Potato Responds to Salt Stress by Increased Activity of Antioxidant Enzymes 1

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Running title: Antioxidant activity of potato under salt stress 3

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Keyvan Aghaei1, 2, Ali Akber Ehsanpour2 and Setsuko Komatsu1* 5

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1 National Institute of Crop Science, Tsukuba 305-8518, Japan 7

2 Department of Biology, Faculty of Sciences, University of Isfahan, Isfahan, Iran 8

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* Author for correspondence: Setsuko Komatsu, National Institute of Crop Science, 10

Kannondai 2-1-18, Tsukuba 305-8518, Japan. Tel.: +81 29 838 7142; Fax: +81 29 838 11

7142. E-mail: skomatsu@affrc.go.jp 12

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Present address of Keyvan Aghaei : Department of Biology, University of Zanjan, 14

Zanjan, Iran 15

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Abbreviations: APX - ascorbate peroxidase; CAT – catalase; GPR – glutathione 17

peroxidase; GR - glutathione reductase; ROS - reactive oxygen species; SOD - super 18

oxide dismutase. 19

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Abstract 27

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To understand the response of potato to salt stress, antioxidant enzyme activities and ion 29

content were analyzed for a sensitive and a tolerant cultivar. Nodal cuttings of the tolerant 30

cultivar, Kennebec, and the sensitive cultivar, Concord, were exposed to media without or 31

with 30, 60, 90, or 120 mM NaCl for 4 weeks. On exposure to NaCl, the length and fresh 32

and dry weight of both shoots and roots of Concord showed greater decreases than those of 33

Kennebec. The decrease in shoot growth was more severe than that of the root for both 34

cultivars. The K+ content of shoots and roots of both cultivars was reduced in a dose-35

dependent manner by exposure to NaCl; the Na+ content increased. Activities of ascorbate 36

peroxidase, catalase, and glutathione reductase were increased in NaCl-exposed shoots of 37

Kennebec; the corresponding activities in NaCl-exposed shoots of Concord were decreased. 38

Roots of both cultivars showed similar changes in the activities of these enzymes on 39

exposure to NaCl. These studies established that enzyme activities in Concord shoots are 40

inversely related to the NaCl concentration, whereas those in Kennebec do not show a dose 41

dependency, which is also the case for the roots of both cultivars. Our findings suggest that 42

an increase in activity of antioxidant enzymes, such as ascorbate peroxidase, catalase, and 43

glutathione reductase, can contribute to salt tolerance in Kennebec, a salt resistant cultivar 44

of potato. 45

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High concentrations of salt in soil are causing large decreases in yields for a wide 53

variety of crops all over the world (Sekmen et al. 2007). Salt stress results in 54

alterations in plant metabolism, including a reduced water potential, ion imbalances 55

and toxicity, and reduced levels of CO2 assimilation (Bohnert and Jensen 1996). 56

Although a wide range of genetic adaptations to saline conditions has been observed 57

and a number of significant physiological responses have been associated with 58

tolerance, underlying mechanisms of salt tolerance in plants are still poorly understood. 59

The effects of various environmental stresses in plants are known to be mediated, 60

at least in part, by an enhanced generation of reactive oxygen species (ROS) 61

including .O2, H2O2, and .OH (Hernandaz et al. 2000, Benevides et al. 2000). These 62

ROS are highly reactive and can alter normal cellular metabolism through oxidative 63

damage to membranes, proteins, and nucleic acids; they also cause lipid peroxidation, 64

protein denaturation, and DNA mutation (Imalay 2003). 65

To prevent damage to cellular components by ROS, plants have developed a 66

complex antioxidant system. The primary components of this system include 67

carotenoids, ascorbate, glutathione, and tocopherols, in addition to enzymes such as 68

superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), 69

peroxidases, and the enzymes involved in ascorbate–glutathione cycle (Foyer and 70

Halliwell 1976), such as ascorbate peroxidase (APX) and glutathione reductase (GR). 71

Many components of this antioxidant defense system can be found in various 72

subcellular compartments (Hernandez et al. 2000). 73

The scavenging of ROS by increased activation of antioxidant enzymes can 74

improve salt tolerance (Alscher et al. 2002). A relationship between salt tolerance and 75

increased activation of antioxidant enzymes has been demonstrated in Plantago 76

(Sekmen et al. 2007), pea (Hernandaz et al. 2000), Arabidopsis, rice (Dionisio-Sese 77

and Tobita 2007), tomato, soybean, and maize (Azevedo-Neto et al. 2006). The potato 78

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plants also respond to other stresses, including heat (Tang et al. 2006) and frost 79

(Martinez et al. 1996), by activation of their antioxidant enzymes. However, there is 80

insufficient information available on the effect of salt stress on this defense system of 81

potato cultivars (Benevides et al. 2000, Tang et al. 2006). Furthermore, there is a 82

significant difference in salt tolerance among potato cultivars (Martinez et al. 1996). 83

Potato has been classified as a moderately salt-sensitive crop (Martinez et al. 84

1996). However, variations in salt sensitivity among various cultivars of potato have 85

been observed (Ochat et al. 1999). The salt tolerance of a few potato cultivars has 86

been evaluated under field and greenhouse conditions (Potluri and Devi Prasad 1994). 87

In vitro evaluation of salt stress is an alternative to costly, labor-intensive, and 88

occasionally problematic field-based evaluations. The most important advantage of in 89

vitro cultures is their rapid regeneration in a controlled environment after exposure to 90

stress (Potluri and Devi Prasad 1994). In this study, we used potato cultivars 91

exhibiting differences in their level of salt tolerance to investigate the effects of salt 92

stress on potato. Physiological parameters, Na+ and K+ contents, and antioxidant 93

enzyme activities were analyzed in shoots and roots of two cultivars: the salt-tolerant 94

cultivar Kennebec and the salt-sensitive cultivar Concord. A comparison of the 95

responses of these cultivars to salt stress may be useful in identifying the mechanisms 96

of salt tolerance in potato. 97

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Results 105

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Effects of salt stress on growth of potato plants 107

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To evaluate the effects of salt stress on the growth of potato, the physiological 109

differences between two potato cultivars grown under saline conditions were 110

determined. The lengths of shoots and roots in Kennebec and Concord cultivars were 111

measured 4 weeks after exposure to 0, 30, 60, 90, or 120 mM NaCl (Fig. 1). NaCl 112

treatment adversely affected the shoot length of both cultivars, and this negative effect 113

was more severe in Concord than in Kennebec. Concord cultivar would not grow after 114

treatment with 120 mM NaCl, whereas this treatment was not lethal to Kennebec (Fig. 115

1A). The shoot length of Kennebec was decreased by 52% and 76% at 90 and 120 mM 116

NaCl, respectively. In Concord, the shoot length decreased by 55% at 90 mM NaCl, 117

and no growth was observed at 120 mM NaCl. The root length of Kennebec was 118

decreased by 40% at 120 mM NaCl. The roots of Concord showed an adverse effect of 119

NaCl at 60 mM NaCl, which decreased the root length by 34%; this decrease 120

continued at 90 mM NaCl, and no root was formed at 120 mM NaCl (Fig. 1B). 121

Four weeks after NaCl treatment, the fresh (Fig. 2A) and dry weights (Fig. 2B) of 122

shoots and roots were measured in the two potato cultivars. Treatment by 60 mM 123

NaCl decreased by 53% fresh and dry weights of the shoot of Kennebec. This adverse 124

effect was more severe in Concord than in Kennebec at 90 mM NaCl, where the root 125

length decreased by 85% in Concord and by 31% in Kennebec. On the basis of the 126

fresh and dry weights of the root, a negative effect of NaCl treatment began at 90 mM 127

for Kennebec with 45% and 30% decreases in the fresh and dry weights, respectively. 128

In Concord, the loss in the fresh and dry weights of the roots started at 60 mM, where 129

reductions of 43% and 40%, respectively, were observed. Root fresh and dry weights 130

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were also less at 90 mM NaCl in Concord, where a decrease of 91% in both weights 131

occurred. The treatment at 120 mM NaCl prevented the root growth in Concord and 132

elicited considerable losses of root weight in Kennebec with decreases of 90% and 133

85% in the fresh and dry weights, respectively. 134

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Effect of salt stress on K+ and Na+ contents 136

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To investigate the effect of salt stress on the K+ and Na+ contents in potato, the 138

concentrations of these ions were measured in shoots (Fig. 3A) and roots (Fig. 3B) of 139

Kennebec and Concord at 0, 30, 60, 90, and 120 mM NaCl after 4 weeks. At 90 mM 140

NaCl concentration, the Na+ content of the shoots of both Kennebec and Concord 141

cultivars increased by 62% (Fig. 3A), whereas Na+ content in the roots of Kennebec 142

and Concord increased by 45% and 64%, respectively (Fig. 3B). The K+ content of the 143

shoots of both cultivars decreased at 60 mM NaCl by 30% and 120 mM NaCl by 63% 144

(Fig. 3A). The two cultivars showed different responses with respect to the K+ content 145

of their roots under NaCl treatment. At 60 mM NaCl, the K+ content of Kennebec root 146

decreased by 35%. This pattern did not change at 90 mM NaCl, and a 72% decrease 147

was observed at 120 mM NaCl (Fig. 3B). In roots of Concord, the K+ content started 148

to decrease at 30 mM NaCl, where a 25% reduction was observed, and this trend 149

continued at 90 mM NaCl, where a 55% decrease occurred (Fig. 3B). The K+ and Na+ 150

contents were not measured at 120 mM NaCl in Concord because the cultivar did not 151

grow at this NaCl concentration. 152

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Effect of salt stress on antioxidant enzyme activities 154

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To evaluate the mechanisms of salt tolerance in potato cultivars, the activities of 156

antioxidant enzymes were measured. In Kennebec, the APX activity, compared with 157

that of untreated plants, increased by 58% on treatment with 60 mM NaCl; this figure 158

remained unchanged at 90 mM NaCl (Fig. 4A). Although the activity of APX was 159

similar in untreated plants of both cultivars, it decreased by 53% and 71% in shoots of 160

Concord at 60 and 90 mM NaCl, respectively. The NaCl dose dependency of APX 161

activity was similar in roots of both potato cultivars. After treatment with 60 mM 162

NaCl, the APX activity in the roots increased three-fold in Kennebec and by 55% in 163

Concord, whereas at 90 mM NaCl, it decreased by 12% and 45% in Kennebec and 164

Concord, respectively (Fig. 4B). The APX activity in Kennebec was higher than that 165

in Concord at 60 mM and 90 mM NaCl. 166

In comparison with untreated plants, the CAT activity in shoots of Kennebec 167

increased by 21% at 60 mM NaCl, but it decreased by 35% at 90 mM NaCl. The CAT 168

activity was decreased in shoots of Concord by 54% and 82% at 60 and 90 mM NaCl, 169

respectively (Fig. 4A). The NaCl dose dependency of the CAT activity in roots was 170

similar in both cultivars. In 60 mM NaCl, the CAT activity increased twofold in 171

Kennebec and by 1.7-fold in Concord; at 90 mM NaCl, the CAT activity in both 172

cultivars decreased (Fig. 4B). 173

The GR activity in shoot of Kennebec increased by 55% and 76% at 60 and 90 174

mM NaCl, respectively, compared with that of untreated plants. However, in shoot of 175

Concord, the GR activity showed a marked decrease on NaCl treatment: this decrease 176

reached 73% at 90 mM NaCl (Fig. 4A). Both cultivars showed similar patterns in 177

terms of the dose-dependent GR activity in their roots in response to NaCl treatments 178

(Fig. 4B). The GR activity in root of Kennebec increased threefold on treatment with 179

60 mM NaCl but showed a decrease at 90 mM. In Concord, the activity increased 1.5-180

fold at 60 mM NaCl and then decreased at 90 mM NaCl (Fig. 4B). 181

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Discussion 182

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Although potato is a commercially important crop, it cannot be grown satisfactorily in 184

arid or semi-arid areas of the world where salt stress is a major problem. Little is 185

known about the salt tolerance or the physiological consequences of salt stress in 186

potato (Backhosen et al. 2005). In field experiments, it has been shown that potato 187

plants lose chlorophyll and protein, and accumulate proline when subjected to salt 188

stress (Heure and Nadler 1998). Concentrations of NaCl above 50 mM were sufficient 189

to cause growth restrictions and decrease tuber yield in various field-grown potato 190

cultivars (Backhosen et al. 2005). These findings indicated that potato plants display 191

the same salt sensitivity as other glycophytic crops. However, all these results come 192

from field experiments in which environmental factors cannot be satisfactorily 193

controlled. 194

Salt stress severely decreased the growth of both the cultivars that we examined, 195

but the adverse effects of salt were much more severe in Concord than in Kennebec in 196

terms of all the physiological characteristics that we measured (Figs. 1 and 2). This 197

indicates that Kennebec is a relatively salt-tolerant cultivar compared with Concord, 198

which is very sensitive to salt. This is in agreement with the findings of Jefferies 199

(1996), who proposed that genetic variations in salt tolerance exist in potato cultivars. 200

The reduction in growth parameters under salt stress in both potato cultivars in our 201

experiments confirmed previous studies that potato was a relatively salt-sensitive crop 202

(Backhosen et al. 2005). In a manner consistent with our results, salt-sensitive 203

cultivars of pea (Hernandaz et al. 2000) and lentil (Bandeoglu et al. 2005) have shown 204

considerably the reduced growth compared with tolerant cultivars under NaCl 205

treatment. It has also been reported that under salt stress, relatively salt-tolerant potato 206

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cultivars accumulated more shoot fresh and dry weights than do salt-sensitive cultivars 207

(Rahnama and Ebrahimzadeh 2004). 208

In this study, Na+ and K+ contents were measured to determine whether the salt-209

induced injuries to potato cultivars are a consequence of Na+ toxicity and K+ 210

deficiency. Increase of Na+ content and decreases of K+ content under salt stress, as 211

identified in this study, have previously been reported for rice (Dionisio and Tobita 212

2007). The K+ content in control plants was, as expected, much higher than the Na+ 213

content in both shoots and roots of the two cultivars, and was markedly reduced after 214

NaCl treatment (Fig. 3). It has been proposed that Na+ competes with K+ for 215

intracellular influx because these cations are transported by the same proteins 216

(Hasegawa et al. 2000). K+ plays a key role in a wide range of physiological processes, 217

such as protein and starch synthesis, enzyme activation, ATP synthesis, osmotic 218

adjustment, and transport of sugars (Rahnama and Ebrahimzadeh 2004). 219

These results suggest that increased levels of Na+ result in decreased levels of K+ 220

in shoots and roots, leading to damaging effects of NaCl in potato. Although 221

halophytes can actively control their uptake of Na+ and Cl–, salt-sensitive plants such 222

as potato or rice cannot control the influx of these ions (Flowers and Yeo 1986). In our 223

experiments, both tolerant and sensitive cultivars of potato showed increased levels of 224

Na+ in shoots and roots in response to salt stress. These results are in contrast with 225

those of Flowers and Yeo (1986), who proposed that there is an inverse relationship 226

between shoot Na+ content and salt tolerance. However, in a manner consistent with 227

our results, Dionosio-Sese and Tobita (2007) have shown that Na+ accumulation in a 228

tolerant variety of rice under salinity stress is similar to that in a salt-sensitive variety. 229

Furthermore NaCl induced a rapid reduction of K+ in roots than in shoots (Fig. 3), 230

suggesting that K+ in roots might be replaced by Na+ and then transported to shoot. 231

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CAT and APX regulate H2O2 levels in plants. The results of our study show that 232

CAT and APX activities in shoots of Kennebec increased under salt stress. Although 233

APX activity decreased at high level of salt, they remained as high as those present in 234

untreated plants. In contrast, the activities of CAT and APX decreased markedly in 235

Concord subjected to salt stress. These results suggest that CAT and APX can play a 236

pivotal role in scavenging H2O2 in potato plants under salt stress. The better growth of 237

Kennebec at high salt concentrations can be explained, at least in part, by its higher 238

activity of CAT and APX (Fig. 4). Similar finding for CAT and APX activities 239

induced in salt-tolerant tomato, sugar beet, rice, and Plantago which are similar to our 240

results have also been reported (Sekmen et al. 2007). 241

GR, one of the important enzymes in ascorbate-glutathion cycle, catalyzes the 242

NADPH- dependent reduction of oxidized glutathione and is important in protecting 243

many plants from oxidative stress caused by salt stress (Foyer et al. 1991). Although 244

salt stress markedly enhanced GR activity in Kennebec, especially in the shoots, the 245

activity of the enzyme decreased in Concord, a salt-sensitive cultivar. It has been 246

shown that a decreased activity of GR contributes to salt-stress sensitivity (Aono et al. 247

1995). Thus, a reduction of the GR activity in Concord shoots may be responsible for 248

the sensitivity of the cultivar to salt stress. A similar result has been observed in 249

maize (Azevedo-Neto et al. 2006). 250

Although Kennebec and Concord showed pronounced differences in terms of the 251

CAT, APX, and GR activities in their shoots, their roots showed similar dose-252

dependent patterns of activities of these enzymes under salt stress. It appears that 253

oxidative stress is less severe in the roots of these cultivars than in their shoots. Our 254

data showed that, in both potato cultivars, the lengths of roots were less severely 255

affected than those of the shoots by increasing salt treatment (Aghaei et al. 2008). 256

Furthermore, the rate of decrease in root length was almost the same in these two 257

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cultivars, except at high salt doses, as a result of the similar dose-dependent pattern of 258

the activities of antioxidant enzymes. Greater activation of these enzymes under salt 259

treatments can explain the better growth of Kennebec root compared with that of 260

Concord. 261

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Materials and Methods 284

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Plant material and culture conditions 286

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Sprouted healthy tubers of 2 potato (Solanum tuberosum L.) cultivars Concord and 288

Kennebec were planted in 500 mL pots containing steam sterilized soil, nodal sections 289

of pot grown plants were used as primary explants. Stem-cuttings consisting of a 290

single node and a leaf were surface sterilized in a solution of 10% (v/v) sodium 291

hypochlorite for 15 min. They were rinsed with sterile distilled water 3 times and were 292

transferred on 35 mL MS medium (Murashige and Skoog 1962). Cultures were 293

maintained and sub-cultured in growth chamber under 16/8 h light/dark photoperiod 294

with 150 µmol m–1 s–1 illumination at 25 ±1ºC. In vitro grown plants were propagated 295

by sub-culturing with 3 weeks interval. Single nodes were transferred to MS media 296

containing 0, 30, 60, 90 and 120 mM NaCl. After 4 weeks, the physiological 297

parameters including shoot and root length, shoot and root dry weight, shoot and root 298

fresh weight were measured. The experiments were repeated 3 times. 299

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Determination of Na+ and K+ contents 301

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Plant materials were dried at 75ºC for 24 h, dissolved in 10 mL of 3% sulfosalicylic 303

acid and incubated over night at 4ºC. The extracts were filtered through Whatman 304

filter paper No. 2 and filtrates were analyzed for Na+ and K+ contents using a flame 305

photometer (M410, Corning, Palo Alto, CA, USA). 306

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Enzyme extraction 308

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A portion (300 mg) of excised potato shoots and a portion (100 mg) of roots were 310

homogenized in 4 mL of 25 mM potassium phosphate buffer (pH 7.8) containing 0.4 311

mM EDTA-4H, 1 mM ascorbic acid and 2% polyvinylpyrrolidone. The homogenate 312

was centrifuged at 15,000 × g for 20 min at 4◦C and the supernatant was filtered 313

through Miracloth (Calbiochem, San Diego, CA, USA). The filtrate was used as an 314

enzyme extract for CAT (EC 1.11.1.6), APX (EC 1.11.1.11) and GR (EC 1.6.4.2) 315

activity assays. 316

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Enzyme activity assays 318

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CAT activity was assayed in a 1 mL reaction mixture containing 50 mM potassium 320

phosphate buffer (pH 7.0), 10 mM H2O2 and 0.05 mL of enzyme extract. The 321

subsequent decomposition of H2O2 was determined at 240 nm (λ=240 nm every 10 sec 322

for 5 min at 22oC, E= 0.0394 mM−1 cm−1) (Sunohara and Matsumoto 2004) using 323

spectrophotometer (DU730, Beckman, Fullerton, CA, USA). 324

APX activity was determined in a 1 mL reaction mixture containing 25 mM 325

potassium phosphate buffer (pH 7.0), 0.25 mM ascorbic acid, 0.1 mM EDTA-4H, 0.1 326

mM H2O2 and 0.05 mL of enzyme extract. The subsequent decrease in ascorbic acid 327

was determined at 290 nm (E = 2.8 mM−1 cm−1) (Nakano and Asada 1987). A unit of 328

ascorbate peroxidase is defined as the amount necessary to oxidize 1 µmol of 329

ascorbate min–1 at 25°C (290 nm extinction coefficient of 2.8 L mmol–1 cm–1). 330

GR activity was assayed in a 1 mL reaction mixture containing 25 mM potassium 331

phosphate buffer (pH 7.8), 0.5 mM oxidized glutathione, 120 µM NADPH and 0.1 332

mL of enzyme extract. The resultant decrease in NADPH was monitored at 340 nm (E 333

= 6.1 mM−1cm−1) (Halliwell and Foyer 1878). A unit of activity is the amount of 334

enzyme that will catalyze the reduction of 1 µmol of GSSG min–1 at 25°C. 335

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Determination of protein concentration 337

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Protein concentrations of the shoot and root extracts were determined using the Bio-339

Rad protein assay kit (Bio-Rad, Hercules, CA, USA) with bovine serum albumin as a 340

standard. 341

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Acknowledgments 343

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The authors are grateful to scholarship section of the Ministry of Science, Research and 345

Technology of I. R. Iran and the Higher Education Department of Isfahan University. 346

We thank to the National Institute of Crop Science of Japan for kind supports. We also 347

thank to Potato Research Institute of Isfahan University and Dr G.R. Balali for his 348

cooperation. 349

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Figure legends 438

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Fig. 1. Effects of salt stress on the lengths of shoot and root. Potato cultivars 440

Kennebec and Concord were planted on MS media containing 0 (control), 30, 60, 90, 441

or 120 mM NaCl (A), and the lengths of shoots (white column) and roots (grey 442

column) were measured at 4 weeks after treatment (B). Three plants in each treatment 443

were used. The experiments were repeated three times and the results show the 444

average ± S.E. Asterisks indicate significant differences between control and treatment 445

(*P<0.05, **P<0.01). SE is denoted by error bars. 446

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Fig. 2. Effects of salt stress on the fresh and dry weights of shoot and root. Potato 448

cultivars Kennebec and Concord were planted on MS media containing 0 (control), 30, 449

60, 90, or 120 mM NaCl. The fresh weight (A) and dry weight (B) of shoots (white 450

column) and roots (grey column) were measured 4 weeks after treatment. Three plants 451

in each treatment were used. The experiments were repeated three times and the 452

results show the average ± S.E. Asterisks indicate significant differences between 453

control and treatment (*P<0.05, **P<0.01). SE is denoted by error bars. 454

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Fig. 3. Effects of salt stress on the Na+ and K+ contents of shoots and roots. Potato 456

cultivars Kennebec and Concord were planted on MS media containing 0 (control), 30, 457

60, 90, or 120 mM NaCl, and the Na+ (square) and K+ (triangle) contents of shoots (A) 458

and roots (B) were measured 4 weeks after treatment. Three plants in each treatment 459

were used. The experiments were repeated three times and the results show the 460

average ± S.E. Asterisks indicate significant differences between control and treatment 461

(*P<0.05, **P<0.01). SE is denoted by error bars. 462

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Fig. 4. Effects of salt stress on the activity of antioxidant enzymes. Potato cultivars 464

Kennebec (triangle) and Concord (square) were planted on MS media containing 0 465

(control), 30, 60, 90, or 120 mM NaCl. The activities of APX, CAT, and GR were 466

measured in shoots (A) and roots (B) 4 weeks after treatment. Three plants in each 467

treatment were used. The experiments were repeated three times and the results show 468

the average ± S.E. Asterisks indicate significant differences between control and 469

treatment (*P<0.05, **P<0.01). SE is denoted by error bars. 470