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Journal of Plant Physiology ] (]]]]) ]]]—]]]
www.elsevier.de/jplph
Potassium nitrate application alleviates
sodium chloride stress in winter wheat
cultivars differing in salt tolerance
Yanhai Zhenga,b, Aijun Jiac, Tangyuan Ningb, Jialin Xud,
Zengjia Lib,1, Gaoming Jiangb,a,
a
Key Laboratory of Vegetation and Environmental Change,
Institute of Botany, the Chinese Academy of Sciences, 20 Nanxincun, Xiangshan, 100093 Beijing, China
b
Key Laboratory of Crop Biology , Shandong Agricultural University, 61 Daizong Street, 271018 Taian, China
c
Dezhou Hengdong Pesticide & Chemical Co. Ltd. 18 Tianqu Industrial Park, 231025 Dezhou, China
d
Dezhou Virescence Institute of Salty & Alkaline Soil, 156 Tianqu East Road, 253016 Dezhou, China
Received 27 July 2007; received in revised form 7 January 2008; accepted 10 January 2008
KEYWORDS
Potassium nitrate;
Salt tolerance;
Sodium chloride
stress;
Stress alleviation;
Winter wheat
Summary
A sand culture experiment was conducted to answer the question whether or not
exogenous KNO3 can alleviate adverse effects of salt stress in winter wheat by
monitoring plant growth, K+/Na+ accumulation and the activity of some antioxidant
enzymes. Seeds of two wheat cultivars (CVs), DK961 (salt-tolerant) and JN17
(salt-sensitive), were planted in sandboxes and controls germinated and raised with
Hoagland nutrient solution (6 mM KNO3, no NaCl). Experimental seeds were exposed
to seven modified Hoagland solutions containing increased levels of KNO3 (11, 16,
21 mM) or 100 mM NaCl in combination with the four KNO3 concentrations (6, 11, 16
and 21 mM). Plants were harvested 30 d after imbibition, with controls approximately 22 cm in height. Both CVs showed significant reduction in plant height, root
length and dry weight of shoots and roots under KNO3 or NaCl stress. However, the
combination of increased KNO3 and NaCl alleviated symptoms of the individual salt
stresses by improving growth of shoots and roots, reducing electrolyte leakage,
malondialdehyde and soluble sugar contents and enhancing the activities of
antioxidant enzymes. The salt-tolerant cultivar accumulated more K+ in both shoots
and roots compared with the higher Na+ accumulation typical for the salt-sensitive
cultivar. Soluble sugar content and activities of antioxidant enzymes were found to
Abbreviations: CAR, carotenoid; CAT, catalase (EC 1.11.1.6); CHL, chlorophyll; cv(s), cultivar(s); EL, electrolyte leakage;
MDA, malondialdehyde; POD, peroxidase (EC 1.11.1.7); SOD, superoxide dismutase (EC 1.15.1.1).
Corresponding author at: Key Laboratory of Vegetation and Environmental Change, Institute of Botany, the Chinese Academy of
Sciences, 20 Nanxincun, Xiangshan, 100093 Beijing, China. Tel.: +86 1062836286; fax: +86 1062830843.
E-mail address: [email protected] (G. Jiang).
1
Also for correspondence.
0176-1617/$ - see front matter & 2008 Elsevier GmbH. All rights reserved.
doi:10.1016/j.jplph.2008.01.001
Please cite this article as: Zheng Y, et al. Potassium nitrate application alleviates sodium chloride stress in winter wheat cultivars
differing in salt tolerance. J Plant Physiol (2008), doi:10.1016/j.jplph.2008.01.001
ARTICLE IN PRESS
2
Y. Zheng et al.
be more stable in the salt-tolerant cultivar. Our findings suggest that the optimal K+/
Na+ ratio of the nutrient solution should be 16:100 for both the salt-tolerant and the
salt-sensitive cultivar under the experimental conditions used, and that the
alleviation of NaCl stress symptoms through simultaneously applied elevated KNO3
was more effective in the salt-tolerant than in the salt-sensitive cultivar.
& 2008 Elsevier GmbH. All rights reserved.
Introduction
Soil salinity, which is a worldwide problem,
severely limits crop production. Traditionally, this
problem has been approached by altering farming
practices to prevent soil salinization and/or by
implementing schemes to remedy salt-stressed
soils, such as plastic foil covers, foliar application
of glycinebetaine or establishing deep-rooted
plantings (Chen et al., 2005). The most promising
solution to overcome the soil salinity problem,
however, might be the use of salt-tolerant species
that show high yields in saline soils and/or decrease
farmland pollution through remediation (Ashraf and
O’Leary, 1996). To achieve this goal, efficient
breeding programs towards more salt-tolerant
plants, including traditional and genetic engineering strategies, have to be developed (Gorham
et al., 1997).
Potassium plays an important role in balancing
membrane potential and turgor, activating enzymes, regulating osmotic pressure, stoma movement and tropisms (Cherel, 2004). To maintain
normal cell metabolism, the K+ content in wheat
cells is kept around 150 mM and the Na+ content at
about 30 mM, resulting in a K+/Na+ ratio of
approximately 5 (Carden et al., 2003). A suitable
K+/Na+ ratio is important for the adjustment of cell
osmoregulation, turgor maintenance, stomatal
function, activation of enzymes, protein synthesis,
oxidants metabolism and photosynthesis (Shabala
et al., 2003). However, overproduction of reactive
oxygen species (ROS) caused by salinity usually
leads to lipid peroxidation and induces K+ leak from
the cell by activating K+ efflux channels (Demidchik
et al., 2003; Cuin and Shabala, 2007). Tester and
Davenport (2003) reported that one of the key
features of plant salt tolerance is the ability of
plant cells to maintain an optimal K+/Na+ ratio.
Under salinity stress, the K+/Na+ ratio shows a
tendency to decrease. This occurs as a result of
either excessive Na+ accumulation in plant tissue or
enhanced K+ leakage from the cell. Potassium
leakage normally happens as a result of NaClinduced membrane depolarization under saline
conditions (Shabala et al., 2003).
Previous studies revealed that supplying low
levels of KNO3 could alleviate the NaCl-induced
decreases in seed germination of certain grass
species (Neid and Biesboer, 2005). However, none
of these studies has focused on the differential
responses of salt-tolerant and salt-sensitive crop
cultivars (cvs) to increased levels of KNO3 in the
absence and presence of NaCl stress. Can increased
levels of KNO3 alleviate damages induced by NaCl
stress? What is the optimal K+/Na+ ratio under
stress conditions? The major objectives of this
study were, therefore, to determine in winter
wheat the extent to which KNO3 can ameliorate the
effect of salt stress, and to compare the responses
of two wheat varieties differing in their degree of
salt tolerance.
Materials and methods
Plant growth conditions and treatments
Seeds of two wheat (Triticum aestivum L.) cvs, DK961
(salt-tolerant) and JN17 (salt-sensitive), were sown in
sandboxes (22 16 5 cm3, length width height) in a
greenhouse. Controls were irrigated with Hoagland
nutrient solution (6 mM KNO3, no NaCl). Experimental
seeds were exposed to seven modified Hoagland solutions
containing increased levels of KNO3 (11, 16, 21 mM) or
100 mM NaCl in combination with the four KNO3 concentrations (6, 11, 16 and 21 mM). Water lost by evapotranspiration was replenished each day. The average day/
night temperature was kept at 16–26 and 10–16 1C,
respectively, with a mean photoperiod being 14 h. All the
treatments were arranged in a randomized complete
block design. Measurements were carried out at 30 d
after treatment.
Plant growth, water and soluble sugar contents
Growth parameters (plant height, root length and dry
weight) were recorded 30 d after treatment. Thirty
individual wheat seedlings were randomly harvested
from each sandbox. Shoots and roots were separated
and carefully washed with distilled water and dried with
tissues before fresh weights were recorded. Fresh
samples were oven-dried at 70 1C to a constant dry
weight before the dry weights were recorded. Water
Please cite this article as: Zheng Y, et al. Potassium nitrate application alleviates sodium chloride stress in winter wheat cultivars
differing in salt tolerance. J Plant Physiol (2008), doi:10.1016/j.jplph.2008.01.001
ARTICLE IN PRESS
Potassium nitrate alleviates sodium chloride stress in winter wheat
contents (WCs) of shoot and root were calculated by the
follow formula:
WCð%Þ ¼ ½ðfreshweight dryweightÞ=fresh weight
100.
Soluble sugar content was measured following the
method described by Yemm and Willis (1954). About
0.05 g ground dry shoots and roots were soaked in 6–7 mL
deionized water. The solution was boiled (100 1C) for
30 min to extract soluble sugar and centrifuged under
4000 rpm for 10 min. The extracts were decanted and the
residue was re-extracted for two more times, with
extracts being completed to 50 mL. In all, 0.1 mL extracts
and 3 mL anthrone reagent (0.15 g anthrone+84 mL oil of
vitriol+16 mL H2O) were mixed and the absorbance of the
mixture was recorded at 620 nm. The content of soluble
sugar was calculated from a standard curve of glucose at
620 nm by colorimetry.
Contents of K+ and Na+ in shoot and root
Oven-dried shoots and roots were finely ground
before passing through a 2-mm sieve. About 0.5 g
samples were soaked for 12 h in digesting tubes with
10 mL concentrated nitric acid and 3 mL perchlorate
acid, and then digested at 300 1C for 6 h. The extractions
were completed to 50 mL with deionized water. The
amount of K+ and Na+ contents was measured using an
atomic absorption spectrophotometer (SP9-400, PYE,
England).
Chlorophyll and carotenoid contents
Chlorophyll (CHL) and carotenoid (CAR) contents of the
shoot were measured by the non-maceration method
(Hiscox and Isrealstam, 1979). Samples (0.05 g) were
incubated into 5 mL dimethyl sulfoxide at 65 1C for 4 h.
The absorbance of the supernatant was recorded at 645,
665 and 470 nm, with CHL and CAR contents being
calculated afterwards.
Membrane permeability, lipid peroxidation and
antioxidant enzymes activities
Membrane permeability of leaves was measured by
electrolyte leakage (EL) following the method described
by Dionisio-Sese and Tobita (1998). Ten pieces of 4 cm
middle section of leaves were placed in test tubes
containing 10 mL distilled deionized water. The tubes
were incubated in a water bath at 32 1C for 2 h
and the initial electrical conductivity of the medium
(EC1) was analyzed using an electrical conductivity
analyzer (KL-220, Xingzhou Company Ltd, China).
The samples were autoclaved at 121 1C for 20 min to
release all electrolytes, cooled to 25 1C, and then
the final electrical conductivity (EC2) was measured. The EL was calculated using the formula:
EL ¼ EC1/EC2 100.
Lipid peroxidation was determined by estimating
the malondialdehyde (MDA) content according to Kramer
3
et al. (1991). Frozen samples (0.5 g) mixed with 5 mL
phosphate buffer (pH 7.8) were crushed into a fine
powder in a mortar and pestle under liquid nitrogen. The
homogenate was centrifuged at 10,000g for 20 min at
4 1C, with the supernatant being used for MDA determination. A mixture of 1 mL extracts (MDA)+2 mL 0.6%
thiobarbituric acid (TBA) (0.6 g TBA+1 M NaOH+10%
trichloroacetic acid complete to 100 mL) was produced,
boiled for 15 min, cooled and centrifuged for 10 min
(4000 rpm). The concentration of MDA was calculated
from the absorbance at 600, 532 and 450 nm, and
MDA contents were determined through the following
formula:
MDAðmmol g1 FWÞ
¼ ð6:45 ðD532 D600 Þ 0:56D450 Þ V=W,
where D532, D600 and D450 are the absorbance at 600, 532
and 450 nm, respectively, and V is the volume of
extraction, W is the fresh weight of sample.
Frozen samples (0.5 g) of shoots were prepared in the
same way as measuring MDA contents. Guaiacol peroxidase (POD) was determined through measuring the
oxidation of guaiacol. The assay mixture contained
50 mM sodium phosphate (pH 6.0), 28 mL guaiacol and
19 mL 30% H2O2. The absorbance was recorded five times
at 470 nm at 30 s intervals. Variation of absorbance per
minute per gram fresh weight (DA470 g1 min1 FW)
stands for enzymes activity. Superoxide dismutase (SOD)
activity was determined following the method of Giannopotitis and Ries (1977). The supernatant was desalted
by Sephadex G-25 gel filtration to remove interfering
materials and used as the crude enzyme extract. One unit
of SOD activity (U) was defined as the amount of crude
enzyme extract that is required for inhibiting the
reduction rate of nitro-blue tetrazolium by 50%. Catalase
(CAT) activity was determined following the method
described by Aebi (1984). Unit of CAT activity
(DA240 g1 min1 FW) was defined as variation of absorbance per minute per gram fresh weight. All spectrophotometric analyses were conducted at 25 1C on an UV/
visible light spectrophotometer (UV-365, SHIMADZU,
Japan).
Statistic analysis
The experiment was designed as a randomized
block consisting of eight treatments containing
four increased levels of KNO3 or 100 mM NaCl
in combination with the four KNO3 concentrations on two winter wheat cvs differing in salt
tolerance. There were three replicates for each
treatment. One-way analysis of variance was
performed to assess the effect of KNO3 under
different treatment levels, and to test for alleviation to NaCl stress on seedling growth for each cvs.
Significant effects and interactions were determined at pp0.05.
Please cite this article as: Zheng Y, et al. Potassium nitrate application alleviates sodium chloride stress in winter wheat cultivars
differing in salt tolerance. J Plant Physiol (2008), doi:10.1016/j.jplph.2008.01.001
ARTICLE IN PRESS
4
Y. Zheng et al.
(Table 1). Conversely, those parameters in JN17
never reached as high as DK961.
Results
Growth parameters
Water content
Plant height, root length and dry weight of
control (6 mM KNO3+0 mM NaCl) plants were measured to be considerably higher in the salt-tolerant
(DK961) than sensitive wheat (JN17). Those parameters displayed significant reductions under increased levels of KNO3 (11, 16, 21 mM) or 100 mM
NaCl (6 mM KNO3+100 mM NaCl) treatments for both
cvs. However, the combination of increased KNO3
(11, 16, 21 mM) and NaCl (100 mM NaCl) alleviated
symptoms of the individual salt stresses (Table 1).
Plant height, root length and dry weight of DK961
showed 21%, 16% and 29% reductions in the
treatment of 6 mM KNO3+100 mM NaCl in comparison with the control. Such reductions decreased to
15%, 11% and 19% in the treatment of 11 mM
KNO3+100 mM NaCl, and even only 4%, 3% and 6%
in the treatment of 16 mM KNO3+100 mM NaCl
treatment. In contrast, those parameters in JN17
displayed 41%, 43% and 32% reductions in 6 mM
KNO3+100 mM NaCl treatment against the control,
while 24%, 28% and 25% reductions in the treatment
of 11 mM KNO3+100 mM NaCl, and only 15%, 15% and
7% reductions were measured in the treatment of
16 mM KNO3+100 mM NaCl. However, those parameters reduced more considerably in both cvs in
the treatment of 21 mM KNO3+100 mM NaCl than
in the treatment of 16 mM KNO3+100 mM NaCl
WC was higher in the salt-tolerant (DK961) than
the salt-sensitive cv (JN17) in control (6 mM
KNO3+0 mM NaCl) plants (Table 1). They reduced
drastically in increased levels of KNO3 (11, 16,
21 mM) or 100 mM NaCl (6 mM KNO3+100 mM NaCl)
treatments in both cvs. Properly increased KNO3
concentrations (11, 16 mM) alleviated those adverse effects under 100 mM NaCl stress, and they
elevated more rapidly in DK961 than JN17. Nevertheless, excessive KNO3 concentration (21 mM) was
harmful for plant growth under 100 mM NaCl stress.
Soluble sugar
The soluble sugar contents in shoot and root of
control (6 mM KNO3+0 mM NaCl) plants were drastically higher (20% and 9%, respectively) in DK961
than in JN17. They had significantly elevated in
both increased levels of KNO3 (11, 16, 21 mM) and
100 mM NaCl (6 mM KNO3+100 mM NaCl) treatments
(Table 2). However, properly increased KNO3 concentration (11, 16 mM) in Hoagland solution with
100 mM NaCl could diminish the damage caused by
salinity. Soluble sugar content in shoot of salttolerant DK961 was not significantly different from
the control plants when the KNO3 concentration
Table 1. Plant height, dry weight (DW) and water content (WC) in shoot and root of salt-tolerant DK961 and saltsensitive JN17 in four levels of KNO3 (6, 11, 16 and 21 mM) treatments or 100 mM NaCl in combination with the four
KNO3 concentrations
Treatment
Shoot
Root
Variety
NaCl
(mM)
KNO3
(mM)
Plant height
(cm)
DW (g)
WC (%)
Root length
(cm)
DW (g)
WC (%)
DK961
0
6 (CK)
11
16
21
6
11
16
21
22.5470.82a
23.0771.25a
19.4572.13b
15.0872.02c
17.8472.36c
19.1670.46b
21.7471.08a
17.1270.90c
0.31070.03a
0.3070.33a
0.2670.36b
0.2170.21c
0.2270.07c
0.2570.04b
0.2970.02a
0.2270.08c
67.5972.36a
67.2272.31a
59.1372.68b
44.3472.32c
52.9871.02c
64.8370.98b
66.6670.86a
64.0370.72b
8.9270.46a
8.6671.51a
7.6571.16b
5.6771.13c
7.4670.25c
7.8870.36b
8.5870.57a
6.5870.32d
0.1970.01a
0.2070.02a
0.1670.09b
0.1270.07c
0.1470.15c
0.1570.02b
0.1970.01a
0.1270.03c
57.5972.57a
57.1272.36a
53.2872.86b
49.3272.93c
53.2170.86c
54.1970.95b
57.1071.06a
53.9471.23c
6 (CK)
11
16
21
6
11
16
21
18.6970.41a
17.5172.11b
16.6273.06c
10.2774.73d
10.9870.55c
14.2270.32b
15.8270.46a
10.6270.77c
0.2870.02a
0.2671.32b
0.2271.14c
0.1870.89d
0.1970.04c
0.2170.03b
0.2670.02a
0.2070.06c
57.4770.76a
58.4670.13a
54.0970.29b
43.5871.09c
41.7170.92c
49.6171.01a
51.0071.23a
47.4071.56b
8.9270.53a
9.0271.26a
6.1272.19b
5.2173.12c
5.0270.23c
6.4070.35b
7.5870.30a
5.2670.36c
0.2470.01a
0.2571.68a
0.2170.43b
0.1670.31c
0.1770.02b
0.2070.01b
0.2470.01a
0.2070.02b
52.5571.27a
51.6672.42a
47.7872.10b
41.7472.32c
42.6671.04c
44.0071.26b
47.6671.37a
36.2671.61d
100
JN17
0
100
Data are the mean7SE (n ¼ 6). Different letters within a column indicate significant differences (Po0.05, t test). CK, control,
Hoagland nutrient solution containing 6 mM KNO3 and 0 mM NaCl.
Please cite this article as: Zheng Y, et al. Potassium nitrate application alleviates sodium chloride stress in winter wheat cultivars
differing in salt tolerance. J Plant Physiol (2008), doi:10.1016/j.jplph.2008.01.001
ARTICLE IN PRESS
Potassium nitrate alleviates sodium chloride stress in winter wheat
5
Table 2. Soluble sugar contents in shoot and root of salt-tolerant DK961 and salt-sensitive JN17 in four levels of KNO3
(6, 11, 16 and 21 mM) treatments or 100 mM NaCl in combination with the four KNO3 concentrations
Treatment
DK961
JN17
NaCl
(mM)
KNO3
(mM)
Soluble sugar
content in
shoot
(mg g1 DW)
Soluble sugar
content in
root
(mg g1 DW)
Shoot/root
ratio
Soluble
sugar
content in
shoot
(mg g1 DW)
Soluble
sugar
content in
root
(mg g1 DW)
Shoot/root
ratio
0
6 (CK)
11
16
21
48.3171.80c
48.7872.23c
57.6173.28b
71.2073.36a
20.5271.39b
20.8671.28b
21.4072.13b
28.0972.12a
2.3570.04b
2.3470.03b
2.6970.05a
2.5370.07a
40.2371.20d
43.6972.16c
50.2673.12b
61.5173.24a
18.7371.39c
19.1571.32b
20.1972.13b
26.4272.80a
2.1670.10b
2.2870.03b
2.4970.06a
2.3370.10a
100
6
11
16
21
69.2072.10a
56.0172.18b
48.5973.01d
49.1571.36c
27.1771.21a
22.3971.32c
19.4372.20d
26.6071.74b
2.5570.01b
2.5070.02a
2.5070.05a
1.8570.04b
51.1272.46a
48.6272.02b
43.5171.26d
45.3771.69c
25.2271.80a
19.4771.23b
19.0471.02b
18.8871.17c
2.4270.09a
2.4970.02a
2.1870.06b
2.4070.03a
Data are the mean7SE (n ¼ 6). Different letters within a column indicate significant differences (Po0.05, t test). CK, control,
Hoagland nutrient solution containing 6 mM KNO3 and 0 mM NaCl.
Table 3. Potassium and sodium concentrations in shoot and root of salt-tolerant DK961 and salt-sensitive JN17 in four
levels of KNO3 (6, 11, 16 and 21 mM) treatments or 100 mM NaCl in combination with the four KNO3 concentrations
Treatment
Shoot
Root
Variety
NaCl
(mM)
KNO3
(mM)
K+ content
(mg g1)
Na+ content
(mg g1)
K+/Na+
K+ content
(mg g1)
Na+ content
(mg g1)
K+/Na+
DK961
0
6 (CK)
11
16
21
6
11
16
21
29.6470.46b
41.1272.15a
47.2573.18a
59.0873.8c
18.9070.64c
39.0770.85a
43.4571.18a
19.0870.61c
6.2570.23d
5.4170.34b
4.7670.39b
4.1270.41c
32.7970.83a
24.4170.74b
12.7670.43b
6.7670.31c
4.7470.06a
7.2271.13a
9.1372.86a
14.3473.23b
1.4870.04c
4.1570.23a
4.9670.11a
2.8270.57b
12.1171.15b
16.1072.35c
32.2072.68b
56.5773.12a
8.9270.16d
11.1070.43c
12.2070.68b
16.5770.96a
10.0270.24b
9.2870.62b
8.1870.79b
7.8170.67c
35.0570.56a
26.2870.39b
10.1870.56b
9.8170.34c
1.2170.06b
1.7370.08c
3.9470.21b
7.2471.12a
0.5970.03d
1.0870.05c
1.2070.09b
1.6970.07a
6 (CK)
11
16
21
6
11
16
21
32.5672.69b
36.5172.11c
50.6273.06a
65.2774.73d
9.7370.88e
34.5071.10c
50.0271.04a
35.3770.72d
7.1870.35d
12.0671.32b
9.9471.14c
6.1770.89e
48.6070.38a
22.1270.30b
12.1570.40c
10.2670.09e
4.5370.10a
2.8670.13c
4.0970.29b
10.5871.09c
0.2070.05d
1.5670.13c
4.1270.36b
3.4570.39c
12.3570.76c
19.2370.62d
33.6272.91b
54.5673.21a
4.3570.08e
29.0073.23d
33.5173.91b
24.9672.22a
8.8670.18c
11.7770.86b
6.4770.32d
6.2470.23d
58.6271.02a
41.7770.86b
22.4770.32d
16.2470.23d
1.3970.07b
1.6370.02c
5.1970.10a
8.7470.13a
0.0770.01d
0.6970.12c
1.4970.11a
1.5470.15a
100
JN17
0
100
Data are the mean7SE (n ¼ 6). Different letters within a column indicate significant differences (Po0.05, t test). CK, control,
Hoagland nutrient solution containing 6 mM KNO3 and 0 mM NaCl.
reached 16 mM under 100 mM NaCl stress. In
contrast, they increased significantly in salt-sensitive JN17 in the combinations of four levels of
KNO3 and 100 mM NaCl treatments compared
with control. The ratio of shoot/root in DK961 and
JN17 still remained higher in the treatments of
11 mM KNO3+100 mM NaCl and 16 mM KNO3+100 mM
NaCl than 6 mM KNO3+100 mM NaCl and 21 mM
KNO3+100 mM NaCl. Nevertheless, the soluble
sugar content in both shoot and root of two cvs
increased in treatment of 21 mM KNO3+100 mM
NaCl, with the shoot/root ratio being seriously
decreased.
Ions and pigments
Saline growth medium had a significant effect on
the concentrations of Na+ and K+ in the shoot and
the root of both cvs (Table 3). The K+ and Na+
contents measured in control (6 mM KNO3+0 mM
NaCl) plants were much lower in the shoot, higher
Please cite this article as: Zheng Y, et al. Potassium nitrate application alleviates sodium chloride stress in winter wheat cultivars
differing in salt tolerance. J Plant Physiol (2008), doi:10.1016/j.jplph.2008.01.001
ARTICLE IN PRESS
6
Y. Zheng et al.
in the root, of salt-tolerant DK961 than in saltsensitive JN17, with the K+/Na+ ratios being pretty
similar in both cvs. Salinity caused significant
decrease in K+ concentration, increase in Na+
concentration in shoot and root, with the K+/Na+
ratio being significantly reduced. The elevation of
KNO3 concentration in Hoagland nutrient solution
decreased Na+ accumulation in shoot of both salttolerant and salt-sensitive cvs, and increased K+
content as well as K+/Na+ ratio under NaCl stress.
Such effect was clearly noted in the cases of 11 and
16 mM KNO3 concentration in the nutrient solution
under 100 mM NaCl condition. However, the above
parameters in both cvs decreased in the treatment
of 21 mM KNO3+100 mM NaCl, although the salttolerant cv showed more stability under 100 mM
NaCl stress and recovered more rapidly than the
salt-sensitive one.
In control (6 mM KNO3+0 mM NaCl) plants, CHL
content and CHL/CAR ratio were lower, but the
CAR content was higher in DK961 than in JN17.
Excessive KNO3 concentration in either non-NaCl
conditions (11, 16, 21 mM) or nutrient solution with
100 mM NaCl (21 mM) caused evident decreases in
CHL and CHL/CAR ratio, and considerable increases
were measured in the CAR content for both cvs
(Figure 1). Proper elevation of KNO3 concentration
(11, 16 mM) in Hoagland solution with 100 mM NaCl
could alleviate the adverse effects caused by
individual salt stress; however, such an alleviation
declined when KNO3 concentration reached 21 mM
in both cvs. The CHL and CAR contents were higher
in DK961 than in JN17 under NaCl stress, with the
CHL/CAR ratio displaying similar tendency as the
CHL content.
KNO3+100 mM NaCl and 21 mM KNO3+100 mM NaCl,
respectively. The MDA content increased by 19%
and 40% in DK961 and JN17 in the treatment of
6 mM KNO3+100 mM NaCl in comparison with control. They reduced to 14% and 32%, 2% and 17%, 16%
and 34% in DK961 and JN17 in the treatments of
11 mM KNO3+100 mM NaCl, 16 mM KNO3+100 mM
NaCl and 21 mM KNO3+100 mM NaCl, respectively.
Antioxidant enzymes activities
Even in control (6 mM KNO3+0 mM NaCl) plants,
the activities of SOD, POD and CAT were significantly higher (21%, 7% and 47%) in salt-tolerant
DK961 than in salt-sensitive JN17. Salt stress
remarkably elevated the activities of those antioxidant enzymes (Figure 3). SOD activities in DK961
and JN17 increased by 22% and 57% in the
treatment of 6 mM KNO3+100 mM NaCl compared
with control. Those parameters increased by only
11% and 28%, 4% and 9%, 11% and 18%, respectively,
in DK961 and JN17 in the treatments of 11 mM
KNO3+100 mM NaCl, 16 mM KNO3+100 mM NaCl
and 21 mM KNO3+100 mM NaCl. POD activities in
DK961 and JN17 were 31% and 70% higher than
control in the treatment of 6 mM KNO3+100 mM
NaCl. Increments of only 11% and 37%, 7% and 12%,
12% and 17% were noted, respectively, in DK961 and
JN17 in the treatments of 11 mM KNO3+100 mM
NaCl , 16 mM KNO3+100 mM NaCl and 21 mM
KNO3+100 mM NaCl. Similar trends existed in CAT
activities of both cvs.
Discussion
Membrane permeability and lipid
peroxidation
It was noted that the EL in flag leaves and MDA
contents in shoot of control (6 mM KNO3+0 mM NaCl)
plants were lower (39% and 5%, respectively) in
salt-tolerant DK961 than in salt-sensitive JN17.
Excessive KNO3 concentration in either non-NaCl
(11, 16, 21 mM KNO3) or 100 mM NaCl (21 mM KNO3)
nutrient solution caused obvious increases in EL and
MDA content in both cvs (Figure 2). However, those
parameters declined in the combination of increased KNO3 (11, 16, 21 mM) and 100 mM NaCl
treatments. The EL increased 100% and 143% in
DK961 and JN17 in the treatment of 6 mM
KNO3+100 mM NaCl in comparison with the control.
Such figures decreased to 75% and 128%, 5% and
85%, 34% and 103% in DK961 and JN17 in the
treatments of 11 mM KNO3+100 mM NaCl, 16 mM
Salt-induced inhibition on plant growth could be
attributed to specific ion toxicity (Huang and
Redmann, 1995). Salinity stress caused a significant
increase in Na+ concentration, and a considerable
decrease in K+ concentration, resulting in drastic
decline in the K+/Na+ ratio (Table 3). The elevation
of KNO3 concentration in the saline nutrient
solution was proven to be effective in increasing
K+/Na+ ratio in shoot and root of winter wheat.
However, excessive K+/Na+ ratio was also harmful
to the growth of wheat plants. Salt-tolerant cv was
slightly affected by salt stress, and it could be
quickly recovered by appropriately increasing KNO3
concentration in saline nutrient solution that was
supplied to the plants. In contrast, growth parameters (plant height, root length, dry weight) of
the salt-sensitive cv decreased more drastically
than the salt-tolerant one under NaCl stress,
and those growth parameters were improved
Please cite this article as: Zheng Y, et al. Potassium nitrate application alleviates sodium chloride stress in winter wheat cultivars
differing in salt tolerance. J Plant Physiol (2008), doi:10.1016/j.jplph.2008.01.001
ARTICLE IN PRESS
Potassium nitrate alleviates sodium chloride stress in winter wheat
18
6
4
2
CHL content (% control, 0mMNaCl)
0
11
16
1.5
1.0
21
-20
-40
-60
DK961
JN17
-80
11
16
21
-20
-40
-60
DK961
JN17
-80
KNO3 concentration (mM)
CAR content (% control, 100mMNaCl)
CHL content (% control, 100mMNaCl)
6
80
60
40
20
DK961
JN17
100
80
60
40
20
6
11
16
21
KNO3 concentration (mM)
DK961
JN17
Cultivars
6
11
16
21
-10
-20
-30
-40
DK961
JN17
-50
11
21
16
6
KNO3 concentration (mM)
KNO3 concentration (mM)
120
0
2
0
DK961
JN17
100
KNO3 concentration (mM)
3
0
JN17
DK961
Cultivars
120
0
4
1
0.5
0.0
JN17
DK961
Cultivars
6
2.0
Control
5
CHL/CAR
8
2.5
CHL/CAR (% control, 0mMNaCl)
10
Control
CHL/CAR (% control, 100mMNaCl)
CAR content (mg g-1 DW)
12
CAR content (% control, 0mMNaCl)
CHL content (mg g-1 DW)
Control
14
0
6
3.0
16
0
7
0
6
11
16
21
-10
-20
-30
-40
DK961
JN17
-50
KNO3 concentration (mM)
Figure 1. Chlorophyll (CHL) and carotenoid (CAR) contents in shoots of salt-tolerant (DK961) and salt-sensitive wheat
(JN17). (A) Plants raised under control conditions (6 mM KNO3+0 mM NaCl), (B) plants raised under increased KNO3
concentrations (11, 16, 21 mM) in the absence (0 mM NaCl) and (C) presence of NaCl (100 mM NaCl). (B) and (C) show
changes of contents in % of control. Vertical bars indicate SE (n ¼ 6).
insufficiently by increasing KNO3 concentration,
even in the optimal K+/Na+ ratio treatment
(16 mM KNO3+100 mM NaCl) (Table 1). Therefore,
we concluded that moderately elevating KNO3
concentration in nutrient solution with NaCl could
be more effective in improving the growth of the
salt-tolerant winter wheat cvs than the saltsensitive one. It is possible that in the presence
Please cite this article as: Zheng Y, et al. Potassium nitrate application alleviates sodium chloride stress in winter wheat cultivars
differing in salt tolerance. J Plant Physiol (2008), doi:10.1016/j.jplph.2008.01.001
ARTICLE IN PRESS
MDA content (µmol g-1 FW, control)
Y. Zheng et al.
14
12
Control
10
8
6
4
2
0
DK961
JN17
Cultivars
EL (% control, 0mM NaCl)
180
160
MDA content (% control, 0mM NaCl)
200
DK961
JN17
140
120
100
80
60
40
20
0
6
11
16
21
KNO3 concentration (mM)
EL (% control, 100mM NaCl)
180
160
140
DK961
JN17
120
100
80
60
40
20
0
21
16
6
11
KNO3 concentration (mM)
MDA content (% control, 100mM NaCl)
Electrolyte leakage (%, control)
8
30
25
Control
20
15
10
5
0
DK961
JN17
Cultivars
50
40
DK961
JN17
30
20
10
0
6
16
21
11
KNO3 concentration (mM)
60
50
DK961
JN17
40
30
20
10
0
21
11
16
6
KNO3 concentration (mM)
Figure 2. Electrolyte leakages in flag leaves and malondialdehyde (MDA) contents in shoots of salt-tolerant (DK961) and
salt-sensitive wheat (JN17). (A) Plants raised under control conditions (6 mM KNO3+0 mM NaCl), (B) plants raised under
increased KNO3 concentrations (11, 16, 21 mM) in the absence (0 mM NaCl) and (C) presence of NaCl (100 mM NaCl). (B)
and (C) show changes of contents in % of control. Vertical bars indicate SE (n ¼ 6).
of high salt concentrations, the amount of naturally
occurring K+ may suppress plant growth (Chen
et al., 2005). Increasing KNO3 concentration in
nutrient solution with NaCl has improved the
possibility of K+ absorbance, and therefore relieves
the adverse saline effects. Sodium accumulation in
the shoot and the root was also noted to be
associated with salt tolerance; other features
include increased plant dry weight and WC under
saline condition.
Carbohydrate accumulation in plant has been
well known for osmotic adjustment under salt
Please cite this article as: Zheng Y, et al. Potassium nitrate application alleviates sodium chloride stress in winter wheat cultivars
differing in salt tolerance. J Plant Physiol (2008), doi:10.1016/j.jplph.2008.01.001
ARTICLE IN PRESS
120
100
80
60
40
20
SOD activity (% control, 100mMNaCl)
DK961
JN17
Cultivars
100
80
POD activity (% control, 0mM NaCl)
SOD activity (% control, 0mMNaCl)
0
DK961
JN17
60
40
20
0
6
11
16
21
KNO3 concentration (mM)
80
60
DK961
JN17
40
20
0
6
11
16
21
KNO3 concentration (mM)
160
Control
140
120
100
80
60
40
20
0
DK961
JN17
60
40
20
0
6
11
16
21
KNO3 concentration (mM)
80
DK961
JN17
60
40
20
0
6
11
16
21
KNO3 concentration (mM)
Control
10
8
6
4
2
0
JN17
DK961
Cultivars
100
80
CAT activity ( A240 g-1 min-1 FW)
140
12
180
CAT activity (% control, 0mM NaCl)
Control
POD activity (% control, 100mM NaCl)
SOD activity (U g-1 FW)
160
9
CAT activity (% control, 100mM NaCl)
180
POD activity ( A470 g-1 min-1 FW)
Potassium nitrate alleviates sodium chloride stress in winter wheat
JN17
DK961
Cultivars
100
80
DK961
JN17
60
40
20
0
6
11
16
21
KNO3 concentration (mM)
80
60
DK961
JN17
40
20
0
6
11
16
21
KNO3 concentration (mM)
Figure 3. Superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities in shoots of salt-tolerant (DK961)
and salt-sensitive wheat (JN17). (A) Plants raised under control conditions (6 mM KNO3+0 mM NaCl), (B) plants raised
under increased KNO3 concentrations (11, 16, 21 mM) in the absence (0 mM NaCl) and (C) presence of NaCl (100 mM
NaCl). (B) and (C) show changes of contents in % of control. Vertical bars indicate SE (n ¼ 6).
stress (Cheeseman, 1988). In our case, the soluble
sugar content in control (6 mM KNO3+0 mM NaCl)
plants was higher in salt-tolerant cv than saltsensitive one, indicating that the salt-tolerant cv
had higher osmotic adjustment ability than the
salt-sensitive one. Sodium chloride stress caused
significant increases in soluble sugar content of
shoot and root in both salt-tolerant (DK961) and
sensitive cvs (JN17) (Table 2). Suitable elevation of
KNO3 concentration (11, 16 mM) in saline nutrient
solution (100 mM NaCl) was found to be effective in
reducing soluble sugar content in both shoot and
Please cite this article as: Zheng Y, et al. Potassium nitrate application alleviates sodium chloride stress in winter wheat cultivars
differing in salt tolerance. J Plant Physiol (2008), doi:10.1016/j.jplph.2008.01.001
ARTICLE IN PRESS
10
root. However, the soluble sugar content increased
again in both cvs in the treatment of 21 mM
KNO3+100 mM NaCl, indicating a serious suppression
of either excessive K+ or Na+. Both excessive and
insufficient levels of the K+/Na+ ratio would inhibit
the growth of wheat. We concluded that the
optimal K+/Na+ ratio in plant-growing medium was
16:100 under the experimental conditions.
Cell membrane stability has been widely used to
differentiate stress-tolerant and susceptible cvs of
many crops, since the stable membrane stability is
closely correlated with the better field performance (Mansour et al., 1994). In the present study,
salinity stress caused considerable membrane
damage, either the lipid peroxidation, measured
as MDA equivalents, or the EL (Figure 2) was found
to be remarkably raised under increased levels of
KNO3 concentration (11, 16, 21 mM) or NaCl stress
(6 mM KNO3+100 mM NaCl). Suitable increase of
KNO3 concentration (11, 16 mM) in the nutrient
solution with 100 mM NaCl (plants grew on sand
material supplied with such solution) showed
alleviatory effects on both cvs. This was realized
by lowering the intensity of lipid peroxidation and
EL caused by salinity stress. However, excessive
KNO3 concentration in non-NaCl (11, 16, 21 mM
KNO3) or 100 mM NaCl (21 mM KNO3) nutrient
solution showed adverse effects to the stability of
cell membrane.
The effects of various environmental stresses on
plants were known to be mediated, at least
partially, by an enhanced generation of ROS (Able
et al., 2003). Plants with higher levels of antioxidants, either constitutive or induced, have been
reported to possess greater resistance to different
types of environmental stresses (Young and Jung,
1999). The general comparison of the examined
antioxidants in the salt-tolerant and salt-sensitive
cvs revealed that even in the control (6 mM
KNO3+0 mM NaCl) plants, the SOD, POD and CAT
activities were significantly higher in the salttolerant than the salt-sensitive cultivar (Figure 3).
Salinity led to an evident increase in those enzymes
activities in shoots of both cultivars. Although SOD,
POD and CAT activities elevated more considerably
in the salt-sensitive cv, they did not reach the high
levels of the salt-tolerant one, which resulted, at
least in part, from the high initial antioxidant
defense of salt-tolerant cvs. These results were in
good agreement with that obtained by Gossett
et al. (1994), who found higher constitutive and
induced levels of antioxidant enzyme activities in
more tolerant barley and sugar beet cvs under
drought and salt stresses.
In conclusion, suitable increment of KNO3 concentration in the plant-growing medium with NaCl
Y. Zheng et al.
could alleviate symptoms of the individual salt
stresses by improving growth of shoots and roots,
reducing EL, malondialdehyde and soluble sugar
contents and enhancing the activities of antioxidant enzymes in both salt-sensitive and tolerant
cvs. The optimal ratio of K+/Na+ in wheat-growing
medium was suggested to be 16:100 under the
experimental conditions.
Acknowledgments
Financial support by National Key Basic Research
Development Project (No. 2007CB106804), West
Proceeding Project of the Chinese Academy
of Science (No. KZCX2-XB2-01) and National
Science and Technology Support Project (No.
2006BAC01A12) is gratefully acknowledged. Thanks
are due to Professor Tao Wang, University of
Wisconsin-Madison, USA, for his kind constructive
advice on the language editing of the manuscript.
References
Able AJ, Sutherland MW, Guest DI. Production of reactive
oxygen species during non-specific elicitation, nonhost resistance and field resistance expression in
cultures of tobacco cells. Funct Plant Biol 2003;30:
91–9.
Aebi H. Invitro catalase. Methods Enzymol 1984;105:
121–6.
Ashraf M, O’Leary JW. Responses of newly developed salttolerant genotype of spring wheat to salt stress: yield
components and ion distribution. Agron Crop Sci
1996;176:91–101.
Carden DE, Walker DJ, Flowers TJ, Miller AJ. Single-cell
measurements of the contributions of cytosolic Na+
and K+ to salt tolerance. Plant Physiol 2003;131:
676–83.
Cheeseman JM. Mechanisms of salinity tolerance in
plants. Plant Physiol 1988;117:547–50.
Chen Z, Newman I, Zhou M, Mendham N, Zhang G,
Shabala S. Screening plants for salt tolerance by
measuring K+ flux: a case study for barley. Plant Cell
Environ 2005;28:1230–46.
Cherel L. Regulation of K+ channel activities in plants:
from physiological to molecular aspects. J Exp Bot
2004;55:337–51.
Cuin TA, Shabala S. Compatible solutes reduce ROSinduced potassium efflux in Arabidopsis roots. Plant
Cell Environ 2007;30:875–85.
Demidchik V, Shabala SN, Coutts KB. Free oxygen radicals
regulate plasma membrane Ca2+- and K+-permeable
channels in plant root cells. J Cell Sci 2003;116:
81–8.
Dionisio-Sese ML, Tobita S. Antioxidant responses of rice
seedlings to salinity stress. Plant Sci 1998;135:1–9.
Please cite this article as: Zheng Y, et al. Potassium nitrate application alleviates sodium chloride stress in winter wheat cultivars
differing in salt tolerance. J Plant Physiol (2008), doi:10.1016/j.jplph.2008.01.001
ARTICLE IN PRESS
Potassium nitrate alleviates sodium chloride stress in winter wheat
Giannopotitis CN, Ries SK. Superoxide dismutase in higher
plants. Plant Physiol 1977;59:309–14.
Gorham J, Bridges J, Dubcovsky J, Dvorak J, Hollington
PA, Luo MC, et al. Genetic analysis and physiology of a
trait for enhanced K+/Na+ discrimination in wheat.
New Phytol 1997;137:109–16.
Gossett DR, Millhollon EP, Lucas MC. Antioxidant response to NaCl stress in salt-tolerant and saltsensitive cultivars of cotton. Crop Sci 1994;34:
706–14.
Hiscox JD, Isrealstam GF. A method for the extraction of
chlorophyll from leaf tissue without maceration. Can J
Bot 1979;57:1332–4.
Huang J, Redmann RE. Salt tolerance of Hordeum and
Brassica species during germination and early seedling
growth. Can J Plant Sci 1995;75:815–9.
Kramer GF, Norman HA, Krizek DT, Mirecki RM. Influence
of UV-B radiation on polyamines, lipid peroxidation
11
and membrane lipids in cucumber. Phytochemistry
1991;30:2101–8.
Mansour MM, Van Hasselt PR, Kuiper PJ. Plasma membrane lipid alterations induced by NaCl in winter
wheat roots. Physiol Plant 1994;92:473–8.
Neid SL, Biesboer DD. Alleviation of salt-induced stress on
seed emergence using soil additives in a greenhouse.
Plant Soil 2005;268:303–7.
Shabala SN, Shabala L, Van Volkenburgh E. Effect of calcium
on root development and root ion fluxes in salinised
barley seedlings. Funct Plant Biol 2003;30:507–14.
Tester M, Davenport R. Na+ tolerance and Na+ transport in
higher plants. Ann Bot 2003;91:503–27.
Yemm EW, Willis AJ. The estimation of carbohydrates in
plant extracts by anthrone. Biochem J 1954:508–14.
Young CB, Jung J. Water-induced oxidative stress and
antioxidant defenses in rice plants. J Plant Physiol
1999;155:255–61.
Please cite this article as: Zheng Y, et al. Potassium nitrate application alleviates sodium chloride stress in winter wheat cultivars
differing in salt tolerance. J Plant Physiol (2008), doi:10.1016/j.jplph.2008.01.001