全 文 ：Received 25 Feb. 2004 Accepted 21 Jun. 2004
Supported by the National Natural Science Foundation of China（30270130）.
* Author for correspondence. E-mail: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (8): 921-927
Effect of Salt Stress on Ammonium Assimilation Enzymes of the Roots of
Rice (Oryza sativa) Cultivars Differing in Salinity Resistance
ZHOU Wei, SUN Qing-Jie, ZHANG Chu-Fu*, YUAN Yong-Ze, ZHANG Ji, LU Bin-Bin
(Key Laboratory of Ministry of Education for Plant Developmental Biology, College of Life Sciences,
Wuhan University, Wuhan 430072, China)
Abstract: Effects of NaCl-stress on ammonia assimilation enzymes and the related parameters were
determined in the roots of three rice (Oryza sativa L.) cultivars differing in salt tolerance. The results
showed that the activities of glutamine synthetase (GS) and NADH-dependent glutamate synthase (NADH-
GOGAT), as well as the levels of soluble protein decreased under high concentration salt. The influence
extent was in accordance with Zao-hua 2 (salt-sensitive), Jin-zhu 1 (normal cultivar) and Jin-dao 779 (salt-
resistant), which was consistent with their salt-tolerance. Nevertheless, under the stress of high salt
concentration, NADH-dependent glutamate dehydrogenase (NADH-GDH) activity of Zao-hua 2 and Jin-zhu
1 was induced significantly, but that of Jin-dao 779 did not increase remarkably. The salt stress led to the
accumulation of total soluble sugar (TSS) in the root of Jin-zhu 1 and Jin-dao 779 in different degree. The
level of TSS in Zao-hua 2 changed with different NaCl concentrations. Among the cultivars observed,
content of proline increased in different degree, but under high salt concentrations, it had a more marked
rise in the roots of salt-sensitive cultivars.
Key word: glutamine synthetase (GS); glutamate synthase (GOGAT); glutamate dehydrogenase (GDH); rice;
salt stress
Salt stress has important effects upon plant growth and
development. It causes the plant tissue to absorb water
difficultly, breeds physiological disorder, and destroys cell
structure, resulting in the decrease in yield and quality of
crops. Under the adverse stress, the production of nitro-
gen-metabolizing enzymes in plants is affected, which re-
sults in NH4+ accumulation and thereby poisons tissue cells.
Because of its critical role in absorption of water and inor-
ganic mineral, root is often the first organ experiencing salt
stress（Chen and Plant, 1999）. It is well-known that some
plant genotypes have relatively stronger tolerance to salt
stress. It is of significance to understand their salt-resis-
tant mechanisms for improving crop yield and quality.
The assimilation of inorganic nitrogen into carbon skel-
eton in the form of ammonium (NH4+) for the production of
amino acid is one of the most important biochemical pro-
cesses in plants. In higher plants, glutamine synthetase
(GS, EC 6.3.1.2) is the main enzyme of ammonia assimilation,
with energy provision by ATP. The enzyme catalyses the
combination of ammonia and glutamate into glutamine，
and transfers inorganic nitrogen into organic nitrogen. The
circle reaction consisting of GS and glutamate synthase
(GOGAT) is the main pathway of ammonium assimilation of
plants (Lam et al., 1996). Previous results show that GSra
and GSrb are two kinds of GS isozymes existing in the root
of rice, and production of GSrb is induced by external
nitrogen (Zhang et al., 1997). Immunological assay showed that
both GSra and GSrb are the cytosolic forms (Lin et al., 2000).
Glutamate dehydrogenase (GDH, EC 1.4.1.2) catalyzes
the amination of glutamate and its catabolism. Having high
Km to NH4+, the role of GDH in the NH4+ assimilation is still
unclear (Lam et al., 1996). Some researches have shown
that with increasing of NH4+ concentration in tissues, the
amount and activity of GDH enzyme increase significantly
to defense the harm of ammonia (Lin et al., 1996; Lutts et
al., 1999).
Osmotic adjustment is one of important mechanisms by
which plant adapts salt and drought stress. Sugar and pro-
line are the main substances of osmotic adjustment in plants
under salt stress. Their contents increase significantly (Delauney
and Verma, 1993; Soussi et al., 1999; Zhao et al., 2001).
To investigate the relationship among ammonium as-
similation enzymes and the levels of NH4+, total soluble
sugar (TSS) and proline, and to explore the mechanisms by
which the salt-resistant rice plant keeps higher activity of
GS, the enzyme activities and the relative parameters of rice
cultivars with different resistance to salt were measured
under the salt stresses with different NaCl concentration.
Acta Botanica Sinica 植物学报 Vol.46 No.8 2004922
1 Materials and Methods
1.1 Plant materials and culture
Rice (Oryza sativa L.) Jin-zhu 1 (normal cultivar),
Jin-dao 779 (salt-resistant) and Zao-hua 2 (salt-sensitive)
seeds were provided by Prof. LIU Jing of Rice Research
Department at Tianjing Agriculture Science and Technol-
ogy Research Institute. Seeds were first surface sterilized
with 0.1% HgCl2 and washed thoroughly with double-dis-
tilled water. The seeds were soaked in distilled water and
placed in an incubator (30 ℃) for 24 h and then were trans-
ferred to a petri dish with several layers of paper towel with
small amount of water, and the petri dish was placed inside
an incubator for another 48 h. The germinated seeds were
sown on the nylon net floating on nutrient solution (Yoshida
et al., 1976) in porcelain pots and cultivate them with pho-
toperiod 14 h (with fluorescent illumination of 100
mmol· m-2· s-1), relative humidity 60%-70%，tempera-
ture 30℃/25℃ (day/night), after 7 d, the plants were sub-
jected to salt stress by adding NaCl to nutrient solution (0,
50, 100 and 150 mmol/L, respectively), the roots of seed-
lings were harvested at 72 h after adding salt, they were
directly used into the extraction of enzymes or stored in the
low temperature refrigerator .
1.2 Extraction and activity determination of enzymes
Enzymes were extracted from the roots of rice seedlings
according to the method introduced by Zhang et al. (1997).
One gram sample was ground to a fine power in precooled
mortar with sand and pestle, and then homogenized in ex-
traction buffer (3 mL/g FW). The homogenates were centri-
fuged 19 000g for 20 min. The supernatant parts were stored
at -70 ℃ prior to assays. The crude extracts were used for
determination of activities of GS, NADH-GOGAT and GDH.
GS activity in extracts was determined according to
Zhang et al. (1997). One unit of GS activity was the enzyme
amount catalyzing the formation 1 µmol g-glutamylhy-
droxamate/min at 37 ℃.
The activity of NADH-GOGAT was determined as de-
scribed by Lin et al. (1996). The total reaction volume was
3 mL. Glutamine was added to initiate reaction as soon as
enzyme extraction was added. The change of absorption
value was detected at 340 nm in 3 min. One activity unit of
NADH-GOGAT was defined as reducing 1 mmol NADH in
reaction mixture/min at 30 ℃.
NADH-GDH activity was determined according to
Loulakakis et al. (1990). One unit of GDH activity was de-
fined as oxidation of 1 mmol NADH/min at 30 ℃.
1.3 Determination of root GS isozymes
The separation of GS isozymes in crude extract of rice
roots was made according to Davis (1964). GS isozyme ac-
tivity bands on the native gel were displayed by adding
acidic FeCl3 solution (Zhang et al., 1997).
1.4 Determination of ammonium level
NH4+ concentration was measured by high performance
liquid chromatography (HPLC) according to the method
introduced by Husted et al. (2000). In the method, the reac-
tion between NH4+ and o-phthalaldehyde (OPA) was per-
formed at 63℃ and at neutral pH with 2-mercaptoethanol
as reducing agent (FN). The flow rate of the carrier stream
was 1 mL/min and the sample volume used was 5 µL. The
derivative was detected at an excitation wavelength of 410
nm and an emission wavelength of 470 nm.
1.5 Analysis of soluble protein content
The content of soluble protein was measured accord-
ing to the method of Bradford (1976).
1.6 Determination of TSS
The content of TSS was determined according to the
method introduced by Irigoyen (1992).
1.7 Determination of proline content
The content of proline was determined according to the
method of Bates (1973) and the absorbance of the toluene
phase was measured at 520 nm.
2 Results
2.1 Changes in biomass and soluble total protein
Salt stress inhibited significantly the growth and devel-
opment of rice. Rice leaves lost water and became withered
quickly under salt stress. As shown in Fig.1A, the biomass
of three rice cultivars showed a similar tendency to decline
with the increase in salt concentration, but the decrease of
Zao-hua 2 (salt-sensitive) was the most marked.
The root soluble protein content of Zao-hua 2 and Jin-
zhu 1 changed slightly at lower concentration (50-100 mmol/L
NaCl), but decreased significantly under high salt concen-
tration (150 mmol/L NaCl). The protein content of Jin-dao
779 (salt-resistant) had minor decline even under high salt
concentration (Fig.1B).
2.2 Changes in total GS activity and its isozymes
Compared with the control (NaCl-free), after 72 h expo-
sure to the lower concentration salt stress (50-100 mmol/L
NaCl), the total GS activity of Zao-hua 2 (salt-sensitive)
decreased in some degree, Jin-dao 779 and Jin-zhu 1, which
have stronger resistance to salt, changed only slightly.
Under the stress of higher concentration salt the total GS
activities of all three cultivars declined, but to different ex-
tent (Fig.2A). The inhibition of the total GS activity of Jin-
dao 779, Jin-zhu 1 and Zao-hua 2 were 15%, 31.2% and
43.4%, respectively (Fig.2A). As shown in Fig.3, under the
ZHOU Wei et al.: Effect of Salt Stress on Ammonium Assimilation Enzymes of the Roots of Rice (Oryza sativa) Cultivars
Differing in Salinity Resistance 923
stress of 150 mmol/L NaCl, the GSrb activity decreased only
slightly in Jin-dao 779, but significantly in Jin-zhu 1, and
the GSrb activity of Zao-hua 2 was almost undetectable.
This decrease was not observed in GSra activity, indicat-
ing the total GS activity decline in the cultivars resulted
mainly from the decrease in GSrb activity.
2.3 Changes in NADH-GOGAT and NADH-GDH activi-
ties
The NADH-GOGAT activities in the roots of three rice
cultivar seedlings decreased in different degree under NaCl
stress for 72 h (Fig.2B), with the most remarkable decrease
in the salt-sensitive rice cultivar (Zao-hua 2), but no signifi-
cant change in the salt-resistant rice cultivar (Jin-dao 779).
The activity changes of three rice cultivars were accordant
to the changes of GS activities.
Contrary to GS and NADH-GOGAT, the NADH-GDH
activity was enhanced in roots of these rice cultivars. As
shown in Fig.2C, the NADH-GDH activities in Jin-zhu 1
and Zao-hua 2 increased slightly under low salt stresses
(50 and 100 mmol/L NaCl), but remarkably under high salt
stress (150 mmol/L NaCl). No significant change in NADH-
GDH was observed in Jin-dao 779.
2.4 Changes in NH4+ concentration
The NH4+ concentration of the roots of three rice culti-
vars were determined using HPLC. As shown in Fig.4, un-
der NaCl stresses, the concentration of NH4+ increased,
but in different degree among them. The more sensitive to
salt, the more significantly the NH4+ concentration in-
creased in the cultivar roots.
2.5 Changes in TSS and proline concentration
The stress of low salt concentration caused the increase
in TSS concentration of the rice roots. Under high salt (150
mmol/L NaCl), TSS concentration of Zao-hua 2 decreased a
little but was still higher than that of the control group.
Fig.1. Changes on the biomass (A) of total plant and the soluble protein content (B) in roots of three rice cultivars exposed for 72 h to
0, 50, 100 and 150 mmol/L NaCl.
Fig.2. Changes in glutamine synthetase (GS) (A), NADH-
GOGAT (B) and NADH-GDH (C) activities in the roots of
three rice cultivars exposed for 72 h to 0, 50, 100 and 150
mmol/L NaCl.
Acta Botanica Sinica 植物学报 Vol.46 No.8 2004924
TSS concentration of Jin-zhu 1 and Jin-dao 779 increased
all along with salt concentration (Fig.5A). Under salt
stresses, the accumulation of TSS in roots of the rice culti-
var seedlings is in correlation with their resistance to salt
stress.
As shown in Fig.5B, salt stress induced increase in pro-
line concentration of roots. The proline concentration of all
three rice cultivars increased with salt concentration, the
degree of increase of salt-sensitive rice was higher than
that of salt-resistant rice.
3 Discussion
Salt stress causes a series of physiological and bio-
chemical changes and affects the growth and development,
finally yield and quality of plants (Greenway and Munns,
1980; Boyer, 1982). In this study, three rice cultivar seed-
lings differing in salinity resistant had been exposed to salt
of different concentrations for three days. It was found
that their biomass and soluble protein decreased with the
increase in salt concentration, and the degree of decrease
is in correlation with the sensitivity of rice to salt. This
observation was similar to those from Lutts et al. (1999),
except that the rice cultivars he studied have different tol-
erance to salt in their midtillering. It was shown that the
stress response of the rice differing in salinity resistance to
salt of different concentrations is similar during the period
of seedling and midtillering. Bourgeais-chaillou et al. (1992)
also observed that salt stress caused the biomass decline
of soybean.
Salt stress affects the activity of GS and other ammonic
assimilation enzymes in plants, and this effect is different
due to the diversity among plants, tissues or the sources of
nitrogen. In our experiment, under stresses of different salt
concentrations, the root GS and NADH-GOGAT activity of
Jin-zhu 1 (normal cultivar) and Zao-hua 2 (salt-sensitive)
decreased, but those of Jin-dao 779 (salt-resistant) retained
at a high level even under high salt concentration (Figs.2A,
B). GS isozymes of rice roots have different responses to
salt stress (Fig.3). Results from native-PAGE and activity
staining indicated that the activities of two GS isozymes in
roots of Jin-dao 779 were not altered significantly, and were
consistent with change of total GS activity. Under high salt
concentration, the GSrb activities of Jin-zhu 1 and Zao-hua
2 decreased significantly, with the GSrb activity of Zao-
hua 2 almost undetectable. This suggests that the decline
of total GS activity in roots of under salt stress was mostly
due to the decrease of GSrb activity. The salt stress experi-
ment of Tai-chung local 1 showed that decline of the root
GS total activity was also due to the GSrb decrease (Li
Fig.3. Native-PAGE and activity staining of glutamine syn-
thetase (GS) isozymes in roots of three rice cultivars exposed to
0, 50, 100 and 150 mmol/L NaCl for 72 h. A, Jin-zhu 1 (normal
cultivar); B, Jin-dao 779 (salt-resistant); C, Zao-hua 2 (salt-
sensitive).
Fig.4. NH4+ concentration in the roots of three rice cultivars
exposed to 0, 50, 100 and 150 mmol/L NaCl for 72 h.
Fig.5. TSS (A) and Proline (B) concentration in the roots of
three rice cultivars exposed for 72 h to 0, 50, 100 and 150
mmol/L NaCl.
ZHOU Wei et al.: Effect of Salt Stress on Ammonium Assimilation Enzymes of the Roots of Rice (Oryza sativa) Cultivars
Differing in Salinity Resistance 925
et al., 1999). The results suggest that GSrb induced by
external nitrogen is more sensitive to the change of the
external condition.
Different from the activity change of GS and NADH-
GOGAT enzymes, the NADH-GDH activity of Jin-zhu 1 and
Zao-hua 2 were enhanced with the increase of salt
concentration，but the activity of NADH-GDH in the root
of Jin-dao 779 was not altered significantly even if it was
under the high concentration salt (Fig.2C). Lutts et al. (1999)
observed that, under salt stress, the NADH-GDH activity
of the rice cultivars differing in salinity resistance all
increased. There was no evident difference between them.
This may be due to their material harvested in the vegeta-
tive growth. It has been shown that the change in NADH-
GDH activity under salt stress is dependent on the method
of treatment. If seedling with budlet was directly planted to
the media containing salt to grow, the enzyme activity would
decrease (Li et al., 1999).
It has been shown that the concentration of NH4+ in
plant tissues increased under salt stress (Bourgeais-
chaillou et al., 1992; Lutts et al., 1999). In present study,
we found that NH4+ accumulation in rice roots was in re-
sponse to the salt-resistance extent of rice cultivars. The
rice cultivars with lower salt-tolerance had higher NH4+ ac-
cumulation (Fig.4). The degree of NH4+ accumulation in the
roots of rice cultivars differing in salinity resistance re-
flected the activity change of ammonia assimilation enzymes
in tissues. NH4+ accumulation level was negatively related
with the change of GS and NADH-GOGAT activity but had
positive correlation with NADH-GDH activity. With the in-
crease of NH4+ accumulation level, the ability of NADH-
GDH assimilating ammonium would enhance in tissues
(Gulati and Jaiwal, 1996; Lutts et al., 1999; Kumar et al.,
2000). Basing on these results, we may deduce that high
GS/GOGAT activity in the roots of salt-tolerant rice cultivar
was why NH4+ concentration can be controlled in the lower
level. In salt-tolerant rice cultivars, GS/GOGAT circle is still
mainly means of ammounium assimilation under salt stress.
It was also shown that NADH-GDH activity was enhanced
with the increase of the NH4+ concentration, alleviating the
poison effect due to the NH4+ accumulation in tissues. In
the salt-sensitive rice cultivars, NADH-GDH may play a
main role in ammounium assimilation and be one of the
important mechanisms by which plant acquires resistance
to salt stress.
The experiment results from Oliveira and Coruzzi (1999)
showed that sugar increased the GS mRNA level and in-
duced the expression of GS gene. Under the salt and
drought stress, the tissues of plants lose water, and the
total soluble sugar has an osmotic adjustment function.
When the water potential outside decrease by the salt stress
and other factors，the concentration of TSS in tissue
would increase and participate in the osmotic adjustment
to prevent for the excessive loss of water in vivo (Irigoyen
et al., 1992; Soussi et al., 1999). The similar results were
observed in our experiment. Under salt stress, the TSS con-
centration in the roots of three rice cultivars increased in
different degree, and that of salt resistant rice increased
more significantly. The GS activity in root of salt-tolerant
rice cultivar kept at higher level as well. This also indicated
that the increase of TSS level would decrease the loss of
water to keep the relative balance of ions in tissue, which
makes the activity of GOGAT and GS enzymes higher.
The accumulation of proline in tissue was often one of
the research targets of negative environment. This ammonic
acid plays a role in osmotic adjustment and stabilizing cell
structure in stressed tissue (Delauney and Verma, 1993；
Hare and Cress, 1997). In our experiments, the proline con-
centration in the roots of three rice cultivars increased un-
der salt stress. The change of proline level is consistent
with that of NH4+ level and NADH-GDH activity. When
there was an excessive NH4+ accumulation, the level of pro-
line increased under the higher salt concentration. The more
tolerant to salt, the less the content of proline, and Lutt
et al. (1999) got similar results. This indicated that the con-
tent of proline might not be the indicator for the degree of
salt-tolerance but tissue damage in rice plant. From above,
it can be deduced that accumulation of excessive NH4+
induces the NADH-GDH activity, so the glutamate (the
precursor of proline) level rises, and in the end, the proline
production increases in the tissue. Moreover, this result
confirms that the increase of proline level not only adjusts
osmotic pressure, but also is a means of assimilating ex-
cess ammounium and has a role in storing and transferring
nitrogen (Ahmad and Hellebust, 1988; Singh, 1993; Hua et
al., 1997; Brugière et al., 1999).
From all above, the rice plants with higher tolerance to
salt can keep the higher activity of GS and GOGAT, which
play an important role in decreasing the level of NH4+ accu-
mulation in the tissues under salt stress.
Acknowledgements: We are grateful to Dr. TAN Xin-Xing
(Department of Biochemistry and Cell Biology, Rice
University, Houston, TX77005, USA) for his critical reviews
of this manuscript.
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