Plant Science 169 (2005) 959–965

www.elsevier.com/locate/plantsci

The expression of the endogenous vacuolar Na+/H+ antiporters in

roots and shoots correlates positively with the salt resistance
of wheat (Triticum aestivum L.)
M. Saqib a,b,*, C. Zörb a, Z. Rengel c, S. Schubert a
a
Institute of Plant Nutrition, Justus Liebig University, Heinrich-Buff-Ring 26-32, 35392-Giessen, Germany
b
Institute of Soil & Environmental Sciences, University of Agriculture, Faisalabad 38040, Pakistan
c
Soil Science & Plant Nutrition, School of Earth & Geographical Sciences, University of Western Australia,
35 Stirling Highway, Crawley, WA 6009, Australia
Received 30 March 2005; received in revised form 1 July 2005; accepted 2 July 2005
Available online 18 July 2005

Abstract

Among the most common effects of salinity is the growth inhibition by Na+ toxicity. Vacuolar Na+/H+ antiporters have been suggested to
be involved in sequestering Na+ into vacuoles, thus preventing toxic effects of Na+ in the cytoplasm. This study reports how the expression of
endogenous vacuolar Na+/H+ antiporters relates to the salt resistance of two wheat (Triticum aestivum L.) genotypes that differ in Na+
translocation from root to the shoot and Na+ accumulation in the young and old leaves. The genotype SARC-1 having the lowest root-to-shoot
Na+ translocation was the most salt-resistant in terms of absolute and relative shoot fresh weight production. However, compared to the salt-
sensitive wheat genotype 7-Cerros, the salt-resistant genotype SARC-1 showed a significantly higher Na+ concentration in young leaves, a
similar Na+ concentration in medium leaves and a significantly lower Na+ concentration in the old leaves. The expression of endogenous
vacuolar Na+/H+ antiporters in roots and shoots was significantly higher in the salt-resistant genotype SARC-1 than in the salt-sensitive
genotype 7-Cerros. However, within a genotype there was little difference in the expression of vacuolar Na+/H+ antiporters between shoots
and roots, and between cortical and stelar root parts. It is suggested that the higher expression of endogenous vacuolar Na+/H+ antiporters in
roots and shoots of the salt-resistant wheat genotype SARC-1 facilitated Na+ exclusion from the cytoplasm of its shoot cells and improved its
salt resistance.
# 2005 Elsevier Ireland Ltd. All rights reserved.

Keywords: Na+/H+ antiporters; Na+ exclusion; Salinity; Salt resistance; Triticum aestivum L.; Wheat

1. Introduction of Na+ compartmentation in leaf tissues and cells [4,5]. The

Na+ is the most abundant cation in saline soils. It is the
primary cause of the ion-specific damage, particularly in
graminaceous crops [1,2]. Na+-specific damage is associated
with the accumulation of Na+ in leaf tissues followed by the
disruption of enzymatic processes and protein synthesis [3].
The reduction in growth and yield then occurs as a result of
the reduced lifespan of individual leaves. The timescale over
which the Na+-specific damage is manifested depends on the
rate of accumulation of Na+ in leaves and the effectiveness
* Corresponding author. Tel.: +49 641 9939164; fax: +49 641 9939169.
E-mail address: drhmsab@yahoo.com (M. Saqib).
initial entry of Na+ from the soil solution into the root
cortical cytoplasm is passive [6] and occurs through various
ion channels and other transporters in the root-cell
plasmalemma [3]. Na+ also enters in the plant by diffusion
into xylem via the apoplast [7].
It has been suggested that the genotypic difference in Na+
exclusion at the root surface is based on a relatively low
passive Na+ permeability of the epidermal and cortical
plasmalemma [8]. Compared with a salt-sensitive maize
genotype, salt-resistant ones show lower Na+ uptake by roots
and less translocation to the shoot [2], the latter possibly
because of an efficient sodium uptake by the xylem
parenchyma cells [1,9]. Similarly, Na+ exclusion correlates

0168-9452/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.plantsci.2005.07.001
960 M. Saqib et al. / Plant Science 169 (2005) 959–965
positively with the salt resistance of wheat [10–12]. A salt-
resistant barley genotype showed 1.5 times lower Na+
concentration in roots and shoots and the shoot Na+
concentration reflected the cytoplasmic concentration in the
root cortical cells [13]. The Na+/H+ antiporters in the root
cortex and stele may be responsible for low translocation of
Na+ to shoots by sequestering Na+ into the vacuoles.
Na+/H+ antiporters are membrane proteins playing an
important role in the cellular pH and Na+ homeostasis. They
are involved in transporting Na+ out of the cytosol into the
vacuole and the apoplast. The acitivity of Na+/H+ antiporters
has been reported a long time ago [14–16]. Recently, the
vacuolar as well as the plasmalemma Na+/H+ antiporters
have been studied at the molecular level [17–19]. The
vacuolar Na+/H+ antiporters sequester Na+ into the vacuole,
and thus save the cytoplasm from damaging effects of high
Na+ concentrations. They also maintain osmotic balance by
accumulating Na+ in the vacuole [20]. While transporting
Na+ into vacuoles, the Na+/H+ antiporters use the pH
gradient generated by the vacuolar H+-translocating
enzymes H+ ATPase and PPiase [21].
The overexpression of vacuolar Na+/H+ antiporters
improved the salt resistance of Arabidopsis [17], tomato
[22], rice [23,24], tobacco [25] and wheat [26]. However,
rare reports are available on the expression of endogenous
vacuolar Na+/H+ antiporters and their relationship with the
salt resistance of different genotypes of a species. Wu et al.
[25] reported that salt-induced accumulation of the GhNHX1
mRNA in cotton was 3–7 times higher in the salt-resistant
than the salt-sensitive genotype. It is not known how salt-
resistant and sensitive wheat genotypes will differ in the
expression of endogenous vacuolar Na+/H+ antiporters in
response to salinity and how this response will correlate with
their Na+ exclusion and salt resistance. This project was
conducted with four wheat genotypes, testing the hypothesis
that the salt-resistant wheat genotype would show higher
expression of endogenous vacuolar Na+/H+ antiporters in
roots and shoots, thereby maintaining low Na+ concentration
in shoots and higher resistance of leaves due to Na+
sequestration into the vacuoles.
2. Materials and methods
2.1. Experiment I
2.1.1. Plant material
The plant material used was four wheat genotypes
(Triticum aestivum L. cv. SARC-1, Inqlab-91, MH-97 and
7-Cerros; SARC-1 obtained from the Institute of Soil
and Environmental Sciences, University of Agriculture,
Faisalabad, Pakistan; Inqlab-91 and MH-97 obtained from
the Ayub Agricultural Research Institute, Faisalabad,
Pakistan and 7-Cerros obtained from Centre for Arid
Zone Studies, University of Wales, Bangor, UK). The
genotypes were chosen on the basis of their previous
history of growth and grain yield production under saline
conditions. SARC-1 and Inqlab-91 [27] were used as
relatively salt-resistant and MH-97 [27] and 7-Cerros (J.
Akhtar, 2000, personal communication) as salt-sensitive
wheat genotypes.
2.1.2. Growth conditions and plant culture
Seeds of the wheat genotypes were soaked in 1 mM
CaSO4 solution for 24 h at 25 8C. The pre-soaked seeds were
then placed between two filter paper sheets supported by two
foam sheets of the same size on outer sides, thus making a
sandwich. These sandwiches were placed in plastic tubs, 1/4
filled with 1 mM CaSO4 in dark. At 1.5 leaf stage, the
seedlings of uniform size were transplanted into pots with
3 L 1/8 Hoagland nutrient solution [28]. Two and 4 day after
transplanting, the concentration of nutrient solution was
increased to 1/4 and 1/2 Hoagland, respectively. The NaCl
addition in the saline treatment was started one day after
the 1/2 Hoagland nutrient solution concentration was
reached and was continued in increments of 25 mM NaCl
every other day till the final level of 125 mM NaCl was
reached. A 1/2 Hoagland nutrient solution containing
1 mM NaCl, added with the first installment of the saline
treatment, was used as a control. The pH of the treatment
solutions was adjusted to 6.0–6.5. The treatments were
run with four replicates and the treatment solutions were
changed twice a week. The experiment was carried out in a
growth chamber with a light intensity of 400 mE m
2 s
1
for 15 h. The light and dark period temperatures were
25 and 18 8C, respectively, and the relative humidity
was 70%.
2.1.3. Plant harvest and analysis of Na+ and K+ in
shoots and roots
After 21 days of growth in the treatment solutions, plants
were harvested and separated into shoots and roots. The
shoot and root fresh weights were recorded and the leaves
were separated into young (immature growing leaves),
medium (mature leaves without salt toxicity symptoms) and
old (mature leaves with salt toxicity symptoms). The plant
material was dried at 70 8C for 48 h, dry-ashed at 520 8C and
digested in 5 M HNO3. The digested plant material was
filtered, diluted with distilled water and analyzed for Na+
and K+ concentrations using an atomic absorption spectro-
photometer (Varian FS 220). The Na+ translocation from
roots to shoots was calculated as follows:
Root-to-shoot Naþ translocation
shoot Naþ content ðmmolÞ
¼
root Naþ content ðmmolÞ
In addition, the resulting values of root-to-shoot Na+
translocation were normalized by dividing them with
the values of shoot:root ratio of the respective wheat
genotypes.
 M. Saqib et al. / Plant Science 169 (2005) 959–965
2.2. Experiment II
This study was conducted with the two wheat genotypes
SARC-1 and 7-Cerros selected from the first experiment as
salt-resistant and sensitive, respectively. The same culturing
procedures, treatments and growth condititions as described
for the first experiment were used in experiment II.
2.2.1. Collection of plant material for molecular
analysis
At the time of harvest, the shoots and roots were rinsed
briefly in distilled water, blotted dry and immediately frozen
in liquid nitrogen. Following Roberts and Tester [29] some
of the main roots were cut into pieces of 5–10 cm and
manipulated with gloved fingers to separate cortical and
stelar tissues. The lateral roots were removed from these
pieces of main roots by means of a sharp stainless steal blade
and a longitudinal blade cut was drawn through the cortical
tissues of each of these main root pieces. The cortical and
stelar tissues were separated with the help of a sharp-edged
foresap on one end of the root piece and pulled gently
towards the opposite end thus completing the separation.
The cortical and stelar tissues were then immediately and
separately frozen in liquid nitrogen.
2.2.2. Isolation of mRNA and synthesis of first-strand
cDNA
The frozen shoot and root tissues were ground in liquid
nitrogen using a pre-cooled mortar and pestle. Total RNA
was isolated from this ground material using phenol–
chloroform according to a modified method [30]. From the
total RNA, poly A+ or mRNA was isolated using the
paramagnetic particle technique and following the instruc-
tions of the manufacturer (Dynal). First-strand cDNA was
synthesized using 1 mg mRNA following the manufacturer’s
instructions in Super ScriptTM-II First-Strand Synthesis
System for RT-PCR (Invitrogen).
2.2.3. Real-time PCR protocols
Real-time PCR assays were performed on the Rotor-Gene
2000/3000 Real-Time Amplification Thermal Cycling Sys-
tem following [9]. The primer pair for the Na+/H+ antiporters
(TaNHX family) was designed in the highly conserved
homologue regions of all available TaNHX isoforms (NCBI
Accession Nos. AY040245, AY040246 and AY296910) from
wheat. The primer pair was 50 -ATC TAC YTS CTC CCK
CCC AT-30 in sense direction and 50 -AGA TAG CYC CAA
TTG CAA GA-30 in anti-sense direction. The primer
amplification length was 208 bp. The primer pair for the
house-keeping gene ubiquitin (NCBI Accession No.
AY297059) was 50 -AAG ACC ATC ACC CTG GAG GT-
30 in sense direction and 50 -GAG ACG GAG CAC CAA GTG
AA-30 in anti-sense direction with an amplification length of
188 bp. The 10 mL reaction mixture for the real-time PCR
contained cDNA (1:10) 1 mL, dNTPs (10 mM Mix, Invitro-
gen) 0.20 mL, taq polymerase (5 units/mL, GeneCraft)
961
0.15 mL, sterile water 6.70 mL, primer pair (100 pmol/mL,
Carl Roth) 0.20 mL, SYBR-green (10,000, Invitrogen)
0.75 mL of 1:2000 dilution and 10 PCR buffer BiothermTM
(+MgCl2, 25 mM, GeneCraft) 1 mL. The real-time PCR
reaction was initiated with denaturization at 94 8C for 3 min.
The cycling protocol consisted of denaturization at 94 8C for
30 s, annealing at 58 8C for 30 s and elongation at 72 8C for
30 s. The PCR reaction was run for 40 cycles. At the end, a
melting curve was run from 72 to 99 8C. The melting
temperatures of TaNHX and ubiquitin were 84 and 89 8C,
respectively. The melting curve prepared using SYBR-green
fluorescence of obtained PCR-sequences detected no hairpin
or loop formation. To verify correct and single bands, real-
time PCR amplification products were checked on DNA
agarose gels. Negative controls with no templates were
carried out with each run and each amplification was repeated
at least twice in independent runs.
2.2.4. Relative quantification of the real-time PCR data
The expression of the Na+/H+ antiporters was quantified
following the relative quantification method with two
standard curves. This method includes a house-keeping
gene as an internal control and measures the expression level
of the gene of interest (in this case Na+/H+ antiporters:
TaNHX) with reference to the internal control. The
expression of the house-keeping gene is not affected by
the treatment. In this study, ubiquitin was used as a house-
keeping gene according to Zhu [31]. Each sample was
separately analyzed with ubiquitin as well as Na+/H+
antiporters. In each case, standard curves were generated
from the dilution series of one of the sample cDNA. The
values of the expression in each sample relative to the
standard curve were calculated. The resulting data were
normalized by dividing the value of expression of Na+/H+
antiporters in a sample with the value of expression of the
house-keeping gene (ubiquitin) in that sample. These
normalized values of relative expression in shoots and
roots of SARC-1 were compared with the values of 7-Cerros.
The values of relative expression of the Na+/H+ antiporters
in the shoots and roots of SARC-1 were arbitrarily set to 100,
and values of the relative expression of the Na+/H+
antiporters in the shoots and roots of 7-Cerros were
expressed as percent of the respective SARC-1 values.
For comparing relative expression of Na+/H+ antiporters in
the stelar tissue with the cortical root tissue and in the shoots
with the roots, the values of expression in the cortical tissue
and in the shoots were arbitrarily set to 100.
2.3. Statistical analysis
The data from experiment I were analyzed for variance
using Sigma Stat 2.0 computer software, the significance of
differences among the genotypes was analyzed using the
Tukey test. The data from experiment II are presented as
percentages and variation between the genotypes was
characterized by standard error.
962 M. Saqib et al. / Plant Science 169 (2005) 959–965

Table 1
Shoot and root fresh weights per plant of four wheat genotypes as affected by salinity
Genotypes Shoot fresh weight per plant (g) Root fresh weight per plant (g)
Control Saline Control Saline
SARC-1 8.2  0.4 b 4.5  0.2 a (55) 6.2  0.2 bc 6.5  0.3 a (105)
Inqlab-91 8.5  0.5 b 3.2  0.2 ab (38) 5.1  0.2 c 3.4  0.2 b (67)
MH-97 11.4  0.7 a 3.0  0.3 ab (26) 6.6  0.4 b 4.0  0.5 b (61)
7-Cerros 13.1  0.8 a 2.5  0.1 b (19) 8.0  0.5 a 2.6  0.1 b (33)
Analysis of variance
*** ***
Salinity (S)
** ***
Genotype (G)
*** ***
SG
Plants were 14 days old when treatments were applied for 21 days. Control, 1 mM NaCl; saline, 125 mM NaCl. Means  standard error (n = 4). Different letters
(a–c) within a column denote significance according to Tukey test (P 0.05). The values in parentheses are percentages of the respective controls.
**
Mean significant at P 0.01.
***
Mean significant at P 0.001.

3. Results 3.2. Shoot and root Na+ accumulation

3.1. Shoot and root growth
Salinity caused a significant reduction in shoot and root
growth of the wheat genotypes tested (Table 1). In saline
treatment (125 mM NaCl), SARC-1 produced the maximum
absolute (4.5 g per plant) and relative (55% of control)
shoot fresh weights but differed significantly only from 7-
Cerros (Table 1). SARC-1 also produced the maximum root
fresh weight (6.5 g per plant) under salinity and differed
significantly from all the other wheat genotypes (Table 1). The
wheat genotypes differed in the number of dead leaves in
saline treatment with extremes being SARC-1 with only 17%
and 7-Cerros having 30% (data not shown). On the basis of
absolute and relative shoot growth in saline conditions,
SARC-1 and 7-Cerros were characterized as the most salt-
resistant and salt-sensitive wheat genotypes, respectively.
Among the most common effects of salinity is the growth
inhibition by Na+ toxicity. Salinity (125 mM NaCl)
significantly increased root-to-shoot Na+ translocation and
Na+ concentration in roots and shoots. However, there was a
significant interaction between salinity and genotype, and
the genotypes differed significantly for these parameters in
saline treatment (Table 2). The wheat genotype SARC-1 had
the lowest Na+ translocation to the shoot (1.4 against 3.3–4.5
in other genotypes). This genotype SARC-1 had the lowest
Na+ concentration in shoots, but the highest in roots.
However, the Na+ uptake per gram plant differed non-
significantly among the genotypes. The distribution pattern
of Na+ among the leaves under salinity was similar in the
four wheat genotypes, with the maximum Na+ concentration
in the old leaves followed by the medium-aged and young
leaves in a descending order (Table 3). There was no

Table 2
Shoot and root Na+ concentrations, root-to-shoot Na+ translocation and Na+ uptake per gram plant of four wheat genotypes under salinity (125 mM NaCl)
Genotypes Shoot Na+ Root Na+ Root-to-shoot Normalized Na+ uptake
(mmol g
1 dry wt) (mmol g
1 dry wt) Na+ translocation root-to-shoot Na+ per g plant
translocation (mmol)
SARC-1 1.3  0.1 b 1.7  0.1 a 1.4  0.1 c 2.0  0.1 c 1.5  0.01 a
Inqlab-91 1.8  0.2 a 1.3  0.1 b 4.5  0.4 a 4.8  0.3 a 1.7  0.13 a
MH-97 1.5  0.1 b 1.1  0.1 b 3.3  0.2 b 4.4  0.1 b 1.4  0.05 a
7-Cerros 1.6  0.1 a 1.1  0.1 b 4.1  0.1 ab 4.3  0.2 ab 1.5  0.05 a
Analysis of variance
*** *** *** *** ***
Salinity (S)
* *** *** ***
Genotype (G) NS
* *** *** ***
SG NS
Plants were 14 days old when salinity was applied for 21 days. Means  standard error (n = 4). Different letters (a–c) within a column denote significance
according to Tukey test (P 0.05). The root-to-shoot Na+ translocation was calculated by dividing the shoot Na+ content (mmol) by root Na+ content (mmol).
The normalized root-to-shoot Na+ translocation was obtained by dividing root-to-shoot Na+ translocation values by the values of shoot:root ratio of the
respective wheat genotypes. NS means non-significant.
*
Mean significant at P 0.05.
***
Mean significant at P 0.001.
 M. Saqib et al. / Plant Science 169 (2005) 959–965 963

Table 3
Na+concentration in different leaves of four wheat genotypes under salinity
(125 mM NaCl)
Genotypes Young leaf Na+ Medium leaf Na+ Old leaf Na+
(mmol g
1 (mmol g
1 (mmol g
1
dry wt) dry wt) dry wt)
SARC-1 0.24  0.02 a 0.59  0.02 a 3.2  0.1 b
Inqlab-91 0.13  0.01 b 0.62  0.05 a 4.7  0.5 a
MH-97 0.17  0.03 b 0.52  0.08 a 3.7  0.1 b
7-Cerros 0.15  0.02 b 0.58  0.05 a 4.1  0.2 a
Plants were 14 days old when salinity was applied for 21 days. Mean-
s  standard error (n = 4). Different letters within a column denote sig-
nificance according to Tukey test (P 0.05).

Fig. 2. Relative expression of endogenous vacuolar Na+/H+ antiporters in

significant difference among the wheat genotypes for Na+
concentration in the medium-aged leaves, but in young
leaves, SARC-1 showed a significantly higher Na+ concen-
tration than the other genotypes. However, in old leaves Na+
concentration of SARC-1 was the lowest and did not
significantly differ from MH-97. SARC-1 had a significantly
higher K+/Na+ ratio in the shoot than in the root, whereas the
other genotypes showed a significantly higher K+/Na+ ratio
in the root than in the shoot (Fig. 1). SARC-1 also differed
from the other genotypes at the root as well as shoot level
(except MH-97) and its K+/Na+ ratio was the lowest in roots
and the highest in shoots.
roots and shoots of two wheat genotypes (SARC-1, salt-resistant; 7-Cerros,
salt-sensitive) under salinity (125 mM NaCl). Plants were 14 days old when
salinity was applied for 21 days. The columns show the relative expression
of the Na+/H+ antiporters. The bars show standard error (n = 4).
8–16% higher expression in the cortical than in the stelar
root tissues (Fig. 3). Similarly, a comparison of the
expression of endogenous vacuolar Na+/H+ antiporters in
roots and shoots showed non-significant differences between
the shoots and roots within both the genotypes (data not
shown).

3.3. Relative expression of the endogenous vacuolar 4. Discussion

Na+/H+ antiporters
+ +
The expression of the endogenous vacuolar Na /H
antiporters was studied in the roots and shoots of salt-
resistant and sensitive wheat genotypes. The expression of
Na+/H+ antiporters in SARC-1 (salt-resistant genotype) was
29% higher in roots and 27% higher in shoots, compared
with 7-Cerros (salt-sensitive genotype) under saline treat-
ment (125 mM NaCl; Fig. 2). However, there was little
difference in the expression of Na+/H+ antiporters between
the cortical and stelar root tissues within the genotypes, with
4.1. Na+ retention in roots, accumulation in shoots and
salt resistance of wheat
Low shoot Na+ accumulation contributes to the salt
resistance of plants, including wheat [32,1,10–12]. In the
present study, the salt-resistant genotype SARC-1 produced
the highest absolute (4.5 g per plant) and relative (55% of
control) shoot fresh weights but had the lowest Na+
concentration in its shoots (1.3 mmol g
1 dry wt) after salt
treatment (Tables 1 and 2). The salt-sensitive genotype 7-
Cerros produced the minimum absolute and relative shoot

Fig. 1. K+:Na+ ratio in shoots and roots of four wheat genotypes under
salinity (125 mM NaCl). Plants were 14 days old when salinity was applied
for 21 days. The columns show the K+:Na+ ratio. The bars show standard
error (n = 4). Different capital letters denote significance between shoot and
root within a genotype and different small letters denote significance among
the genotypes within shoot or root, according to Tukey test (P 0.05).
Fig. 3. Relative expression of endogenous vacuolar Na+/H+ antiporters in
the cortical and stelar root tissues in two wheat genotypes under salinity
(125 mM NaCl). Plants were 14 days old when salinity was applied for 21
days. The columns show the relative expression of the Na+/H+ antiporters.
The bars show standard error (n = 4).
964 M. Saqib et al. / Plant Science 169 (2005) 959–965
fresh weights but had higher Na+ concentration in shoots.
However, the difference in shoot Na+ concentration
between these genotypes was relatively small to be able
to explain major growth differences. In sensitive genotypes,
more Na+ accumulates in the older transpiring leaves,
causing their premature senescence and reducing the
photosynthetic capacity of the plants to a level that cannot
sustain further growth [4]. The Na+ concentrations in the old
leaves of SARC-1 (3.2 mmol g
1 dry wt) and 7-Cerros
(4.1 mmol g
1 dry wt) support this argument (Table 3). As
a result, SARC-1 maintained more green leaf area (data not
shown), which provided a high photosynthetic area and
ultimately contributed to higher shoot fresh weight of this
genotype. The higher Na+ concentration in young and
similar Na+ concentration in medium leaves of SARC-1
compared to 7-Cerros point to an effective sequestration of
Na+ into the vacuoles and/or tolerance of high cytoplasmic
Na+ concentration by this salt-resistant wheat genotype.
Compared with salt-sensitive maize genotypes, salt-
resistant genotypes show less translocation of Na+ from
roots to shoots because of efficient sodium retention by the
xylem parenchyma cells [1,2]. The study presented here
demonstrates that root-to-shoot translocation of Na+ by the
salt-resistant genotype SARC-1 was more than two times
lower than by the salt-sensitive 7-Cerros (Table 2). This
shows a significant retention of Na+ by the salt-resistant
wheat genotype in the roots. This is also obvious from the
K+:Na+ ratios of shoots and roots with SARC-1 having
higher K+:Na+ ratio in shoots and lower in roots compared
with the other genotypes (Fig. 1). Such a finding may be due
to more sequestration of Na+ into the vacuoles by the salt-
resistant wheat genotype in the root cortical and stelar
tissues. In addition, a higher root-to-shoot ratio of the
salt-resistant genotype may also help to retain more Na+ in
roots.
4.2. Relationship of endogenous vacuolar Na+/H+
antiporters with Na+ accumulation and salt resistance of
wheat
In SARC-1, the relative expression of the endogenous
vacuolar Na+/H+ antiporters under saline conditions was
29% higher in the roots and 27% higher in the shoots
compared with 7-Cerros (Fig. 2). A relatively higher
expression of the endogenous vacuolar Na+/H+ antiporters
in shoots of the salt-resistant wheat genotype (SARC-1) may
have enabled it to sequester more Na+ into the vacuoles, thus
keeping its cytoplasm safe from the toxic effects of Na+.
This seems to have helped this genotype to tolerate high Na+
concentrations in its young and medium leaves (Table 3) and
to use these high Na+ concentrations to maintain osmotic
balance in these leaves [20]. These results are in agreement
with the earlier findings [23,24], where the salt-resistant
transgenic rice genotypes did not differ significantly from
the salt-sensitive wild-type for leaf Na+ concentration. These
data are also in agreement with previous studies, which show
that higher expression of the Na+/H+ antiporters in
transgenic plants helps to avoid Na+ stress [17,22,24–26].
The higher expression of the endogenous vacuolar Na+/
+
H antiporters in the roots of SARC-1 seems to have
decreased the Na+ translocation from its roots to shoots by
sequestering Na+ into cortical and stelar root cell vacuoles.
Earlier physiological studies [13,33] report that the salt-
resistant barley genotypes maintain low cytosolic and
higher vacuolar Na+ in root cortical tissues, pointing to the
presence of endogenous vacuolar Na+/H+ antiporters. Our
results support this hypothesis and further show that
endogenous vacuolar Na+/H+ antiporters are also present
in the stelar root tissues with no significant difference in
quantitative expression between stelar and cortical root
tissues (Fig. 3). However, relatively less differences between
the genotypes for the expression of endogenous vacuolar
Na+/H+ antiporters than the differences in Na+ retention at
root level, and the salt resistance of the genotypes show the
possibility of further genetic differences in the expression of
endogenous vacuolar Na+/H+ antiporters at post transcrip-
tional level or other resistance mechanisms. There may also
be a genotypic difference in the specific functionality of the
antiporters as the antiporters may have diverse functions,
such as K+/H+ antiport activity, as has been reported for
Arabidopsis [34].
5. Conclusions
Wheat genotypes differ in the expression of endogenous
vacuolar Na+/H+ antiporters in roots and shoots. The
genotype with higher expression of these Na+/H+ antiporters
in roots and shoots retains more Na+ at the root level and is
able to withstand high Na+ concentration in its leaves, thus
becoming more salt-resistant in terms of absolute and
relative shoot growth. Therefore, salt resistance in wheat
correlates positively with the expression of endogenous
vacuolar Na+/H+ antiporters in roots and shoots.
Acknowledgement
M. Saqib gratefully acknowledge the support of DAAD
(Deutscher Akademischer Austauschdienst) Germany, in the
form of a fellowship that enabled him to carry out this work.
References
[1] R. Fortmeier, S. Schubert, Salt tolerance of maize (Zea mays L.): the
role of sodium exclusion, Plant Cell Environ. 18 (1995) 1041–1047.
[2] A. Sümer, C. Zörb, F. Yan, S. Schubert, Evidence of sodium toxicity
for the vegetative growth of maize (Zea mays L.) during the first phase
of salt stress, J. Appl. Bot. 78 (2004) 135–139.
[3] M. Tester, R. Davenport, Na+ tolerance and Na+ transport in higher
plants, Ann. Bot. 91 (2003) 503–527.
 M. Saqib et al. / Plant Science 169 (2005) 959–965
[4] R. Munns, Physiological processes limiting plant growth in saline
soils: some dogmas and hypotheses, Plant Cell Environ. 16 (1993) 15–
24.
[5] R. Munns, Comparative physiology of salt and water stress, Plant Cell
Environ. 25 (2002) 239–250.
[6] J.M. Cheeseman, Pump-leak sodium fluxes in low salt corn roots, J.
Membr. Biol. 70 (1982) 157–164.
[7] A.R. Yeo, M.E. Yeo, T.J. Flowers, The contribution of an apoplastic
pathway to sodium uptake by rice roots in saline conditions, J. Exp.
Bot. 38 (1987) 1141–1153.
[8] S. Schubert, A. Läuchli, Sodium exclusion mechanisms at the root
surface of two maize cultivars, Plant Soil 123 (1990) 205–209.
[9] C. Zörb, A. Noll, S. Karl, K. Leib, F. Yan, S. Schubert, Molecular
characterization of Na+/H+ antiporters (ZmNHX) of maize (Zea mays
L.) and its expression under salt stress, J. Plant Physiol. 162 (2005)
55–66.
[10] R. Munns, R.A. James, Screening methods for salinity tolerance: a
case study with tetraploid wheat, Plant Soil 253 (2003) 201–218.
[11] M. Saqib, J. Akhtar, R.H. Qureshi, Pot study on wheat growth in saline
and waterlogged compacted soil II. Root growth and leaf ionic
relations, Soil Till. Res. 77 (2004) 179–187.
[12] M. Saqib, J. Akhtar, R.H. Qureshi, Na+ exclusion and salt resistance of
wheat (Triticum aestivum) in saline-waterlogged conditions are
improved by the development of adventitious nodal roots and cortical
root aerenchyma, Plant Sci. 169 (2005) 125–130.
[13] T.J. Flowers, M.A. Hajibagheri, Salinity tolerance in Hordeum vul-
gare: ion concentrations in root cells of cultivars differing in salt
tolerance, Plant Soil 231 (2001) 1–9.
[14] E. Blumwald, R.J. Poole, Na+/H+ antiport in isolated tonoplast vesicles
from storage tissue of Beta vulgaris, Plant Physiol. 78 (1985) 163–
167.
[15] J. Garbarino, F.M. DuPont, Rapid induction of Na+/H+ exchange
activity in barley root tonoplast, Plant Physiol. 89 (1989) 1–4.
[16] M. Staal, F.J.M. Maathuis, T.M. Elzenga, J.H.M. Overbeek, H.B.A.
Prins, Na+/H+ antiport activity in tonoplast vesicles from roots of the
salt-tolerant Plantago maritima and the salt-sensitive Plantago media,
Physiol. Plant 82 (1991) 179–184.
[17] M.P. Apse, G.S. Aharon, W.A. Snedden, E. Blumwald, Salt tolerance
conferred by overexpression of a vacuolar Na+/H+ antiport in Arabi-
dopsis, Science 285 (1999) 1256–1258.
[18] H. Shi, B.H. Lee, S.J. Wu, J.K. Zhu, Overexpression of a plasma
membrane Na+/H+ antiporter gene improves salt tolerance in Arabi-
dopsis thaliana, Nat. Biotech. 21 (2003) 81–85.
[19] Q. Qiu, B.J. Barkla, R. Vera-Estrella, J. Zhu, K.S. Schumaker, Na+/H+
exchange activity in the plasma membrane of Arabidopsis, Plant
Physiol. 132 (2003) 1041–1052.
965
[20] E. Glenn, J.J. Brown, E. Blumwald, Salt tolerance and crop potential
of halophytes, Crit. Rev. Plant Sci. 18 (1999) 227–255.
[21] E. Blumwald, A. Gelli, Secondary inorganic ion transport in plant
vacuoles, Adv. Bot. Res. 25 (1997) 401–407.
[22] H.X. Zhang, E. Blumwald, Transgenic salt-tolerant tomato plants
accumulate salt in foliage but not in fruit, Nat. Biotech. 19 (2001)
765–768.
[23] M. Ohta, Y. Hayashi, A. Nakashima, A. Hamada, A. Tanaka, T.
Nakamura, T. Hayakawa, Introduction of a Na+/H+ antiporter gene
from Atriplex gmelini confers salt tolerance to rice, FEBS Lett. 532
(2002) 279–282.
[24] Y. Fukuda, A. Nakamura, A. Tagiri, H. Tanaka, A. Miyao, H.
Hirochika, Y. Tanaka, Function, intracellular localization and the
importance in salt tolerance of a vacuolar Na+/H+ antiporter from
rice, Plant Cell Physiol. 45 (2004) 146–159.
[25] C.A. Wu, G.D. Yang, Q.W. Meng, C.C. Zheng, The cotton GhNHX1
gene encoding a novel putative tonoplast Na+/H+ antiporter plays an
important role in salt stress, Plant Cell Physiol. 45 (2004) 600–607.
[26] Z.Y. Xue, D.Y. Zhi, G.P. Xue, H. Zhang, Y.X. Zhao, G.M. Xia,
Enhanced salt tolerance of transgenic wheat (Triticum aestivum L.)
expressing a vacuolar Na+/H+ antiporter gene with improved grain
yields in saline soils in the field and a reduced level of leaf Na+, Plant
Sci. 167 (2004) 849–859.
[27] M. Saqib, Selection and characterization of wheat genotypes against
salinity and waterlogging, Ph.D Thesis, Department of Soil Science,
University of Agriculture, Faisalabad, Pakistan, 2002.
[28] D.R. Hoagland, D.I. Arnon, The water culture method for growing
plants without soil, Calif. Agric. Exp. Stat.Cir. No. 347 (1950) 39.
[29] S.K. Roberts, M. Tester, Inward and outward K+-selective currents in
the plasma membrane of protoplasts from maize root cortex and stele,
Plant J. 8 (1995) 811–825.
[30] K.H. Cox, R.B. Goldberg, Isolation of total RNA, in: C.H. Shaw (Ed.),
Plant Molecular Biology, IRL Press, Oxford, 1988, pp. 2–8.
[31] Y. Zhu, Adaptation of plasma membrane H+ ATPase of proteoid roots
of white lupin (Lupinus albus L.) under phosphorous deficiency, Ph.D.
Dissertation, Inst. Plant Nut. Justus Liebig University, Giessen, Ger-
many, 2004.
[32] J. Gorham, Salt tolerance in Triticeae: K+/Na+ discrimination in
synthetic hexaploid wheat, J. Exp. Bot. 41 (1990) 623–627.
[33] D.E. Carden, D.J. Walker, T.J. Flowers, A.J. Miller, Single cell
measurements of the contributions of cytosolic Na+ and K+ to salt
tolerance, Plant Physiol. 131 (2003) 676–683.
[34] M.P. Apse, J.B. Sottosanto, E. Blumwald, Vacuolar cation/H+
exchange, ion homeostasis, and leaf development are altered in a
T-DNA insertional mutant of AtNHX1, the Arabidopsis vacuolar Na+/
H+ antiporter, Plant J. 36 (2003) 229–239.