Plant Physiol. (1983) 73, 1065-1066
0032-0889/83/73/1 065/02/$00.50/0
Short Communication
Effect of Water Stress on the Reduction of Nitrate and Nitrite by
Soybean Nodules'
Received for publication August 3, 1983 and in revised form September 26. 1983
CHUNG-LI CHEN AND JIH-MIN SUNG
Department ofAgronomy, National Chung Hsing University, Taiching, Taiwan, Republic of China
MATERIALS AND METHODS
ABSTRACT
The effects of water stress on nitrate reductase and nitrite reductase
Plant Materials and Growth Conditions. Soybean (GlIvcine
activities in symbiotic nodules were examined in field-grown soybean max L Merr. cv Clark) plants were grown in the field plots at
plants (Glycine max L Merr. cv Clark). The in vitro assays of enzyme National Chung Hsing University as previously described (14).
activity indicated that the nodule cytosol and bacteroids contained both After seedling emergence, half of the research plots were subnitrate reductase and nitrite reductase activities. The reduction of nitrate jected to various stress regimes (indicated by nodule water potenin bacteroids increased significantly as nodule water potential declined tial), by withholding irrigation. The control plots continued to
from -0.6 to -1.4 megapascals, and then decreased when -1.8 mega- receive normal irrigation so that nodule water potential of the
pascals water potential was reached. On the contrary, the reduction of soybean plants was maintained at -0.6 MPa. The nodules were
nitrate in nodule cytosol was inhibited as water stress progressed. In- sampled at 25 to 30 d after planting.
Preparation of Bacteroids and Plant Cytosol Fractions. Decreases in water stress intensity also caused a significant inhibition in
nitrite reductase activities of bacteroids and nodule cytosol within soybean tached nodules (I g) were crushed by a pestle and mortar at 4°C
nodules. The results show that nitrate reduction occurred both in the with 10 ml of K-phosphate (pH 7.5) containing 5 mM cysteine,
cytosol and bacteroids of water-stressed soybean nodules. The reduction 5 mM dithionite, and 2% (w/v) PVP. The resulting homogenate
of nitrate functioned at different physiological modes in these two frac- was filtered through four layers of cheese cloth and centrifuged
for 2 min at 500g to remove plant debris. The supernatant was
tions.
then centrifuged at 6000g for 10 min to pellet the bacteroids.
The bacteroids were rinsed twice in K-phosphate and resuspended in the same extraction buffer. The supernatant fraction
was used as host plant cytosol extract. The resulting bacteroids
and cytosol fractions were used for in vitro assays of NRA and
Soybean plant can utilize both soil NO3- and atmospheric N2. NiRA.
Assays of NRA and NiRA. In vivo NRA of intact nodule was
Although the majority of absorbed N03- was reduced in the assayed
according to the procedure detailed by Kleeper et al. (6)
aerial portion of bean plant, NO3- reduction was also detectable with some
modifications (15). In vivo NiRA was assayed accordhave
in symbiotic nodules (1, 2, 4, 8, 13). Various groups
ing
to the procedure as described by Srivastava et al. ( 1). NiRa
disputed the physiological role of NO3- reduction in soybean was measured as the amount of NO2- depleted in incubation
nodules. Daniel and Appleby (2), Riguad et al. (8), and Zablo- medium at a given time interval. In vitro NRA was assayed by
towicz et al. (16) indicated that the utilization of NO3- by the method of Deane-Drummond and Clarkson (3). In vitro
soybean bacteroids was dissimilatory. Nevertheless, Randall et NiRA was determined using a dithionite-methyl viologen elecal. (7), Russell et al. (9), and Ishizawa (4) suggested that the tron donor system and following the disappearance of NOreduction of NO3- in soybean nodules could also be assimilatory, from the assay media (5). The enzyme activities presented should
and, thus contributed to the nitrogen economy of the bean plant. be considered as relative and not absolute rates.
The NO2-, produced as a result of NO- reduction, is further
reduced by NiR2. Daniel and Appleby (2) were unable to detect
RESULTS AND DISCUSSION
NiRA in bacteroids of soybean nodules, even though enzyme
The influence of stress intensity, as indicated by the nodule
activity could be found in Rhizobium japonicum grown anaerobically. However, NiR existed in the cytosol fraction of soybean water potential, on in vivo nodule NRA and NiRA are shown in
Figure 1. NRA decreased considerably as nodule water potential
nodules ( 12).
It is well documented that the NRA in the leaves of higher decreased from -0.6 to -1.8 MPa. A similar pattern was noticed
plants is very sensitive to changes in the water status of the plant. with respect to the NiRA reponse to nodule water potential but
But little attention seems to have been given to the effect of water with different variation in magnitude (Fig. 1).
The in vitro assays of NRA and NiRA ofbacteroids and nodule
stress on NRA in root nodules. Aparicio-Tejo and Sanchez-Diaz
are shown in Figure 2. The NRA in bacteroids increased
(I) reported that the nodule NRA in Medicago sativa L. was cytosol
nodule water potential declined from -0.6 to -1.4 MPa and
stimulated by water stress. There are no reports on the relation- as
decreased when -1.8 MPa was reached. Nevertheless, the
ship between nodule NRA and water stress in the soybean plant. then
obtained from the nodules with -1.8 MPa
The present study reports the effect of water stress on NRA and bacteroids'wasNRA
still maintained at the equivalent level as compared
potential
NiRA using detached soybean nodules.
to that in the control (-0.6 MPa). The reduction of NO3- by NR
in the bacteroids has been described as an assimilatory reduction
'Supported by grants from National Science Council of the Republic (4, 7, 9). But, bacteroids could also utilize NO3- as electron
of China.
acceptor for nitrate respiration if 02 was limited resulting in the
-Abbreviations: NiR, nitrite reductase; NiRA, nitrite reductase activ- production of ATP for the support of N2 fixation (2, 4, 8). In a
previous paper ( 14), we reported that the O2 uptake by soybean
ity: NR, nitrate reductase, NRA, nitrate reductase activity.
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1065
Copyright © 1983 American Society of Plant Biologists. All rights reserved.
1066
Plant Physiol. Vol. 73, 1983
CHEN AND SUNG
has been attributed to the reduced enzyme synthesis or enzyme
induction. In nodule, the exact effect of water stress on nodule
100I1
NR is still unknown. Since the nodule cytosol NR has its origin
in the host plant, the possibility that water stress could prevent
enzyme synthesis as well as enzyme induction should not be
801
eliminated.
U
Several researchers (2, 12) reported that the NiR was not
I
m
a* so
in Rhizobium japonicum bacteroids. In this study, an
presented
U
active NiRA was obtained in the bacteroids of symbiotic nodules
4- 4AO
(Fig. 2). This might be due to the use of the artificial electron
e2
carrier (methyl viologen) instead of succinate. In vitro NiRa of
* Nil
bacteroids was decreased slightly (only 3% inhibition) with in20
OUR
creasing stress intensity up to -1.0 MPa and then decreased
considerably at lower nodule water potentials. At -1.8 MPa
I,
I
a
I
potential, the degree of inhibition in NiRA was approximately
0.6 1.0 1.4 1.8
70% as compared to that in the control. On the other hand, a
50% of inhibition in NiRA was observed in the cytosol of nodules
WI, -Epa
with -1.0 MPa potential. An additional inhibition of 20% would
FIG. 1. The effect of nodule water potential (An) on in vivo NRA and be expected if the stress was increased up to -1.8 MPa. The
NiRA of soybean nodules. The data were expressed as percentage of inhibition in NiRA might be related to the inadequate supplies
NRA (8.19 ± 0.03 umol g-' fresh weight nodule h-' and NiRA (26.10 ± of energy and reducing power. Although the NiRA obtained
2.9 ,mol g-' fresh weight nodule h-' at -0.6 MPa.
from cytosol was higher than the corresponding activity of NR
(Fig. 2), the relationship between NR and NiR in situ may differ,
since NO- accumulated within the nodules. The accumulation
of NO2 was decreased slightly by imposed water stress but not
150
statistically significant (data not given). The average of N02
accumulated in the nodule was 2.30 ,g/g fresh weight nodule.
In conclusion, the data presented indicate that the N03
reduction system occurred both in cytosol and bacteroid fractions
. 166
of water-stressed soybean nodules. The physiological role of
NO03 reduction might differ in the two fractions. The NiRA was
-a
£1
also detectable in bacteroids and cytosol and it was inhibited by
water stress.
-
I
0-
I'
L
aa50
20
* lactereld
LITERATURE CITED
1. APARICIO-TEJO P, M SANCHEZ-DIAZ 1982 Nodule and leaf nitrate reductase
and nitrogen fixation in Medicago saliva L. under water stress. Plant Physiol
0.6 1.0 1.4 1.8 0.6 1.0 1.4 1.
,P -"Pa
FIG. 2. The effect of nodule water potential (4n) on in vilro NRA
and NiRA of soybean bacteroids and nodule cytosol. The data were
expressed as percentage of NRA (23.35 ± 1.28 and 18.74 ± 1.02 ,umol
g-' fresh weight nodule h-' for bacteroids and nodule cytosol, respectively) and NiRA (42.00 ± 3.08 and 81.80 ± 6.79 jumol g-' fresh weight
nodule h-' for bacteroids and nodule cytosol, respectively) at -0.6 MPa.
nodules was depressed by water stress. Sprent and Gallacher (10)
indicated that the reduced 02 uptake in the nodule was attributed
to the increases in 02 diffusive barrier within the nodule. Thus,
it is possible that the reduced 02 uptake caused by water stress
might increase the 02 tensin within the nodule, and result in an
anaerobic environment which is favorable for the nitrate respiration. Give this condition, it is reasonable to postulate that the
increases in NRA in water-stressed nodule bacteroids might be
functioning in a dissimilatory role and serving as an alternative
energy generating process to support N2 fixation. A similar
conclusion was also drawn for alfalfa nodules (1).
It has been reported that cytosol of the nodule also contained
NRA (12, 13). The results in this study confirmed this finding.
The response of cytosol NRA followed a pattern similar to that
observed in the in vivo assay of the detached nodule (Fig. 2).
Stephens and Neyra (12) have concluded that the NR in the
nodule cytosol was derived from the host plant. The different
NRA and stress intensity relationship between cytosol and bacteroids favored their hypothesis.
In higher plants, the inhibitory effect of water stress on NRA
69: 479-482
2. DANIEL RM, CA APPLEBY 1972 Anaerobic nitrate, symbiotic and aerobic
growth of Rhizobium japonictim: Effects on cytochrome P4so, other haemoproteins, nitrate and nitrite reductase. Biochim Biophys Acta 275: 347-354
3. DEANE-DRUMMOND CE, DT CLARKSON 1979 Effect of shoot removal and
malate on the activity of nitrate reductase assayed in vivo in barley roots
(Hordeutm vul(gare cv Midas). Plant Physiol 64: 660-662
4. ISHIZAWA S 1980 Note on nitrate reduction in Rhizobiuim. Soil Sci Plant Nutr
26: 447-450
5. JAIN A, HS SRIVASTAVA 1981 Effect of salicylic acid on nitrate reductase and
glutamate dehydrogenase activities in maize roots. Physiol Plant 53: 285288
6. KLEEPER L, D FLESHER, RH HAGEMAN 1971 Generation of reduced nicotinamide adenine dinucleotide for nitrate reduction in green leaves. Plant
Physiol 48: 580-590
7. RANDALL DD, WJ RUSSELL, DR JOHNSON 1978 Nodule nitrate reductase as a
source of reduced nitrogen in soybean, Glvcine mar. Physiol Plant 44: 325328
8. RIGAUD J, FJ BERGERSON, GL TURNER, RM DANIEL 1973 Nitrate dependent
anaerobic acetylene-reduction and nitrogen-fixation by soybean bacteroids.
J Can Microbiol 77: 137-144
9. RUSSELL WJ, DR JOHNSON, DD RANDALL 1974 Nitrate reduction in soybean
nodules and leaves in relation to N2(C2H2)-fixation. Agron Abstr. p 76
10. SPRENT JI, A GALLACHER 1976 Anaerobiosis in soybean root nodules under
water stress. Soil Biol Biochem 8: 317-320
1 1. SRIVASTAVA RC, B BOSE, D MUKERJI, SN MATHUR, HS SRIVASTAVA 1979
Measurement of nitrite reductase in leaf tissue of Vigna mungo. A new
method. Planta 147: 196-198
12. STEPHENS BD, CA NEYRA 1983 Nitrate and nitrite reduction in relation to
nitrogenase activity in soybean nodules and Rhizobiuim japonicum bacteroids. Plant Physiol 71: 731-735
13. STREETER JG 1982 Synthesis and accumulation of nitrite in soybean nodules
supplied with nitrate. Plant Physiol 69: 1429-1434
14. SUNG JM 1982 The effect of water stress on nitrogen fixation efficiency of
field-grown soybean. J Agric Assoc China 117: 15-24
15. YANG CW, JM SUNG 1980 Relations between nitrate reductase activity and
growth in rice seedlings. J Agric Assoc China II 1: 15-23
16. ZABLOTOwICZRM, DL ESKEW, DD FOCHT 1978 Denitrification in Rhizobiwn.
Can J Microbiol 24: 757-760
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Copyright © 1983 American Society of Plant Biologists. All rights reserved.