. The lack of response to pO2 after
exposure of the roots to ureides for 5 d is similar to the results
of Serraj and Sinclair (1996b) on plants that had low activity after
being subjected to prolonged osmotic stress. Consistent with their
conclusion, O2 limitations seem to be less
important in limiting nodule activity in the case of severe stages of
water-deficit stress or exposure to ureide. Recent evidence (Gordon et
al., 1997) indicates that a limitation on carbon metabolism in the
nodule may become important under prolonged drought stress.
In summary, ureides were shown to accumulate in nodules as a result of
a water-deficit treatment and to be potentially important in a feedback
inhibition of N2-fixation activity. The results of these experiments indicated that ureide accumulation may trigger the
accumulation of an intermediate compound, with Asn being a potential
candidate. The importance of ureides being involved in the high
sensitivity of ureide-transporting legumes to water deficits may result
from decreased phloem flow after water deficit and a resulting increase
in ureide concentrations in the plant. Accumulating ureides and Asn may
feed back into the nodules to inhibit nitrogenase activity.
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FOOTNOTES |
1
This research was supported in part by the
United Soybean Board Project (grant no. 8206).
2
Permanent address: Laboratoire de Physiologie
Végétale, Département de Biologie, Faculté des
Sciences-Semlalia, BP S 15 Marrakech, Morocco.
*
Corresponding author; e-mail trsincl{at}nervm.nerdc.ufl.edu; fax
1-352-392-6139.
Received July 6, 1998;
accepted October 5, 1998.
3
Mention of a trademark or proprietary product
does not constitute a guarantee or warranty of the product by the U.S.
Department of Agriculture and does not imply approval or the exclusion
of other products that may also be suitable.
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ABBREVIATIONS |
Abbreviations:
Alac, allantoic acid.
Aln, allantoin.
ARA, acetylene reduction activity.
Po, nodule
permeability to O2.
pO2, partial pressure of
O2.
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