全 文 ：Received 9 Jun. 2003 Accepted 7 Oct. 2003
* Author for correspondence. Tel: +86 (0)25 84396671; E-mail: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (4): 415-422
Nitric Oxide Modulates the Activities of Plasma Membrane H+-ATPase
and PPase in Wheat Seedling Roots and Promotes the Salt
Tolerance Against Salt Stress
RUAN Hai-Hua, SHEN Wen-Biao*, XU Lang-Lai
(College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China)
Abstract : Effects of exogenous sodium nitroprusside (SNP), a nitric oxide (NO) donor, on the salt
tolerance of wheat (Triticum aestivum L.) seedlings indicated that NO donor significantly alleviated the
growth inhibition, water loss and the decay of chlorophyll in wheat seedlings caused by 150 mmol/L NaCl
salt stress, thus led to the promotion of salt tolerance against salt stress. Combined with 1 mg/mL
hemoglobin treatment reverted the above SNP actions by restoring the growth of wheat seedlings and
chlorophyll content to the level found in untreated wheat seedlings under salt stress. The specific role of
NO in regulating the salt tolerance of wheat seedlings under salt stress was confirmed by using NaNO2 and
K3[Fe(CN)6] as control. Further investigation showed that the effect of both which might be related to the
induction of plasma membrane H+-ATPase and H+-PPase (H+-pyrophosphatase) activities by NO in the roots
of wheat seedling under salt stress. NO obviously enhanced the hydrolylic activities of H+-ATPase and H+-
PPase, but did not affect the H+ transport ability across plasma membrane in wheat seedling roots under
salt stress. Treatment with exogenous CaSO4 and EGTA also showed that Ca2+ was vital to the NO induced
activities of H+-ATPase and H+-PPase respectively in the roots of wheat seedling under salt stress.
Investigation of NO on the content of Na+ and K+ in the roots of wheat seedlings illustrated that NO did not
obviously affect the content of Na+, but significantly elevated the content of K+ as well as leading to the
increase the ratio of K+ to Na+ in the roots of wheat seedling under salinity conditions. This was also
important to believe that NO induced the adaptive abilities of wheat seedlings against NaCl salt stress.
Key words: Triticum aestivum ; nitric oxide; salt tolerance; plasma membrane H+-ATPase; H+-PPase;
K+/Na+
In addition to its function as a signal in defense against
biotic stresses, NO had also been shown to be involved in
the plant defenses against abiotic stresses, such as drought
(Mata and Lamat tina, 2001), salinity (Ruan e t al ., 2002;
Uchida et a l., 2002), UV-B radiat ion (Mackerness e t al .,
2001) and apoptosis (Pedroso et al., 2000). For example, our
previous work confirmed that NO could elevate the activi-
ties of reactive oxygen species (ROS) scavenging enzymes
and eliminate the ROS overproduced by salt stress, thus
protect the wheat seedling leaves from oxidative damages
induced by NaCl (Ruan et al., 2002).
However, plant growth responding to salt stress at least
follows a complex pattern as the results of combined ef-
fects of two main components: the low water potential
(osmotic stress) and physiological disturbances in plant
cells caused by an ionic imbalance in the cytoplasm of the
plant cells (ionic stress) (Nakamura et al., 1992). And the
plasma membrane H+-ATPase in plant roots makes a criti-
cal contribution in the physiology of plants responding
to salinity conditions. They are thought to be playing a key
role in the control of the cell cycle, turgor regulation, os-
motic balance and the active transport of Na+, K+, Ca2+ and
small organic molecules such as amino acids and sugars
across the plasma membrane (Kalampanayil and Wimmers,
2001). These ions and small molecules supply necessary
nourishments for plant growth under normal or stress
conditions. It is well known that the influx of Na+ and efflux
of K+ in plant tissues are the major reasons for ion toxicity
under salinity conditions (Serrano et al., 1999; Zhu, 2001)
due to the difficult discrimination between these two ions
by transport proteins (Blumwald et al., 2000). Contrary to
Na+ toxicity, K+ plays a central role in several physiological
processes (Wada et al., 1992), and the degree to which
plants tolerate salt stress is correlated with their capacity
to maintain a high K+ to Na+ ratio (Santa-Maria and Epstein,
2001). However, the molecular mechanism of NO as a signal
regulating the salt tolerance of wheat seedlings is still limited.
Here, the effects of NO donor on the act ivities of plasma
Acta Botanica Sinica 植物学报 Vol.46 No.4 2004416
membrane H+-ATPase, H+-PPase and Na+, K+ assimilation
in wheat seed ling roots under salt stress were carefu lly
inves tigated and tried to find at the possible pathway of
NO signaling in wheat seedlings during salt stress.
1 Materials and Methods
1.1 Materials cultivation and treatment
The p lan t material used in this research was wheat
(Tri ticum aestivum L. cv. Yangmai 158). Seeds o f wheat
were put in 0.1% HgCl2, and then was hed with dis tilled
water and germinated at 25 ℃ in the dark. Identical sprouts
were selected and cultured in Hoagland solution and grown
in growth chamber under natural ligh t unt il the second
fully expanded leaves appeared . The Hoagland solu tion
(pH 5.5) contained 5 mmol/L (CaNO3)2.4H2O, 5 mmol/L
KNO3, 2 mmol/L MgSO4.7H2O, 1 mmol/L KH2PO4, 4.5 mmol/L
(NH4)H2PO4, 0.06 mmol/L EDTA, 0.1 mmol/L FeSO4.7H2O,
46 mmol/L H3 BO4 , 8 mmol/L MnSO4.4H2O, 0.3 mmol/L
CuSO4. 5H2O and 0.7 mmol/L ZnSO4.7H2O.
SNP ( [Na2 Fe(CN)5].NO, Merck, Darmstad t, Germany)
was used as NO donor. Besides NO, NO2- and Fe(CN)63-
were the major products when SNP d isso lved in water.
Therefore, both NaNO2 and K3[Fe(CN)6] were us ed to ex-
amine the specific function of NO respectively. Hemoglobin,
a specific scavenger of NO (Takahashi and Yamasaki, 2002),
was purchased from Shanghai Boao, China. The pH of stock
solutions of EGTA (Amresco, America) were ad justed to
5.5 with NaOH. Meanwhile, 150 mmol/L NaCl with or with-
out 0.1 mmol/L SNP treatments were chosen according to
our prev ious work (Ruan et a l., 2002). All the so lutions
were prepared by adding the above chemicals or stock so-
lutions directly to Hoagland solutions. Also , all solutions
were renewed every day to main tain the iden t ical
concentrations. Wheat seedling roots were harvested 0, 2,
4, 6, 8 d after treatment and used immediately.
1.2 Measurement of fresh weight, growth rate and rela-
tive water content of whole wheat seedlings
The wheat seedlings were washed with water three times
and weighed for the fresh weigh t. The g rowth rate and
relative water content were obtained as described by Zhao
et al. (2002), Mata and Lamattina (2001), respectively.
1.3 Determination of chlorophyll and Na+, K+ content
Content of ch lorophyll was determined by using the
methods of Beligni and Lamattina (1999), that of Na+ and
K+ was according to the method of Zhao et al. (2002).
1.4 Extrusion of protons from intact roots
The wheat seedlings with the second leaf fully expanded
were divided into four groups and transferred to the treat-
ment solutions. Before experiments, the pH of all treatment
solutions were adjusted to 5.5 using HCl, then the values
of pH of treatment solutions were recorded after 2, 4, 6, 8 d
treatments. The treatment s olutions were renewed every
day. Therefore, the pH of new treatment solutions must be
adjusted to the s ame pH value of the s olution which had
been replaced. The decrease in the pH of external medium
was measured with pH meter (PHS-3C model, Shanghai Leici
Instrument).
1.5 Plasma membrane preparation
A membrane fraction enriched in p las ma membrane
vesicles was prepared as described by Yu et al. (1997).
1.6 Measurement of H+-ATPase and H+-PPase hydroly-
sis activity
H+-ATP hydrolysis assay was performed as described
(Blumwald and Poole, 1987). ATPase activity was expressed
by the amount of Pi librated and the Pi was assayed accord-
ing to the method of Ohnish i et a l. (1975). Assay o f H+-
PPase hydrolysis activity was similar to that o f ATPase
except using 0.3 mmol/L NaPPi instead of ATP-Na2.
1.7 Protein determination
Protein in the membrane vesicles was quantified by the
method of Bradford et al. (1976) with BSA as the standard.
2 Results
2.1 Exogenous NO enhances the adaptive responses of
wheat seedlings against salt stress
A pos sible effect of NO donor on growth o f wheat
Table 1 Effects of NO donor on the fresh weight, growth rate and relative water content of whole wheat seedlings under 150 mmol/L
NaCl salt stress
T reatment Fresh weight (mg) Growth rate (cm/d) Relative water content (%)
period (d) － SNP ＋ SNP － SNP ＋ SNP － SNP ＋ SNP
0 64± 5.1 64± 5.1 1.32± 0.19 1.32± 0.19 92.5± 3.27 92.5± 3.27
2 74± 6.7 82± 7.3* 1.02± 0.14 1.19± 0.21* 73.6± 4.12 82.5± 4.44*
4 78± 9.1 91± 10.2** 0.76± 0.11 0.93± 0.29** 63.4± 5.64 78.3± 4.98**
6 80± 8.6 95± 11.0** 0.54± 0.19 0.79± 0.20** 52.7± 5.31 74.1± 6.62**
8 83± 7.4 99± 7.31** 0.37± 0.18 0.62± 0.29** 46.9± 6.66 64.5± 7.34**
*, significant (P < 0.05); **, highly significant (P < 0.01). SNP, sodium nitroprusside combined treatments with 0.1 mmol/L SNP (＋SNP) or
without SNP (-SNP) were chosen in this experiments. All values represent the means of 50 individual wheat seedlings per treatment repeated
in three independent experiments.


RUAN Hai-Hua et al.: Nitric Oxide Modulates the Activities of Plasma Membrane H+-ATPase and PPase in Wheat Seedling
Roots and Promotes the Salt Tolerance Against Salt Stress 419
treatment (Fig.3C). Yet 0.1mmol/L SNP was demonstrated
to be with no effect on K+ content in wheat leaves (data not
shown). Further experiments using NaNO2 and K3[Fe(CN)6]
as cont rol (as s hown in Fig.3D) displayed that although
0.1 mmol/L NaNO2 and K3[Fe(CN)6 ] t reatment both par-
tially enhanced K+ content in wheat roots under salt stress,
1 mmol/L hemoglob in treatment obviously cut the rise of
K+ level induced by SNP, which further confirmed the spe-
cific role of NO in the regulation of K+ in wheat seedling
roots under salinity conditions.
3 Discussion
Salinity is one of the major factors limiting agricultural
production around the world. Results of our study showed
that NO donor treatments protected wheat seedlings against
salt stress, including promoting the whole p lant growth,
alleviating water loss and counteracting the degradation of
chlorophyll in wheat seedling leaves (Table 1 and Fig.1),
which were als o correlated with the ability of exogenous
NO eliminating the ROS overproduced by salinity (Ruan et
al., 2002; Uchida et al., 2002). However, the byproducts of
SNP when diss olved in water, NO2- and Fe(CN)63- both
dis played differen t levels o f growth inhibition of wheat
s eed lings under s alt s t res s . Moreover, complete
reversib ility of 1 mg/mL hemoglobin combined with NO
treatment in wheat seedlings further confirmed the specific
role of NO in achieving the ability of wheat s eedlings to
tolerate salt.
The plasma membrane H+-ATPase plays a central role in
the growth and the development of plants and is subjected
to the modulation of many environment factors, including
toxins, light, injury, mineral nutrients and o ther biotic and
abiotic constrains (Ballesteros et al., 1998). In our results,
NO sign ificantly induced the H+-ATPase and H+-PPase
hydrolytic activity in wheat seedling roots and met the needs
of energy for wheat seedlings growth under salt stress.
However, NO combined with NaCl treatment did not lead to
the decrease of pH of external medium compared with NaCl
Fig.3. Effects of NO on the content of Na+ (A), K+ (B) and Na+ to K+ ratio (C) in the roots of wheat seedling under 150 mmol/L NaCl
salt stress. D. Different treatments on the content of K+ in wheat seedling roots after 2 d treatment. Number 1-9 rep resent the same
treatments as shown in Fig.1A. Each value is the mean ± SE of three replicates. The data marked with * and ** is different with NaCl
stress alone at a level of significance of P < 0.05 and P < 0.01 respectively.
Acta Botanica Sinica 植物学报 Vol.46 No.4 2004420
salt stress, which implicated that the NO induced ATP hy-
drolytic activity did not obviously affect H+ extrusion across
plasma membrane under salt stress. Furthermore, 150 mmol/
L NaCl salt stress inhibited the H+-ATPase hydrolytic ac-
tivity (Fig.2B), but this inhibition did not counteract the H+
transport activity from wheat seedling roots during NaCl
salt stress (Fig.2A). The contradiction that the reduction in
ATPase activity was not proportional to the decrease in H+
pumping activity may be accounted for an increased cou-
pling efficiency of plasma membrane H+-ATPase under sa-
linity conditions (Ballesteros et al., 1998). Previous works
on plasma membrane H+-ATPas e reported that the d iffer-
ent and contrad ictory results were due to plant species,
tissues, intens ity and duration of the treatment and, even
methods of preparation. H+ transport in response to saline
conditions was reported to be due to an increased perme-
ability for chloride in plasma membrane (Sze, 1985). Gong et
al. (1994) also reported that except the H+ extrusion prompted
by energy of ATP hydrolysis, the formation of DH+ across
membrane also depended on the electron transport system
in plasma membrane. Moreover, NaCl stress enhanced the
activity of H+-PPase, which also might partially be contrib-
uted to the H+ extrusion under salt stress in wheat seedlings.
Further detection using EGTA and CaSO4 t reatments
showed that Ca2+ was a pivotal factor to NO induced H+-
ATPase and H+-PPase activity under salt stress (Fig.2B, C).
This was consistent with the reports that Ca2+ mediates the
activity of H+-ATPas e in plant cells under salinity condi-
tions (Reddy, 2001; Zheng et al, 2001), and also confirmed
that Ca2+ was an important component in NO signaling
pathway (Durner et al., 1998).
Detection of Na+ and K+ content in wheat seedling roots
responding to NO under salt stress confirmed that SNP did
not obvious ly affect the Na+ levels in roots, but signifi-
cantly elevated the content of K+ and decreased the Na+ to
K+ rat io in wheat seedling roo ts. Recently , Carden et al.
(2003) reported that it was K+ to Na+ ratio rather than the
absolute Na+ concentration to be critical for salt tolerance
in plants. NO was effective mainly by means of reinforcing
the up take of K+ and decreas ing the Na+ to K+ ratio in
avoiding the injury caused by NaCl salt stress. Evidence of
Fisher et a l. (1970) confirmed that the plasma membrane
H+-ATPase was st imulated by K+. Therefore, the NO in-
duced K+ increase in roots under salt s tress might be an
important reason for the enhanced H+-ATPase act ivity.
Whereas, the enhanced H+-ATPase activity also promoted
H+/K+ exchange cont ributing to K+ uptake into plant cell
(Briskin and Hanson 1992; Briskin and Gawienowski, 1996).
As the res ults o f K+ up take, the H+ was ext ruded out of
roots, which might explain the obvious acidification by NO
treatment under salt stress at 2 d in contrast with salt stress
alone (Fig.2A). Additionally, cGMP was proved to be con-
tributing to sign ificant increase in K+ level in Zea mays
(Pharmawati et al., 1999), and NO had been tested directly
leading to the burst of cGMP (Durner et al., 1998). Thus the
possibility of K+ modulation induced by NO treatment in
wheat seedling roots under salt stress via cGMP pathway
could no t be ruled out . Meanwhile, the increment of K+
level also facilitates the accumulation of proline during water
deficiency (Tang et al., 1984). Our previous resu lts (Ruan
et al., 2002) had confirmed that NO stimulated the proline
accumulation under salt stress, which might be partially
due to the fact that NO could induce the increment of K+ in
wheat seedling roots under salinity conditions.
Acknowledgements: We wish to thank Dr. ZHENG Qing-
Song and Ms CHEN Ming at Nanjing Agricultural Univer-
sity for their kindly help in using of flame photometer and
assistance in experiments.
References:
Ballesteros E, Kerkeb B, Donaire J P, Belver A. 1998. Effects of
salt stress on H+-ATPase activit y of plasma membrane-en-
riched vesicles isolated from sunflower roots. Plant Sci, 134:
181 – 190.
Beligni M V, Lamattina L. 1999. Nitric oxide counteracts cyto-
toxic processes mediated by reactive oxygen species in plant
tissues. Planta, 208:337 – 344.
Blumwald E, Aharon G S, Apse M P. 2000. Sodium transport in
plant cells. Biochim Biophys Acta, 1465:140 – 151
Blumwald E, Poole R J. 1987. Salt tolerance in suspens ion cul-
tures of sugar beet : induction of Na+/H+ antip ort activity at
the tonoplast by growth in salt. Plant Physiol, 83:884 – 887.
Bradford M M. 1976. A rapid and sens itive met hod for t he
quantitation of microgram quant ities of protein utilizing the
principle of protein-dye binding. Anal Biochem, 72:248 – 254.
Briskin D P, Gawienowski M C. 1996. Role of the plasma mem-
brane H+-ATPase in K+ transport. Plant Physiol, 111:1199 –
1207.
Briskin D P, Hanson J B. 1992. How does the plant plasma
membrane H+-ATPase pump prot ons? J Exp Bot, 43:269 –
289.
Carden D E. Walker D J, Flowers T J, Miller A J. 2003. Single-cell
measurements of the contributions of cytosolic Na+ and K+ to
salt tolerance. Plant Physiol, 131:676 – 683.
Durner J, Wendehenne D, Klessig D F. 1998. Defence gene induc-
tion in tobacco by nitric oxide, cyclic GMP, and cyclic ADP-
ribose. Proc Natl Acad Sci USA, 95:10328 – 10333.
Fisher J B, Hansen D, Hodges T K. 1970. Correlation between
RUAN Hai-Hua et al.: Nitric Oxide Modulates the Activities of Plasma Membrane H+-ATPase and PPase in Wheat Seedling
Roots and Promotes the Salt Tolerance Against Salt Stress 421
ion fluxes and ion-stimulated adenosine triphosphatase activ-
ity of plant roots. Plant Physiol, 46:812 – 814.
Gong X-Q , Jing J-H , Wang H. 1994. The investigation on rela-
tionship between maize leaf elongation and PMH+-ATPase in
the growing zone under osmotic st ress . Acta Bot Boreali-
Occidentalia Sin, 14:67 – 72. (in Chinese with English abstract)
Kalampanayil B D, Wimmers L E. 2001. Identification and char-
acterization of a salt-stress-induced plasma membrane H+ -
ATPase in tomato. Plant Cell Environ, 24:999 –1005.
Mackerness S A H, John C F, Jordan B, Thomas B. 2001. Early
signaling components in ultraviolet-B responses: distinct roles
for different reactive oxygen species and nitric oxide. FEBS
Lett, 489:237 – 242.
Mata C G, Lamattina L. 2001. Nitric oxide induces stomatal clo-
sure and enhances the adaptive plant responses against drought
stress. Plant Physiol, 126:1196 – 1204.
Nakamura Y, Kasamo K, Shimosato N, Sakata M, Ohta E. 1992.
Stimulation of the extrusion of protons and H+-ATPase ac-
tivities with the decline in p yrophosphatase activity of the
tonoplast in intact mung bean roots under high-NaCl stress
and it s relation to external levels of Ca2+ ions . Plant Cell
Physiol, 33:139 – 149.
Ohnishi T, Gall R S, M ayer M L. 1975. An improved assay of
inorganic phosphate in the presence of extralabile phosphate
compounds: application to the ATPase assay in the presence
of phosphocreatine. Anal Biochem, 69:261-267.
Pedroso M C, Magalhacs J R, Durzan D. 2000. A nitric oxide
burst precedes apop tosis in angiosperm and gy mnosp erm
callus cells and foliar tissues. J Exp Bot, 51:1027 – 1036.
Pharmawati M, Shabala S N, Newman I A, Gehring C A. 1999.
Natriuretic pept ides and cGMP modulate K+, Na+, and H+
fluxes in Zea mays roots . Mol Cell Biol Res Commun, 2:53 –
57.
Reddy A S N. 2001. Calcium: silver bullet in signaling. Plant Sci,
160:381 – 404.
Ruan H-H , Shen W-B, Ye M-B, Xu L-L . 2002. Protective effects
of nitric oxide on salt stress -induced oxidat ive damages to
wheat (Triticum aestivum) leaves. Chin Sci Bull, 47:677 – 681.
Santa-María G E, Epstein E. 2001. Potassium:sodium selectivity
in wheat and the amphiploid cross wheat X Lophopyrum
elongatum. Plant Sci, 160:523 – 534.
Serrano R, Mulet J M, Rios G, Marquez J A, de Larrinoa I F,
Leube M P, Mendizabal I, Pascual-Ahuir A, Proft M, Ros R,
Montesinos C. 1999. A glimp se of the mechanisms of ion
homeostasis during salt stress. J Exp Bot, 50:1023 – 1036.
Sze H. 1985. H+ -translocat ing ATPases: advances using mem-
brane vesicles. Annu Rev Plant Physiol, 36:175 – 208.
Takahashi S, Yamasaki H. 2002. Reversible inhibition of photo-
phosphory lation in chloroplasts by nitric oxide. FEBS Lett,
512:145 – 148.
Tang Z-C, Wang Y-Q, Wu X-H, Wang H-C. 1984. Role of potas-
sium in t he accumulation of proline associated with water
stress in sorghum seedlings. Acta Phytophysiol Sin, 10: 209 –
215. (in Chinese with English abstract)
Uchida A, Jagendorf A T, Hibino T, Takabe T, Takabe T. 2002.
Effects of hydrogen peroxide and nitric oxide on both salt and
heat stress tolerance in rice. Plant Sci, 163:515 – 523.
Wada M, Urayama O, Satoh S, Hara Y, Ikawa Y, Fujii T. 1992. A
marine algal Na+-activated ATPase possesses an immunologi-
cally identical epitop e to Na+, K+-ATPase. FEBS Lett, 309:
272 – 274.
Wang L-J, Liu Y-L, Ma K . 2000. The roles of the activities of
p lasmalemma and t onoplast H+-ATPase on salt induced
proline accumulation of Ficus carica cells. Acta Phytophysiol
Sin , 26:232 – 236. (in Chinese with English abstract)
Yu B-J, Gong H-M, Li M-W , Liu Y-L. 1997. Comp arison of
dextran T70 and sucrose densit y gradients centrifugation for
preparing membrane vesicles. J Nanjing Agric Univ , 20:14 –
18. (in Chinese with English abstract)
Zhao F-G , Liu Y-L, Zhang W-H. 2002. Proline metabolism in the
leaves of barley seedlings and its relation to salt t olerance. J
Nanjing Agric Univ, 25:7 – 10. (in Chinese with English
abstract)
Zheng Q-S, Wang R-L, Liu Y-L. 2001. Effect s of Ca2+ on t he
absorp tion and distribut ion of ions in salt-t reat ed cott on
seedlings. Acta Phytophysiol Sin, 27:325 – 330. (in Chinese
with English abstract)
Zhu J K. 2001. Plant salt tolerance. Trends Plant Sci, 6:66 – 71.
(Managing editor: HE Ping)