Potassium nitrate application alleviates sodium chloride stress in winter wheat cultivars differing in salt tolerance

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1 Journal of Plant Physiology ] (]]]]) ]]] ]]] Potassium nitrate application alleviates sodium chloride stress in winter wheat cultivars differing in salt tolerance Yanhai Zheng a,b, Aijun Jia c, Tangyuan Ning b, Jialin Xu d, Zengjia Li b,1, Gaoming Jiang b,a, a Key Laboratory of Vegetation and Environmental Change, Institute of Botany, the Chinese Academy of Sciences, 2 Nanxincun, Xiangshan, 193 Beijing, China b Key Laboratory of Crop Biology, Shandong Agricultural University, 1 Daizong Street, Taian, China c Dezhou Hengdong Pesticide & Chemical Co. Ltd. 18 Tianqu Industrial Park, Dezhou, China d Dezhou Virescence Institute of Salty & Alkaline Soil, 15 Tianqu East Road, 2531 Dezhou, China Received 27 July 27; received in revised form 7 January 28; accepted 1 January 28 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 KNO 3 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), (salt-tolerant) and (salt-sensitive), were planted in sandboxes and controls germinated and raised with Hoagland nutrient solution ( mm KNO 3, no NaCl). Experimental seeds were exposed to seven modified Hoagland solutions containing increased levels of KNO 3 (11, 1, 21 mm) or 1 mm NaCl in combination with the four KNO 3 concentrations (, 11, 1 and 21 mm). Plants were harvested 3 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 KNO 3 or NaCl stress. However, the combination of increased KNO 3 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 ); CHL, chlorophyll; cv(s), cultivar(s); EL, electrolyte leakage; MDA, malondialdehyde; POD, peroxidase (EC ); SOD, superoxide dismutase (EC ). Corresponding author at: Key Laboratory of Vegetation and Environmental Change, Institute of Botany, the Chinese Academy of Sciences, 2 Nanxincun, Xiangshan, 193 Beijing, China. Tel.: ; fax: address: jianggm@12.com (G. Jiang). 1 Also for correspondence /$ - see front matter & 28 Elsevier GmbH. All rights reserved. doi:1.11/j.jplph differing in salt tolerance. J Plant Physiol (28), doi:1.11/j.jplph

2 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 1:1 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 KNO 3 was more effective in the salt-tolerant than in the salt-sensitive cultivar. & 28 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., 25). 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, 199). 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, 24). To maintain normal cell metabolism, the K + content in wheat cells is kept around 15 mm and the Na + content at about 3 mm, resulting in a K + /Na + ratio of approximately 5 (Carden et al., 23). 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., 23). 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., 23; Cuin and Shabala, 27). Tester and Davenport (23) 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., 23). Previous studies revealed that supplying low levels of KNO 3 could alleviate the NaCl-induced decreases in seed germination of certain grass species (Neid and Biesboer, 25). However, none of these studies has focused on the differential responses of salt-tolerant and salt-sensitive crop cultivars (cvs) to increased levels of KNO 3 in the absence and presence of NaCl stress. Can increased levels of KNO 3 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 KNO 3 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, (salt-tolerant) and (salt-sensitive), were sown in sandboxes (22 1 5cm 3, length width height) in a greenhouse. Controls were irrigated with Hoagland nutrient solution ( mm KNO 3, no NaCl). Experimental seeds were exposed to seven modified Hoagland solutions containing increased levels of KNO 3 (11, 1, 21 mm) or 1 mm NaCl in combination with the four KNO 3 concentrations (, 11, 1 and 21 mm). Water lost by evapotranspiration was replenished each day. The average day/ night temperature was kept at 1 2 and 1 1 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 3 d after treatment. Plant growth, water and soluble sugar contents Growth parameters (plant height, root length and dry weight) were recorded 3 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 7 1C to a constant dry weight before the dry weights were recorded. Water differing in salt tolerance. J Plant Physiol (28), doi:1.11/j.jplph

3 Potassium nitrate alleviates sodium chloride stress in winter wheat 3 contents (WCs) of shoot and root were calculated by the follow formula: WCð%Þ ¼½ðfreshweight dryweightþ=fresh weightš 1. Soluble sugar content was measured following the method described by Yemm and Willis (1954). About.5 g ground dry shoots and roots were soaked in 7mL deionized water. The solution was boiled (1 1C) for 3 min to extract soluble sugar and centrifuged under 4 rpm for 1 min. The extracts were decanted and the residue was re-extracted for two more times, with extracts being completed to 5 ml. In all,.1 ml extracts and 3 ml anthrone reagent (.15 g anthrone+84 ml oil of vitriol+1 ml H 2 O) were mixed and the absorbance of the mixture was recorded at 2 nm. The content of soluble sugar was calculated from a standard curve of glucose at 2 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.5 g samples were soaked for 12 h in digesting tubes with 1 ml concentrated nitric acid and 3 ml perchlorate acid, and then digested at 3 1C for h. The extractions were completed to 5 ml with deionized water. The amount of K + and Na + contents was measured using an atomic absorption spectrophotometer (SP9-4, 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 (.5 g) were incubated into 5 ml dimethyl sulfoxide at 5 1C for 4 h. The absorbance of the supernatant was recorded at 45, 5 and 47 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 1 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 (EC 1 ) was analyzed using an electrical conductivity analyzer (KL-22, Xingzhou Company Ltd, China). The samples were autoclaved at 121 1C for 2 min to release all electrolytes, cooled to 25 1C, and then the final electrical conductivity (EC 2 ) was measured. The EL was calculated using the formula: EL ¼ EC 1 /EC 2 1. Lipid peroxidation was determined by estimating the malondialdehyde (MDA) content according to Kramer et al. (1991). Frozen samples (.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 1,g for 2 min at 4 1C, with the supernatant being used for MDA determination. A mixture of 1 ml extracts (MDA)+2 ml.% thiobarbituric acid (TBA) (. g TBA+1 M NaOH+1% trichloroacetic acid complete to 1 ml) was produced, boiled for 15 min, cooled and centrifuged for 1 min (4 rpm). The concentration of MDA was calculated from the absorbance at, 532 and 45 nm, and MDA contents were determined through the following formula: MDAðmmol g 1 FWÞ ¼ð:45 ðd 532 D Þ :5D 45 ÞV=W, where D 532, D and D 45 are the absorbance at, 532 and 45 nm, respectively, and V is the volume of extraction, W is the fresh weight of sample. Frozen samples (.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 5 mm sodium phosphate (ph.), 28 ml guaiacol and 19 ml 3% H 2 O 2. The absorbance was recorded five times at 47 nm at 3 s intervals. Variation of absorbance per minute per gram fresh weight (DA 47 g 1 min 1 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 5%. Catalase (CAT) activity was determined following the method described by Aebi (1984). Unit of CAT activity (DA 24 g 1 min 1 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-35, SHIMADZU, Japan). Statistic analysis The experiment was designed as a randomized block consisting of eight treatments containing four increased levels of KNO 3 or 1 mm NaCl in combination with the four KNO 3 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 KNO 3 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 pp.5. differing in salt tolerance. J Plant Physiol (28), doi:1.11/j.jplph

4 4 Results Growth parameters Plant height, root length and dry weight of control ( mm KNO 3 + mm NaCl) plants were measured to be considerably higher in the salt-tolerant () than sensitive wheat (). Those parameters displayed significant reductions under increased levels of KNO 3 (11, 1, 21 mm) or 1 mm NaCl ( mm KNO 3 +1 mm NaCl) treatments for both cvs. However, the combination of increased KNO 3 (11, 1, 21 mm) and NaCl (1 mm NaCl) alleviated symptoms of the individual salt stresses (Table 1). Plant height, root length and dry weight of showed 21%, 1% and 29% reductions in the treatment of mm KNO 3 +1 mm NaCl in comparison with the control. Such reductions decreased to 15%, 11% and 19% in the treatment of 11 mm KNO 3 +1 mm NaCl, and even only 4%, 3% and % in the treatment of 1 mm KNO 3 +1 mm NaCl treatment. In contrast, those parameters in displayed 41%, 43% and 32% reductions in mm KNO 3 +1 mm NaCl treatment against the control, while 24%, 28% and 25% reductions in the treatment of 11 mm KNO 3 +1 mm NaCl, and only 15%, 15% and 7% reductions were measured in the treatment of 1 mm KNO 3 +1 mm NaCl. However, those parameters reduced more considerably in both cvs in the treatment of 21 mm KNO 3 +1 mm NaCl than in the treatment of 1 mm KNO 3 +1 mm NaCl (Table 1). Conversely, those parameters in never reached as high as. Water content WC was higher in the salt-tolerant () than the salt-sensitive cv () in control ( mm KNO 3 + mm NaCl) plants (Table 1). They reduced drastically in increased levels of KNO 3 (11, 1, 21 mm) or 1 mm NaCl ( mm KNO 3 +1 mm NaCl) treatments in both cvs. Properly increased KNO 3 concentrations (11, 1 mm) alleviated those adverse effects under 1 mm NaCl stress, and they elevated more rapidly in than. Nevertheless, excessive KNO 3 concentration (21 mm) was harmful for plant growth under 1 mm NaCl stress. Soluble sugar Y. Zheng et al. The soluble sugar contents in shoot and root of control ( mm KNO 3 + mm NaCl) plants were drastically higher (2% and 9%, respectively) in than in. They had significantly elevated in both increased levels of KNO 3 (11, 1, 21 mm) and 1 mm NaCl ( mm KNO 3 +1 mm NaCl) treatments (Table 2). However, properly increased KNO 3 concentration (11, 1 mm) in Hoagland solution with 1 mm NaCl could diminish the damage caused by salinity. Soluble sugar content in shoot of salttolerant was not significantly different from the control plants when the KNO 3 concentration Table 1. Plant height, dry weight (DW) and water content (WC) in shoot and root of salt-tolerant and saltsensitive in four levels of KNO 3 (, 11, 1 and 21 mm) treatments or 1 mm NaCl in combination with the four KNO 3 concentrations Treatment Shoot Root Variety NaCl KNO 3 Plant height (cm) DW (g) WC (%) Root length (cm) DW (g) WC (%) (CK) a.317.3a a a.197.1a a a.37.33a a a.27.2a a b.27.3b b b.17.9b b c c c c.127.7c c c.227.7c c c c c b.257.4b b b.157.2b b a.297.2a.7.8a a.197.1a a c.227.8c b d.127.3c c (CK) a.287.2a a a.247.1a a b b a a a a c c b b b b d d c c.17.31c c c.197.4c c c.177.2b c b.217.3b a.47.35b.27.1b b a.27.2a a a.247.1a a c.27.c b c.27.2b d Data are the mean7se (n ¼ ). Different letters within a column indicate significant differences (Po.5, t test). CK, control, Hoagland nutrient solution containing mm KNO 3 and mm NaCl. differing in salt tolerance. J Plant Physiol (28), doi:1.11/j.jplph

5 Potassium nitrate alleviates sodium chloride stress in winter wheat 5 Table 2. Soluble sugar contents in shoot and root of salt-tolerant and salt-sensitive in four levels of KNO 3 (, 11, 1 and 21 mm) treatments or 1 mm NaCl in combination with the four KNO 3 concentrations Treatment NaCl KNO 3 Soluble sugar content in shoot (mg g 1 DW) Soluble sugar content in root (mg g 1 DW) Shoot/root ratio Soluble sugar content in shoot (mg g 1 DW) Soluble sugar content in root (mg g 1 DW) Shoot/root ratio (CK) c b b d c b c b b c b b b b a b b a a a a a a a a a b a a a b c a b b a d d a d b b c b b c c a Data are the mean7se (n ¼ ). Different letters within a column indicate significant differences (Po.5, t test). CK, control, Hoagland nutrient solution containing mm KNO 3 and mm NaCl. Table 3. Potassium and sodium concentrations in shoot and root of salt-tolerant and salt-sensitive in four levels of KNO 3 (, 11, 1 and 21 mm) treatments or 1 mm NaCl in combination with the four KNO 3 concentrations Treatment Shoot Root Variety NaCl KNO 3 K + content (mg g 1 ) Na + content (mg g 1 ) K + /Na + K + content (mg g 1 ) Na + content (mg g 1 ) K + /Na + (CK) b d a b b b a b a c b c a b a b b b c c b a c a c a c d a.597.3d a b a c b c a b a b b b c.77.31c b a c a (CK) b d a c c b c b c d b c a c b b d a d e c a d a e a.27.5d e a.77.1d c b c d b.97.12c a c b b d a d e c a d a Data are the mean7se (n ¼ ). Different letters within a column indicate significant differences (Po.5, t test). CK, control, Hoagland nutrient solution containing mm KNO 3 and mm NaCl. reached 1 mm under 1 mm NaCl stress. In contrast, they increased significantly in salt-sensitive in the combinations of four levels of KNO 3 and 1 mm NaCl treatments compared with control. The ratio of shoot/root in and still remained higher in the treatments of 11 mm KNO 3 +1 mm NaCl and 1 mm KNO 3 +1 mm NaCl than mm KNO 3 +1 mm NaCl and 21 mm KNO 3 +1 mm NaCl. Nevertheless, the soluble sugar content in both shoot and root of two cvs increased in treatment of 21 mm KNO 3 +1 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 ( mm KNO 3 + mm NaCl) plants were much lower in the shoot, higher differing in salt tolerance. J Plant Physiol (28), doi:1.11/j.jplph

6 in the root, of salt-tolerant than in saltsensitive, 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 KNO 3 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 1 mm KNO 3 concentration in the nutrient solution under 1 mm NaCl condition. However, the above parameters in both cvs decreased in the treatment of 21 mm KNO 3 +1 mm NaCl, although the salttolerant cv showed more stability under 1 mm NaCl stress and recovered more rapidly than the salt-sensitive one. In control ( mm KNO 3 + mm NaCl) plants, CHL content and CHL/CAR ratio were lower, but the CAR content was higher in than in. Excessive KNO 3 concentration in either non-nacl conditions (11, 1, 21 mm) or nutrient solution with 1 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 KNO 3 concentration (11, 1 mm) in Hoagland solution with 1 mm NaCl could alleviate the adverse effects caused by individual salt stress; however, such an alleviation declined when KNO 3 concentration reached 21 mm in both cvs. The CHL and CAR contents were higher in than in under NaCl stress, with the CHL/CAR ratio displaying similar tendency as the CHL content. Membrane permeability and lipid peroxidation It was noted that the EL in flag leaves and MDA contents in shoot of control ( mm KNO 3 + mm NaCl) plants were lower (39% and 5%, respectively) in salt-tolerant than in salt-sensitive. Excessive KNO 3 concentration in either non-nacl (11, 1, 21 mm KNO 3 ) or 1 mm NaCl (21 mm KNO 3 ) nutrient solution caused obvious increases in EL and MDA content in both cvs (Figure 2). However, those parameters declined in the combination of increased KNO 3 (11, 1, 21 mm) and 1 mm NaCl treatments. The EL increased 1% and 143% in and in the treatment of mm KNO 3 +1 mm NaCl in comparison with the control. Such figures decreased to 75% and 128%, 5% and 85%, 34% and 13% in and in the treatments of 11 mm KNO 3 +1 mm NaCl, 1 mm KNO 3 +1 mm NaCl and 21 mm KNO 3 +1 mm NaCl, respectively. The MDA content increased by 19% and 4% in and in the treatment of mm KNO 3 +1 mm NaCl in comparison with control. They reduced to 14% and 32%, 2% and 17%, 1% and 34% in and in the treatments of 11 mm KNO 3 +1 mm NaCl, 1 mm KNO 3 +1 mm NaCl and 21 mm KNO 3 +1 mm NaCl, respectively. Antioxidant enzymes activities Even in control ( mm KNO 3 + mm NaCl) plants, the activities of SOD, POD and CAT were significantly higher (21%, 7% and 47%) in salt-tolerant than in salt-sensitive. Salt stress remarkably elevated the activities of those antioxidant enzymes (Figure 3). SOD activities in and increased by 22% and 57% in the treatment of mm KNO 3 +1 mm NaCl compared with control. Those parameters increased by only 11% and 28%, 4% and 9%, 11% and 18%, respectively, in and in the treatments of 11 mm KNO 3 +1 mm NaCl, 1 mm KNO 3 +1 mm NaCl and 21 mm KNO 3 +1 mm NaCl. POD activities in and were 31% and 7% higher than control in the treatment of mm KNO 3 +1 mm NaCl. Increments of only 11% and 37%, 7% and 12%, 12% and 17% were noted, respectively, in and in the treatments of 11 mm KNO 3 +1 mm NaCl, 1 mm KNO 3 +1 mm NaCl and 21 mm KNO 3 +1 mm NaCl. Similar trends existed in CAT activities of both cvs. Discussion Y. Zheng et al. 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 KNO 3 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 KNO 3 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 differing in salt tolerance. J Plant Physiol (28), doi:1.11/j.jplph

7 Potassium nitrate alleviates sodium chloride stress in winter wheat 7 CHL content (mg g -1 DW) CHL content (% control, mmnacl) Control Control Control CAR content (mg g -1 DW) CAR content (% control, mmnacl) CHL/CAR CHL/CAR (% control, mmnacl) CHL content (% control, 1mMNaCl) CAR content (% control, 1mMNaCl) CHL/CAR (% control, 1mMNaCl) Figure 1. Chlorophyll (CHL) and carotenoid (CAR) contents in shoots of salt-tolerant () and salt-sensitive wheat (). (A) Plants raised under control conditions ( mm KNO 3 + mm NaCl), (B) plants raised under increased KNO 3 concentrations (11, 1, 21 mm) in the absence ( mm NaCl) and (C) presence of NaCl (1 mm NaCl). (B) and (C) show changes of contents in % of control. Vertical bars indicate SE (n ¼ ). insufficiently by increasing KNO 3 concentration, even in the optimal K + /Na + ratio treatment (1 mm KNO 3 +1 mm NaCl) (Table 1). Therefore, we concluded that moderately elevating KNO 3 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 differing in salt tolerance. J Plant Physiol (28), doi:1.11/j.jplph

8 8 Y. Zheng et al. Electrolyte leakage (%, control) EL (% control, mm NaCl) Control MDA content (µmol g -1 FW, control) MDA content (% control, mm NaCl) Control EL (% control, 1mM NaCl) MDA content (% control, 1mM NaCl) Figure 2. Electrolyte leakages in flag leaves and malondialdehyde (MDA) contents in shoots of salt-tolerant () and salt-sensitive wheat (). (A) Plants raised under control conditions ( mm KNO 3 + mm NaCl), (B) plants raised under increased KNO 3 concentrations (11, 1, 21 mm) in the absence ( mm NaCl) and (C) presence of NaCl (1 mm NaCl). (B) and (C) show changes of contents in % of control. Vertical bars indicate SE (n ¼ ). of high salt concentrations, the amount of naturally occurring K + may suppress plant growth (Chen et al., 25). Increasing KNO 3 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 differing in salt tolerance. J Plant Physiol (28), doi:1.11/j.jplph

9 Potassium nitrate alleviates sodium chloride stress in winter wheat 9 SOD activity (% control, mmnacl) SOD activity (U g -1 FW) POD activity ( A 47 g -1 min -1 FW) 18 Control 1 Control 1 Control POD activity (% control, mm NaCl) CAT activity ( A 24 g -1 min -1 FW) CAT activity (% control, mm NaCl) SOD activity (% control, 1mMNaCl) POD activity (% control, 1mM NaCl) CAT activity (% control, 1mM NaCl) Figure 3. Superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities in shoots of salt-tolerant () and salt-sensitive wheat (). (A) Plants raised under control conditions ( mm KNO 3 + mm NaCl), (B) plants raised under increased KNO 3 concentrations (11, 1, 21 mm) in the absence ( mm NaCl) and (C) presence of NaCl (1 mm NaCl). (B) and (C) show changes of contents in % of control. Vertical bars indicate SE (n ¼ ). stress (Cheeseman, 1988). In our case, the soluble sugar content in control ( mm KNO 3 + 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 () and sensitive cvs () (Table 2). Suitable elevation of KNO 3 concentration (11, 1 mm) in saline nutrient solution (1 mm NaCl) was found to be effective in reducing soluble sugar content in both shoot and differing in salt tolerance. J Plant Physiol (28), doi:1.11/j.jplph

10 1 root. However, the soluble sugar content increased again in both cvs in the treatment of 21 mm KNO 3 +1 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 1:1 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 KNO 3 concentration (11, 1, 21 mm) or NaCl stress ( mm KNO 3 +1 mm NaCl). Suitable increase of KNO 3 concentration (11, 1 mm) in the nutrient solution with 1 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 KNO 3 concentration in non-nacl (11, 1, 21 mm KNO 3 ) or 1 mm NaCl (21 mm KNO 3 ) 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., 23). 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 ( mm KNO 3 + 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 KNO 3 concentration in the plant-growing medium with NaCl 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 1:1 under the experimental conditions. Acknowledgments Financial support by National Key Basic Research Development Project (No. 27CB184), West Proceeding Project of the Chinese Academy of Science (No. KZCX2-XB2-1) and National Science and Technology Support Project (No. 2BAC1A12) 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 Y. Zheng et al. 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 23;3: Aebi H. Invitro catalase. Methods Enzymol 1984;15: 121. 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 199;17: Carden DE, Walker DJ, Flowers TJ, Miller AJ. Single-cell measurements of the contributions of cytosolic Na + and K + to salt tolerance. Plant Physiol 23;131: Cheeseman JM. Mechanisms of salinity tolerance in plants. Plant Physiol 1988;117: 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 25;28: Cherel L. Regulation of K+ channel activities in plants: from physiological to molecular aspects. J Exp Bot 24;55: Cuin TA, Shabala S. Compatible solutes reduce ROSinduced potassium efflux in Arabidopsis roots. Plant Cell Environ 27;3: Demidchik V, Shabala SN, Coutts KB. Free oxygen radicals regulate plasma membrane Ca 2+ - and K + -permeable channels in plant root cells. J Cell Sci 23;11: Dionisio-Sese ML, Tobita S. Antioxidant responses of rice seedlings to salinity stress. Plant Sci 1998;135:1 9. differing in salt tolerance. J Plant Physiol (28), doi:1.11/j.jplph

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