Interactive effects of selenium and arsenic on growth, antioxidant system, arsenic and selenium species of Nicotiana tabacum L.

Accepted Manuscript Title: Interactive effects of selenium and arsenic on growth, antioxidant system, arsenic and selenium species of Nicotiana tabacum L. Author: Dan Han Shuanglian Xiong Shuxin Tu Jinchang Liu Cheng Chen PII: DOI: Reference:

S0098-8472(15)00081-7 http://dx.doi.org/doi:10.1016/j.envexpbot.2015.04.008 EEB 2927

To appear in:

Environmental and Experimental Botany

Received date: Revised date: Accepted date:

15-11-2014 4-4-2015 14-4-2015

Please cite this article as: Han, Dan, Xiong, Shuanglian, Tu, Shuxin, Liu, Jinchang, Chen, Cheng, Interactive effects of selenium and arsenic on growth, antioxidant system, arsenic and selenium species of Nicotiana tabacum L.Environmental and Experimental Botany http://dx.doi.org/10.1016/j.envexpbot.2015.04.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1

Interactive effects of selenium and arsenic on growth, antioxidant system, arsenic and

2

selenium species of Nicotiana tabacum L.

3 4

First name: Dan; Family name: Hana, b, c

5

First name: Shuanglian; Family name: Xionga, b, c

6

First name: Shuxin; Family name: Tua, b, c

7

First name: Jinchang; Family name: Liua, b, c

8

First name: Cheng; Family name: Chena, b, c

9 10

a

11

430070, China

12

b

13

China

14

c

15

Yangtze River), Ministry of Agriculture, Wuhan 430070, China

College of Resources and Environment, Huazhong Agricultural University, Wuhan

Microelement Research Center, Huazhong Agricultural University, Wuhan 430070,

Key Laboratory of Arable Land Conservation (Middle and Lower Reaches of

16 17

Corresponding author

18

Shuanglian Xiong; College of Resources and Environment, Huazhong Agricultural

19

University, Wuhan 430070, China; Tel.: +86 027 87282137 fax: +86 027 87288618.

20

E-mail: [email protected] (S. Xiong).

21

Shuxin Tu; College of Resources and Environment, Huazhong Agricultural University,

22

Wuhan 430070, China. E-mail: [email protected] (S. Tu).

1

23

Highlights

24

●

The moderate dose of Se/As in solution promoted the growth of FCT.

25

●

Different Se/As levels in solution affects the ratios of Se/As species of FCT.

26

●

Se mitigates As toxicity by improving the antioxidant capacity.

27

●

Se mitigates As toxicity by influencing on the Se/As species of FCT changes.

28 29

Abstract

30

This paper is aimed to study separate and interactive effects of selenium (Se) (selenite,

31

0-5 mg L-1) and arsenic (As) (arsenate, 0-5 mg L-1) on the growth and the antioxidant

32

system of flue-cured tobacco (FCT) as well as the contents and species of As and Se

33

in FCT through a hydroponic experiment and clarify the possible mechanism how Se

34

alleviates the toxicity of As. The results were: single addition of Se (≤ 1 mg L-1) or As

35

(1 mg L-1) by a low dose could stimulate the growth of FCT, but the growth of FCT

36

would be inhibited when Se (5mg L-1) or As (5mg L-1) was added by a high dose. Low

37

As levels stimulated the uptake of Se but high levels of As posing the opposite effects

38

with the low Se dosage. However, the addition of As always inhibited the uptake of Se

39

with high Se levels. Moreover, Se showed dual effects on the uptake of As. At the low

40

As dose (1mg L-1), the addition of Se inhibited the growth of FCT, but significantly

41

promoted the activity of superoxide dismutase (SOD) and peroxidase (POD) enzymes

42

as well as the content of MDA. Meanwhile, the percentages of organic Se and As(III)

43

in the leaves of FCT declined with the increasing Se dose. However, the addition of

44

Se by a moderate dose (0.1mg L-1) alleviated the toxicity of the high As dose (5mg L-1 )

45

and promoted the growth of FCT by elevating the ability of anti-oxidative stress of

46

FCT and reducing the contents of MDA and As in FCT. The Se species in the leaves

47

of FCT existed in organic ones (SeCys and SeMet) (100%), while the major As

48

speciation was As(III) (75%). Likewise, the addition of As counteracted the toxicity of

49

high Se dose (5 mg L-1) and promoted the growth of FCT slightly as it reduced the

50

formation of organic Se or failed to transform excess inorganic Se species into organic

51

ones and depressed the contents of Se in the roots and leaves of FCT. In a word, the

52

low Se dose (0.1mg L-1) alleviated of the toxicity of the high As dose and the addition 2

53

of As counteracted the toxicity of high Se dose (5 mg L-1), as a result of which the

54

promotion of the growth of FCT were realized.

55 56

Keywords: Arsenate; Selenite; Interaction; Antioxidant system; Species.

57

Abbreviations: Se, selenium; As, arsenic; MDA, malondialdehyde; SOD, superoxide dismutase;

58

POD, peroxidase; AsA, ascorbate acid; GSH, glutathione; As(V),arsenate; As(III), arsenite; ROS,

59

reactive oxygen species; MMA, monomethylarsonicacid; DMA, dimethylarsinic acid; Cd,

60

cadmium; Hg, mercury; Ni, nickel; Pb, lead; GR, glutathione reductase; Se(IV), selenite; Se(VI),

61

selenate; SeCys, selenocysteine; SeMet, selenomethionine; AR, arsenate reductase; PC,

62

phytochelatins.

3

63

1. Introduction

64

Arsenic (As) is a severe pollutant which is highly toxic to animals and plants and

65

widely distributes in the environment. Arsenic exists in the forms of arsenate [As(V)],

66

arsenite [As(III)], monomethylarsonic acid [MMA] and dimethylarsinic acid [DMA],

67

but As(V) and As(III) are the main species (Tripathi et al., 2007; Zheng et al., 2013),

68

which mainly come from mining, coal ash, dust, off gas, pesticides and sewage

69

sluge(Meunier et al., 2011; Saunders et al., 2010). The accumulation of As in soil

70

doesn’t only can affect the growth and development of plants, but also pose a threat to

71

human health via the food chain. Arsenic impedes the photosynthesis and reduces the

72

contents of essential nutrients, thus inhibits the growth of crops and even causes them

73

death (Garg and Singla, 2011). For human, arsenic may lead to cancerous and

74

non-cancerous diseases, including bladder cancer, lung cancer, liver cancer and so on

75

(Farnese et al., 2014).

76

Although there is no evidence that selenium (Se) is an essential element for plants so

77

far, it is an essential microelement for human and animals. An appropriate dose of Se

78

can improve the antioxidant capacity, scavenge excessive oxygen free radicals,

79

decrease lipid peroxidation, defer senescence, promoting the growth of plants (Lin et

80

al., 2012), and detoxify heavy metals (metalloids) in plants, for example arsenic (As)

81

(Malik et al., 2012), cadmium (Cd)(Feng et al., 2012; Lin et al., 2012; Saidi et al.,

82

2014), mercury (Hg) (Zhang et al., 2012a), nickel (Ni) (Gajewska et al., 2013) and

83

lead (Pb) (Mroczek-Zdyrska and Wojcik, 2012). Currently, the researches on the

84

mechanism how Se mitigates the toxicity of As are mainly concentrated on the

85

elimination of reactive oxygen species (ROS), the suppression of lipid peroxidation

86

and the enhancement of the antioxidant capacity of plants(Kramárová et al., 2012;

87

Malik et al., 2012). Se exists mainly in the forms of Se(IV), selenate Se (VI),

88

selenocysteine (SeCys) and selenomethionine (SeMet) in plants (Zhu et al., 2009).

89

After absorbed by plants, Se(IV) or Se(VI) is converted into other forms like

90

selenocysteine (SeCys) and selenomethionine (SeMet) (Zhu et al., 2009). Moreover, 4

91

Se can replace sulphur in the amino acids as SeMet and SeCys due to their

92

physicochemical similarity. And the organic Se species (SeCys and SeMet) can be

93

incorporated into proteins, replacing cysteine (Cys) and methionine (Met),

94

respectively, which can result in toxicity in plants (Navarro-Alarcon and

95

Cabrera-Vique, 2008; White et al., 2004). However, the toxicity of soluble inorganic

96

As is greater than that of organic As, and the toxicity of As(III) is higher than that of

97

As(V) in the environment (Yamauchi, 1994; Yin et al., 2013). Having taken up by the

98

roots of plants via the phosphate transport pathway (Wu et al., 2011b; Zhao et al.,

99

2010), As(V) can be rapidly reduced to As (III) by arsenate reductase (AR) (Zhao et

100

al., 2009). As(III), which can also be taken up by the roots of plants mainly through

101

silicic acid transport protein (Ma et al., 2008), is chelated with polypeptides like GSH

102

and PCs and finally stored in the vacuoles of roots to detoxify As(Liu et al., 2010; Ye

103

et al., 2011; Zhang et al., 2012c). A previous study showed that Se could mitigate the

104

toxicity of As via antagonistic effects in Pteris vittata (Feng et al., 2009).

105

However, the changes of As and Se species in plants under the exposure of As and Se

106

are still unclear. We infer that Se can mitigate the toxicity of As by regulating the

107

antioxidant system and simultaneously affecting As species in plants.

108

Tobacco leaf, which contains abundant and high-quality soluble proteins (Teng and

109

Wang, 2012), is considered as an ideal material to produce Se-rich protein. In this

110

study, Nicotiana tabacum L. was chosen as the test material to investigate: (1) the

111

responses of the growth and antioxidant systems of FCT to different doses of Se and

112

As; (2) the relation among the variations of As species and the detoxification of As

113

after Se supplementation.

114

2. Materials and methods

115

2.1. Seedling cultivation

116

The cultivar was N. tabacum K326, and the floating cultivation method was adopted

117

for the cultivation of seedlings. Later, the seedlings of similar sizes were transplanted 5

118

into plastic pots containing 10 L nutrient solution which was composed of 4 mmolL-1

119

Ca(NO3)2·4H2O, 5 mmolL-1 KNO3, 1mmolL-1 NH4H2PO4, 2 mmolL-1 MgSO4·7H2O,

120

0.01 mmolL-1 EDTA-Fe, 0.046 mmolL-1 H3BO3, 0.0008 mmolL-1 ZnSO4·7H2O and

121

0.0003 mmolL-1 CuSO4·5H2O. The plants grew in a greenhouse with natural light at

122

25-30℃.

123

2.2. Experimental design and implementation

124

A hydroponic experiment of four Se levels, i.e. 0, 0.1, 1.0 and 5.0 mg L-1, and three

125

As levels, i.e. 0, 1.0 and 5.0 mg L-1, was designed. As and Se were added in the forms

126

of Na3AsO4.12H2O and Na2SeO3, respectively. A randomized complete block design

127

was employed. There was a total of 12 treatments, namely CK (without the addition

128

of Se and As), Se0.1As0, Se1As0, Se5As0, Se0As1, Se0.1As1, Se1As1, Se5As1,

129

Se0As5, Se0.1As5, Se1As5 and Se5As5, and each treatment was repeated for three

130

times.

131

Four-leaf FCT seedlings were transplanted to 1/4 strength Hoagland-Arnon nutrient

132

solution at first and three days later, the solution was replaced by 1/2 strength nutrient

133

and the nutrient solution was renewed every five days. After forty days, the seedlings

134

of even size were transplanted to the plastic cases (22×16×7cm) and subject to the

135

above-mentioned Se and/or As treatments with full strength Arnon-Hoagland nutrient

136

solution, and each pot contained 2 plants. The plants were harvested after fourteen

137

days. After rinsed carefully with tap water and deionized water successively, the

138

separated fresh leaves and roots were weighed and divided into two parts. One was

139

immersed in N2 liquid immediately and stored at -80℃ to determine the Se and As

140

species and the indices of the antioxidant systems later. The other was over-dried at

141

105°C for 15 min to de-enzyme at first, and then at 65°C for 48 h and finally

142

pulverized to determine the contents of As and Se.

6

143

2.3. Determination of Se and As contents and species

144

The contents of Se and As were determined with a hydride generation atomic

145

fluorescence spectrometer (AFS8220, Beijing Titan Instruments Co., China) (Feng et

146

al., 2009a) after the tissues of FCT were digested with concentrated HNO3-HClO4.

147

The accuracy of elemental analysis was verified by standard reference materials

148

[GBWO7602 (GSV-1)] from the Center for Standard Reference of China.

149

A methanol: water (1:2) method was used to extract Se species in leaves or roots of

150

FCT plants. The separation of SeIV, SeVI, SeMet and SeCys in the green parts of FCT

151

was carried out with a anion exchange chromatography in which the column was

152

connected to a ultraviolet treatment-hydride generation atomic fluorescence

153

spectrometry (UV-HG-AFS) detection system (Han et al., 2013) online.

154

The extraction of As species was similar to that of Se species. Because no organic

155

arsenic in FCT were detected in a preliminary experiment, only As(V) and As(III)

156

were measured in this study. The separation of As(V) and As(III) in leaves and roots

157

of FCT plants was conducted with the anion exchange chromatography in which the

158

column was also connected to the HG-AFS detection system (Zhang et al., 2002)

159

online.

160

The HPLC system consisted of a SHIMADZU 10ATvp Plus liquid chromatography

161

pump (SHIMADZU, Tokyo, Japan), a Rheodyne 7725i injector (Rheodyne, Cotati,

162

USA) and a Hamilton PRP-X100 column (Hamilton, Reno, NV). The mobile phase

163

for HPLC was 15 mmol L-1(NH4)2HPO4 (pH 6.0, 1.0 mL min-1). As for the HG phase,

164

the reduction agents were 1.5% KBH4 (m/v)+0.5% KOH (m/v) and the carrier

165

solution was 7% HCl (v/v). The detection phase was AFS8220, with the Se hollow

166

cathode lamp current (General Research Institute for Nonferrous Metals, Beijing,

167

China) of 50 mA, the negative high voltage of photomultiplier tube of 270V, the flow

168

rate of carrier gas of 400 mL min-1 and the flow rate of makeup gas of 600 mL min-1.

7

169

2.4. Assay of enzymatic and non-enzymatic antioxidants

170

The activity of superoxide dismutase (SOD) was determined with the method put

171

forward by Zhang et al.(Zhang et al., 2012b). In brief, 0.5g fresh FCT leaves was

172

grounded in 5mL extraction buffer containing 50 mM potassium phosphate (pH7.8) at

173

first, and then the homogenate was centrifuged at 10,000×g for 15 min at 4°C. 3mL

174

reaction mixture contained 13 mM methionine, 75 μM NBT, 2 μM riboflavin, 0.1 mM

175

EDTA and 100μL enzyme extract. The reaction mixture was illuminated for 15 min.

176

The sample absorbance was determined at 560 nm, and the unit SOD activity was

177

defined as the amount of enzyme to inhibit 50% of the NBT reduction.

178

The activity of peroxidase (POD) was measured with the method described by Zhang

179

et al. (Zhang et al., 2012b). In brief, 0.5g fresh leaves of FCT were extracted with 5

180

mL 50mM potassium phosphate buffer (pH 5.5). Afterwards, the extracts were

181

centrifuged at 10,000×g for 15min at 4°C. The reaction mixture contained 1 mL

182

extraction buffer, 5μL 30% H2O2, 5μL guaiacol and 15μL supernatant. The molar

183

extinction coefficient of 26.6 mM−1cm−1 was used for the calculation of the enzyme

184

activity.

185

Ascorbate (AsA) was extracted from 0.5g FCT leaves with 10g L−1 oxalic acid and

186

determined spectrophotometrically with 2,4-dinitrophenylhydrazine colorimetry

187

method proposed by Zhang et al. (Zhang et al., 2012b). 0.5g samples were

188

homogenized with 3% metaphosphoric acid at first, then the homogenate was

189

centrifuged for 10min at 10,000×g and finally the supernatant was used for

190

glutathione (GSH) assays (Zhang et al., 2012b).

191

The MDA content was assayed with 2.5 mL solution of 20% (w/v) trichloroacetic acid,

192

which included 0.5% (w/v) thiobarbituric acid and 1.5 mL enzyme extract. The

193

solution was kept in boiling water for 20min bath at first and then cooled quickly.

194

After refrigeration, the homogenate was centrifuged at 5,000 g for 10 min at 25°C.

195

The absorbances of supernatant were recorded at 532 nm and 600 nm, respectively.

196

The absorbance at 600 nm was subtracted from that at 532 nm. The MDA content was

197

calculated based on the extinction coefficient of MDA, namely 155 mM−1cm−1(Feng 8

198

et al., 2009b).

199

2.5. Statistical analysis

200

All data were subject to two-way ANOVA analysis and Tukey's multi-comparisons

201

test (P≤0.05). The results were expressed as the means and the corresponding standard

202

errors. All statistical analyses were completed using the SAS 8.1 software.

203

3. Results

204

3.1. Growth of FCT

205

Without the addition of As, low Se levels (≤ 1mg L-1Se) stimulated the growth of FCT,

206

but high Se levels (5 mg L-1) had the opposite effects on and inhibited the growth of

207

FCT compared with the CK treatment (Fig. 1AB). The fresh weights of leaves and

208

roots of FCT in Se5As0 treatment were 32% and 43% of those of the CK treatment,

209

respectively (Fig. 1AB). Similarly, in the absence of Se, 1mg L-1 As stimulated the

210

growth of FCT, but 5mg L-1As significantly hindered the growth of FCT, especially

211

the roots of FCT (Fig. 1AB). The fresh weights of leaves and roots of FCT in Se0As1

212

treatment were 22% and 24% higher than those in the CK treatment, respectively. The

213

fresh weight of roots in Se0As5 treatment was 24% lower than that in the CK

214

treatment (Fig. 1AB).

215

Under low As treatments (1 mg L-1 As), the addition of Se inhibited the growth of FCT

216

(Fig. 1AB). The fresh weights in Se0.1As1, Se1As1 and Se5As1 treatments were 91%,

217

68%, 38% (for leaves) and 88%, 108%, 35% (for roots) of those in Se0As1 treatment,

218

respectively. However, at the high As level (5mg L-1), the low Se level (0.1mg L-1Se)

219

enhanced but high Se level (5mg L-1Se) obviously depressed the growth of FCT (Fig.

220

1AB). For example, the fresh weights of shoots and roots in Se0.1As5 treatment were

221

1.1 times and 1.5 times of those in Se0As5 treatment, respectively. Nevertheless, the

222

fresh weights of shoots and roots in Se5As5 treatment were 40% and 75% of those in

9

223

Se0As5 treatment, respectively (Fig. 1AB).

224

3.2. Uptake, distribution and species of Se or As of FCT

225

At a certain As dose, the contents of Se in the leaves and roots of FCT went up along

226

with the increasing Se level (Fig. 2AB).

227

Se and As had significant interactive effects on the contents of Se in leaves and roots

228

of FCT (F=79.26 and 81.06, P<0.01, respectively). The effects of As on the uptake of

229

Se depended on the levels of Se and As in solution. At the low Se level (≤ 1 mg L-1), 1

230

mg L-1 As promoted the uptake of Se by the leaves and roots of FCT, but 5 mg L-1As

231

had negative effects on the uptake of Se.

232

At the high Se level (5 mg L-1), As showed antagonistic effects on the uptake of Se in

233

the leaves and roots of FCT. The contents of Se in the leaves and roots of FCT in

234

Se5As1 and Se5As5 treatments were 82% and 69% (for roots), 66% and 61% (for

235

leaves) of those in Se5As0 treatment, respectively (Fig. 2AB).

236

At a certain Se level, the contents of As in the leaves and roots of FCT increased

237

along with elevation of the As level significantly (Fig. 3AB), and in particular the

238

content of As in roots was far higher than that in the leaves (Fig. 3AB).

239

The interaction of Se and As had significant effects on the uptake of As by FCT

240

(F=34.57 and 26.63, P<0.01, respectively). At 1 mg L-1 As level, the elevation of the

241

Se levels raised the As content in the leaves (Fig. 3A) but failed to enhance the As

242

content in the roots of FCT. However, at 5 mg L-1 As level, the growth of Se level

243

remarkably reduced the contents of As in leaves and roots of FCT (Fig. 3AB).

244

Only SeCys and Se(IV) were detected in the roots, whereas SeCys, SeMet and Se(IV)

245

were observed in the leaves of FCT (Fig. 4AB). In 0.1 mg L-1 Se treatment, main Se

246

species in the leaves of FCT were organic ones (SeCys and SeMet) (58%-100%),

247

which gradually increased with the growth of the As level (Fig. 4A). Similarly, the

248

proportion of organic Se species (SeCys) in the roots of FCT went up from 30% to

249

69% with the growth of the As level (Fig. 4B).

250

In 1 mg L-1 Se treatment, the percentage of organic Se species declined in the leaves 10

251

but increased in the roots of FCT along with the elevation of the As level. To be

252

specific, the proportions of organic Se species (SeCys and SeMet) in the leaves

253

decreased from 68% (Se1As1) to 42% (Se1As5), while the percentage of organic Se

254

species (SeCys) in the roots of FCT went up from 47% (Se1As1) to 84% (Se1As5).

255

In 5 mg L-1 Se treatment, the percentages of organic Se species in the leaves and roots

256

of FCT decreased along with the elevation of the As level.

257

In the leaves of FCT, As (V) was the main speciation at the low As level of 1mg L-1

258

(63%-94%), whereas As(III) was the major As speciation at the high As level of 5mg

259

L-1 (63%-78%) (Fig.5A). At a certain As level, the proportion of As(III) in the leaves

260

of FCT declined along with the growth of the Se level. For example, the proportion of

261

As(III) in the leaves dropped from 37% to 6% at 1mg L-1 As but decreased from 74%

262

to 63% at 5mg L-1 As along with the elevation of the Se level.

263

As(III) was the main As speciation in the roots of FCT (Fig. 5B). At the low As level

264

(1mg L-1), 0.1mg L-1 Se reduced the proportion of As(III) in the roots of FCT but the

265

other Se levels raised the proportion of As(III) in comparison with the Se0As1

266

treatment. At the high As level (5mg L-1), the elevation of the Se level had no

267

significant effects on the proportions of As(III) and As (V) (Fig. 5B).

268

3.3. Antioxidant system of FCT under As/Se stress

269

Se and As treatments had great influences on the activities of SOD and POD (P<0.01).

270

Regardless of the addition of As, the addition of Se markedly increased the activities

271

of SOD and POD of the leaves of FCT (Fig. 6AB, P<0.05). However, whether Se is

272

added into the solution or not, the elevation of the As level significantly enhanced the

273

SOD activity but generally decreased the POD activity of the leaves of FCT except

274

that the POD activity was significantly enhanced at the Se level of 1 mg L-1 and along

275

with the growth of the As level (Fig. 6AB).

276

As and Se had significant influences on the GSH content in the leaves of FCT

277

(F=15.50, P<0.01). Without the addition of As in the solution, the growth of the Se

278

level significantly enhanced the GSH content in the leaves of FCT (Fig. 6C). 11

279

At a low As level of 1 mg L-1, the GSH content was also enhanced by increasing the

280

Se level except for the GSH content was reduced by 15% at the Se level of 1 mg L-1

281

compared with the Se0As1 treatment (Fig.6C). At the high As level of 5 mg L-1, the

282

GSH content did not significantly change with the increasing Se level (Fig. 6C).

283

When Se was not added in the solution, the increase of the As level significantly

284

enhanced the GSH content in the leaves of FCT. However, when Se was present in the

285

solution, the addition of As appeared to show insignificant effects on or reduce the

286

GSH content in FCT (Fig. 6C).

287

The interaction of Se and As significantly affected the AsA content in the leaves of

288

FCT (F=60.07, P<0.01). At a certain As level, the increasing Se levels significantly

289

raised the AsA content in the leaves of FCT. When no Se or 0.1 mg L-1 Se was added

290

in the solution, 1 mg L-1 As reduced but 5 mg L-1 As recovered or increased the AsA

291

content compared with the CK and Se0.1As0 treatments, respectively (Fig. 6D).

292

However, when 1 or 5 mg L-1 Se was added in the solution, 5 mg L-1 As significantly

293

enhanced the AsA content (Fig. 6D).

294

Se and As treatments could dramatically affect the MDA content (P<0.01). When no

295

As was added in the solution, 0.1 mg L-1 Se significantly reduced the MDA content in

296

the leaves of FCT but 1 and 5 mg L-1 Se enhanced the MDA content compared with

297

the CK treatment (Fig. 7). However, when Se was absent from the solution, 1 mg L-1

298

As reduced the MDA content in the leaves of FCT but 5 mg L-1 As resulted in the

299

reduction of the MDA content to the level in the CK treatment (Fig. 7).

300

When 1 mg L-1 As was added into the solution, the increasing Se level significantly

301

enhanced the MDA content in the leaves of FCT. However, with 5 mg L-1 As in the

302

solution, only 5 mg L-1 Se significantly enhanced the MDA content. In the presence of

303

0.1 mg L-1 Se in the solution, the addition of As did not significantly affect the MDA

304

content in FCT. However, when 1 or 5 mg L-1 Se was added in the solution, 1 mg L-1

305

As significantly enhanced but 5 mg L-1 significantly reduced the MDA content in the

306

leaves of FCT.

12

307

4. Discussion

308

This study attempted to illuminate the mechanism how Se detoxifies As via regulating

309

the antioxidant systems of FCT and the variations of species of As and Se in FCT. As

310

expected, the addition of Se can detoxify As, showing as the stimulated leaf and root

311

biomass of FCT exposed to high levels of As. However, this alleviation process might

312

be dependent on the dosages of Se and As, because the high Se level (5 mg L-1) had

313

already showed toxic effects on the plant growth whether As was added into the

314

solution or not. Furthermore, when plants were exposed to 1 mg L-1 As, the addition

315

of Se did not show beneficial but negative effects on the growth of FCT because the

316

addition of Se might counteract the beneficial effects of low As dosages on the growth

317

of FCT. Similar results have been reported by Malik et al. (2012) on mung bean

318

(Phaseolus aureus Roxb.) and Zhao et al. (2013b) on garlic (Allium sativum). With

319

increase in arsenic (As) concentration, a marked inhibition in the growth of plants was

320

observed. The plants treated 10 uM As (0.7 mg L-1) and supplemented with 2.5uM Se

321

(0.2 mg L-1) showed 22% growth improvement in shoots and 16% in roots compared

322

to those growing in As alone(Malik et al., 2012). Low-level exposure to Se(≤0.1 mg

323

L-1) or Hg (≤0.01 mg L-1) is beneficial for garlic growth, whereas Se(＞1 mg L-1) or

324

Hg (＞0.1 mg L-1) levels above certain threshold limits can be harmful. As for Se-Hg

325

co-exposed garlic, the stimulation of garlic growth by low levels of Se is also

326

enhanced by HgCl2 at low levels (≤0.1 mg L-1 Hg), by contrast, an antagonistic effect

327

between low levels of Se and higher Hg levels (1 mg L-1 or 10 mg L-1) is

328

observed(Zhao et al., 2013b).

329

Like the effects of As and Se of different levels on the growth of plant, the Se uptake

330

might also depend on the levels of As and Se in the solution. Low As levels stimulated

331

the uptake of Se but high levels of As appeared to bring about the opposite effects

332

when the plants were subject to low dosages of Se. However, when plants were

333

exposed to high Se levels, the addition of As always inhibited the uptake of Se.

334

Similar results about the uptake of As on Se have been reported on P. vittata (Feng et

13

335

al., 2009a) and garlic (Allium sativum) (Zhao et al., 2013a). The increased Se uptake

336

in FCT might be used to synthesize some important substances, such as GSH-Px

337

(glutathione peroxidase), and rebalance the excessive ROS, during which GSH will be

338

needed in huge doses, like the significant growth of the GSH content in this study (Fig.

339

6C). However, when As and Se was added in excess, some vital substances (for

340

example GSH) might not satisfy the metabolism demands of As and Se. Therefore,

341

there was a competition between As and Se to integrate with GSH, and they showed

342

the mutually antagonistic effects. The results above matched well with the hypothesis

343

of Feng et al. (2013). Interestingly, Se showed dual effects on the uptake of As in this

344

study. The exact reasons behind the stimulation of the As uptake in the leaves of FCT

345

subject to low As doses by Se were unknown (Fig. 3A). However, similar inhibition

346

effects of Se on the uptake of As were also reported on Phaseolus aureus Roxb.

347

(Malik et al., 2012) and Pteris vittata (Feng et al., 2009a). Se (SeO32- or SeO42-) at

348

high exposure levels (＞1mg L-1) also significantly inhibited the uptake of Hg in

349

garlic when Hg2+ levels were higher than 1mg L-1 in the culture media (Zhao et al.,

350

2013a). Since As(V) and Se(IV) were taken up by roots via the phosphate transport

351

pathway(Wu et al., 2011a; Zhu et al., 2009), the antagonistic effects between Se and

352

As on their mutual uptakes might be related to their competition for binding sites.

353

The detoxification of As by Se might be also closely with the changes of As and Se

354

species. In the leaves of FCT, the addition of Se resulted in more accumulation of

355

As(V) and less accumulation of As(III) (Fig. 5A), but the opposite trends were

356

observed in the roots (Fig. 5B). It is well established that As(III) is more toxic than

357

As(V) in plants. Therefore, a conclusion could be drawn that Se could reduce the

358

toxicity of As since it reduced the transfer of As(III) from the roots to the

359

above-ground parts of plants.

360

When Se was added at low levels in the solution, organic Se species (SeCys and

361

SeMet) were predominant in the leaves of FCT, and the increasing As levels enhanced

362

the transformation of inorganic Se into organic Se (Fig. 4A), which might be related

363

with the antioxidative effects of low Se dosages (Feng et al., 2013). It was reported

364

that organic Se could directly quench ROS, especially hydroxylic free radical (OH•) 14

365

(Feng et al., 2013). Therefore, the enhancement of organic Se in the leaves of FCT

366

was conducive to the rebalance of ROS of plants. However, the proportion of

367

inorganic Se [Se(IV)] increased when FCT was subjected to the high levels of As and

368

Se. Se was considered as a pro-oxidant at high levels, and synthesized proteins in

369

place of S(Feng et al., 2013; Terry et al., 2000). Therefore, the more accumulation of

370

inorganic Se after the addition of high levels of Se and As might suggest that to avoid

371

the toxicity of high levels Se, plant will reduce the formation of organic Se or cannot

372

transform excess inorganic Se into organic over a short period of time. In addition, the

373

competition between As and Se to integrate with GSH might also a key influential

374

factor to the formation of organic Se, which was realized with the help of GSH (Feng

375

et al., 2013). Similar results have been reported by Han et al. (2013) on Nicotiana

376

tabacum L.. In the leaves and roots of FCT, the addition of Se resulted in more

377

accumulation of organic Se species at low Se levels and more accumulation of

378

inorganic Se species at high Se levels(Han et al., 2013). Moreover, Compared with

379

roots, shoots of all the three plants (alfalfa, maize, and soybean) had a higher

380

percentage of organic Se (especially SeCys) by using Se(IV)-spiked soil(Yu et al.,

381

2011).

382

As expected, Se could affect the antioxidant system and regulate the toxicity of As,

383

judged from the increased activities of SOD and POD and the enhanced contents of

384

GSH and AsA, suggesting that these antioxidants play important roles in the

385

detoxification of As by Se. However, Se also showed toxicity to FCT, as it increase

386

the MDA content (Fig.7), decreased the fresh weights of leaves and roots at high Se

387

levels (Fig.1AB). In line with the increased fresh weight of leaves (Fig.1A), the

388

decreased Se contents in the leaves and roots (Fig.2AB) and organic Se proportion

389

(Fig.4AB) as well as the enhanced SOD activity and contents of GSH and AsA, the

390

addition of As seemed to exert detoxification effects on the high Se level (5 mg L-1)

391

(Fig.4ACD).

15

392

5. Conclusion

393

The low Se level (0.1mg L-1Se) alleviated the toxicity of high As level (5 mg L-1) and

394

thus promoted the growth of FCT by regulating the antioxidant system and reducing

395

the transfer of As(III) from the roots to the above-ground parts of FCT. Likewise, the

396

addition of As counteracted the toxicity of high Se level (5 mg L-1) and promoted the

397

growth of FCT slightly because it reduced the formation of organic Se, failed to

398

transform excess inorganic Se into organic Se over a short period of time and

399

depressed the Se contents in roots and leaves of FCT.

400

Acknowledgments

401

This research was partially supported by National Science Foundation of China

402

(41101464) and National Special Fund for Agro-scientific Research in the Public

403

Interest (201303106).

404

405

406

407

408

409

References

410

Farnese, F.S., Oliveira, J.A., Farnese, M.S., Gusman, G.S., Silveira, N.M., Siman, L.I.,

411

2014. Uptake arsenic by plants: Effects on mineral nutrition, growth and

412

antioxidant capacity. IDESIA (Chile) 32, 99-106. 16

413 414 415

Feng, R., Wei, C., Tu, S., 2013. The roles of selenium in protecting plants against abiotic stresses. Environ. Exp. Bot. 87, 58-68. Feng, R., Wei, C., Tu, S., Ding, Y., Song, Z., 2012. A Dual Role of Se on Cd Toxicity:

416

Evidences from the Uptake of Cd and Some Essential Elements and the Growth

417

Responses in Paddy Rice. Biol. Trace Elem. Res., 1-9.

418

Feng, R., Wei, C., Tu, S., Sun, X., 2009a. Interactive effects of selenium and arsenic

419

on their uptake by Pteris vittata L. under hydroponic conditions. Environ. Exp.

420

Bot. 65, 363-368.

421 422

Feng, R., Wei, C., Tu, S., Wu, F., 2009b. Effects of Se on the uptake of essential elements in Pteris vittata L. Plant Soil 325, 123-132.

423

Gajewska, E., Drobik, D., Wielanek, M., Sekulska-Nalewajko, J., Goclawski, J.,

424

Mazur, J., Sklodowska, M., 2013. Alleviation of nickel toxicity in wheat

425

(Triticum aestivum L.) seedlings by selenium supplementation. Biological Letters

426

50, 63-76.

427 428 429

Garg, N., Singla, P., 2011. Arsenic toxicity in crop plants: physiological effects and tolerance mechanisms. Environmental Chemistry Letters 9, 303-321. Han, D., Li, X., Xiong, S., Tu, S., Chen, Z., Li, J., Xie, Z., 2013. Selenium uptake,

430

speciation and stressed response of Nicotiana tabacum L. Environ. Exp. Bot. 95,

431

6-14.

432

Kramárová, Z., Fargašová, A., Molnárová, M., Bujdoš, M., 2012. Arsenic and

433

selenium interactive effect on alga Desmodesmus quadricauda. Ecotoxicol.

434

Environ. Saf. 86, 1-6.

435

Lin, L., Zhou, W., Dai, H., Cao, F., Zhang, G., Wu, F., 2012. Selenium reduces

436

cadmium uptake and mitigates cadmium toxicity in rice. J. Hazard. Mater.

437

235-236, 343-351.

438

Liu, W.J., Wood, B.A., Raab, A., McGrath, S.P., Zhao, F.J., Feldmann, J., 2010.

439

Complexation of arsenite with phytochelatins reduces arsenite efflux and

440

translocation from roots to leaves in Arabidopsis. Plant Physiol. 152, 2211-2221.

441 442

Ma, J.F., Yamaji, N., Mitani, N., Xu, X.Y., Su, Y.H., McGrath, S.P., Zhao, F.J., 2008. Transporters of arsenite in rice and their role in arsenic accumulation in rice grain. 17

443 444

Proceedings of the National Academy of Sciences 105, 9931-9935. Malik, J.A., Goel, S., Kaur, N., Sharma, S., Singh, I., Nayyar, H., 2012. Selenium

445

antagonises the toxic effects of arsenic on mungbean (Phaseolus aureus Roxb.)

446

plants by restricting its uptake and enhancing the antioxidative and detoxification

447

mechanisms. Environ. Exp. Bot. 77, 242-248.

448

Meunier, L., Koch, I., Reimer, K.J., 2011. Effect of particle size on arsenic

449

bioaccessibility in gold mine tailings of Nova Scotia. Sci. Total Environ. 409,

450

2233-2243.

451

Mroczek-Zdyrska, M., Wojcik, M., 2012. The Influence of Selenium on Root Growth

452

and Oxidative Stress Induced by Lead in Vicia faba L. minor Plants. Biological

453

Trace Element Research 147, 320-328.

454 455 456

Navarro-Alarcon, M., Cabrera-Vique, C., 2008. Selenium in food and the human body: A review. Sci. Total Environ. 400, 115-141. Saidi, I., Chtourou, Y., Djebali, W., 2014. Selenium alleviates cadmium toxicity by

457

preventing oxidative stress in sunflower (Helianthus annuus) seedlings. J. Plant

458

Physiol. 171, 85-91.

459

Saunders, J.R., Knopper, L.D., Koch, I., Reimer, K.J., 2010. Arsenic transformations

460

and biomarkers in meadow voles (Microtus pennsylvanicus) living on an

461

abandoned gold mine site in Montague, Nova Scotia, Canada. Sci. Total Environ.

462

408, 829-835.

463

Teng, Z., Wang, Q., 2012. Extraction, identification and characterization of the

464

water-insoluble proteins from tobacco biomass. J. Sci. Food Agric. 92,

465

1368-1374.

466 467

Terry, N., Zayed, A., De Souza, M., Tarun, A., 2000. Selenium in higher plants. Annu. Rev. Plant Biol. 51, 401-432.

468

Tripathi, R.D., Srivastava, S., Mishra, S., Singh, N., Tuli, R., Gupta, D.K., Maathuis,

469

F.J.M., 2007. Arsenic hazards: strategies for tolerance and remediation by plants.

470

Trends Biotechnol. 25, 158-165.

471

White, P., Bowen, H., Parmaguru, P., Fritz, M., Spracklen, W., Spiby, R., Meacham,

472

M., Mead, A., Harriman, M., Trueman, L., 2004. Interactions between selenium 18

473

and sulphur nutrition in Arabidopsis thaliana. J. Exp. Bot. 55, 1927-1937.

474

Wu, C., Ye, Z., Shu, W., Zhu, Y., Wong, M., 2011a. Arsenic accumulation and

475

speciation in rice are affected by root aeration and variation of genotypes. J. Exp.

476

Bot. 62, 2889-2898.

477

Wu, Z., Ren, H., McGrath, S.P., Wu, P., Zhao, F.J., 2011b. Investigating the

478

contribution of the phosphate transport pathway to arsenic accumulation in rice.

479

Plant Physiol. 157, 498-508.

480 481 482

Yamauchi, H., 1994. Toxicity and metabolism of inorganic and methylated arsenicals. Arsenic in the enviroment, 35-53. Ye, W.L., Khan, M., McGrath, S.P., Zhao, F.J., 2011. Phytoremediation of arsenic

483

contaminated paddy soils with Pteris vittata markedly reduces arsenic uptake by

484

rice. Environ. Pollut. 159, 3739-3743.

485 486 487

Yin, Y., Liu, J., Jiang, G., 2013. Recent advances in speciation analysis of mercury, arsenic and selenium. Chin. Sci. Bull. 58, 150-161. Yu, Y., Zhang, S., Wen, B., Huang, H., Luo, L., 2011. Accumulation and Speciation of

488

Selenium in Plants as Affected by Arbuscular Mycorrhizal Fungus Glomus

489

mosseae. Biol. Trace Elem. Res. 143, 1789-1798.

490

Zhang, H., Feng, X., Zhu, J., Sapkota, A., Meng, B., Yao, H., Qin, H., Larssen, T.,

491

2012a. Selenium in soil inhibits mercury uptake and translocation in rice (Oryza

492

sativa L.). Environ. Sci. Technol. 46, 10040-10046.

493

Zhang, M., Hu, C., Zhao, X., Tan, Q., Sun, X., Cao, A., Cui, M., Zhang, Y., 2012b.

494

Molybdenum improves antioxidant and osmotic-adjustment ability against salt

495

stress in Chinese cabbage (Brassica campestris L. ssp. Pekinensis). Plant Soil 355,

496

375-383.

497 498

Zhang, W., Cai, Y., Tu, C., Ma, L.Q., 2002. Arsenic speciation and distribution in an arsenic hyperaccumulating plant. Sci. Total Environ. 300, 167-177.

499

Zhang, X., Uroic, M.K., Xie, W.Y., Zhu, Y.G., Chen, B.D., McGrath, S.P., Feldmann,

500

J., Zhao, F.-J., 2012c. Phytochelatins play a key role in arsenic accumulation and

501

tolerance in the aquatic macrophyte Wolffia globosa. Environ. Pollut. 165, 18-24.

502

Zhao, F.J., McGrath, S.P., Meharg, A.A., 2010. Arsenic as a food chain contaminant: 19

503

mechanisms of plant uptake and metabolism and mitigation strategies. Annu. Rev.

504

Plant Biol. 61, 535-559.

505 506 507

Zhao, F., Ma, J., Meharg, A., McGrath, S., 2009. Arsenic uptake and metabolism in plants. New Phytol. 181, 777-794. Zhao, J., Gao, Y., Li, Y.F., Hu, Y., Peng, X., Dong, Y., Li, B., Chen, C., Chai, Z.,

508

2013a. Selenium inhibits the phytotoxicity of mercury in garlic (Allium sativum).

509

Environ. Res. 125, 75-81.

510

Zhao, J., Hu, Y., Gao, Y., Li, Y., Li, B., Dong, Y., Chai, Z., 2013b. Mercury modulates

511

selenium activity via altering its accumulation and speciation in garlic (Allium

512

sativum). Metallomics 5, 896-903.

513

Zheng, M.Z., Li, G., Sun, G.X., Shim, H., Cai, C., 2013. Differential toxicity and

514

accumulation of inorganic and methylated arsenic in rice. Plant Soil 365,

515

227-238.

516

Zhu, Y.G., Pilon-Smits, E.A.H., Zhao, F.J., Williams, P.N., Meharg, A.A., 2009.

517

Selenium in higher plants: understanding mechanisms for biofortification and

518

phytoremediation. Trends Plant Sci. 14, 436-442.

519 520 521 522 523 524 525 526 527 528 529 530 531 532 20

533

Fig.1. The biomass in shoots (A) and roots (B) of flue-cured tobacco treated with As

534

and Se for 14 days under hydroponic conditions. The values are mean ± SE (n = 3).

535

Different letters above bars indicate significant difference at P<0.05 (Capital letters

536

denote significance within the same arsenic level at different doses of selenium;

537

lowcase letters denote significance within the same selenium level at different doses

538

of arsenic).

539

Fig.2. The Se concentration in leaves (A) and roots (B) of flue-cured tobacco treated

540

with As and Se for 14 days under hydroponic conditions. The values are mean ± SE (n

541

= 3). Different letters above bars indicate significant difference at P<0.05 (Capital

542

letters denote significance within the same arsenic level at different doses of selenium;

543

lowcase letters denote significance within the same selenium level at different doses

544

of arsenic).

545

Fig.3. Effect of addition of As and Se in the solution on the uptake of As by flue-cured

546

tobacco after being grown for 14 days under hydroponic conditions. Symbols and

547

vertical lines in the curves are means and standard error of means. Each of the curves

548

was drawn based on the related single factor experiment. Fig 3A and 3B illustrate the

549

effects of As and Se on the uptake of As in the leaves and roots. Bars indicate

550

standard error of the mean. Different letters above bars indicate significant difference

551

at P<0.05 (Capital letters denote significance within the same arsenic level at different

552

doses of selenium; lowcase letters denote significance within the same selenium level

553

at different doses of arsenic).

554

Fig.4. Effect of addition of As and Se in the solution on the percentage of Se species

555

[SeCys, SeMet, Se(IV) and Se(VI)] in leaves (A) and roots (B) of flue-cured tobacco.

556

The Se species of leaves and roots treated with no selenium addition were not

557

detected.

558

Fig.5. Effect of addition of As and Se in the solution on the percentage of As species

559

[As(Ⅲ) and As(V)] in leaves (A) and roots (B) of flue-cured tobacco. The As species

560

of leaves and roots treated with no As addition were not detected.

561

Fig.6. Effect of addition of As and Se in the solution on the activities of SOD (A) and

562

POD (B) as well as GSH (C) and AsA (D) contents of flue-cured tobacco’s leaves. 21

563

The values are mean ± SE (n = 3). Different letters above bars indicate significant

564

difference at P<0.05 (Capital letters denote significance within the same arsenic level

565

at different doses of selenium; lowcase letters denote significance within the same

566

selenium level at different doses of arsenic.

567

Fig.7. Effect of addition of As and Se in the solution on MDA content of flue-cured

568

tobacco’s leaves. The values are mean ± SE (n = 3). Different letters above bars

569

indicate significant difference at P<0.05 (Capital letters denote significance within the

570

same arsenic level at different doses of selenium; lowcase letters denote significance

571

within the same selenium level at different doses of arsenic).

572 Fig 1

60

4

Shoot fresh weight (g plant-1)

A

a A a b B C

50

a a AA

B

Se0

a b A B

b B

a A

Se0.1

a AB

Se1 Se5

a ab B C

40 c C

a C

30 20

a A

3

a A b B

ab D

b D

a A a B

a D

2 a B

a C

1

10 0

0 As0

As1

As5

As0

As1

As treatments (mg L-1)

574 575

Root fresh weight (g plant-1)

573

As5

As treatments (mg L-1)

Fig. 2

576 577

Se content in leaves (mg kg-1)

A 10 8

a A

b A

a

a D

a C

a C a D

As1

300 200

a C

a C a D

400

100

c B

4 2

B

b B

b A

a B

b B

As0

578

c A

a B

Se0 Se0.1 Se1 Se5

6

0

b A

a A

B

20 b C

b C a

a C

Se content in roots (mg kg-1)

12

b C

C

0 As5

As treatments (mg L-1)

As0

As1

As5

As treatments (mg L-1)

22

579

Fig. 3

580

As content in leaves(mg kg-1)

Se 0 Se 0.1 Se 1 Se 5

15

a AB a

A

400

B a

a

B a

C

C a C

b b A A

b A

10

5 B

0

B

c

c Ac B B

As0

c C

As1

As5

c c c A B A

As0

As treatments (mg L-1)

581

300

100

b b b b A b AB AB B c

582

A

a

20

500

a

B

A

As content in roots (mg kg-1)

25

0 As1

As5

As treatments (mg L-1)

Fig.4

583

100

100

SeCys SeMet Se(IV) Se(VI)

80

60

60

40

40

20

20

0

0 s0 s1 s5 s0 s1 s5 s0 s1 s5 s0 s1 s5 0 A 0A 0A .1A .1A .1A 1A 1A 1A 5A 5 A 5A S e Se Se Se0 Se0 Se0 Se Se Se Se S e Se

584

Se(IV) and Se(VI)

Percentage of SeCys, SeMet, Se(IV) and Se(VI)

80

Percentage of SeCys, SeMet,

B

A

Treatments (mg kg-1)

s0 s1 s5 s0 s1 s5 s0 s1 s5 s0 s1 s5 0A 0A 0A .1A .1A .1A e1A e1A e1A e5A e5A e5A Se S e Se Se0 Se0 Se0 S S S S S S

Treatments (mg kg-1)

585 586 587 588 589 590 591 592 593 594 23

595

Fig.5

596

100

100 B

As(III) As(V)

80

80

60

60

40

40

20

20

0

598

0 s 0 s0 s0 s0 s1 s1 s1 s1 s 5 s5 s5 s5 0A 1A 1A 5A 0A 1A 1A 5A 0A 1A 1A 5A Se Se0. Se Se Se Se0. Se Se Se Se0. Se Se Treatments (mg kg-1)

597

Percentage of As(III) and As(V)

Percentage of As(III) and As(V)

A

s0 s0 s0 s0 s1 s1 s1 s1 s5 s5 s5 s5 0A 1A 1A 5A 0A 1A 1A 5A 0A 1A 1A 5A Se Se0. Se Se Se Se0. Se Se Se Se0. Se Se Treatments (mg kg-1)

Fig.6

599

500

a ab A A

Se0 Se0.1 Se1 Se5

400

a

b A

A

b B

a

C

A

a a b A b AB BC C

a b B C

a A

a a a A AB a B B

b D

POD activity (U g-1 min-1 FW-1 )

300 250

a B

200

300 150

a C

b B

c

200

C

100

b C

b D

100

50

0

0 a A

B

A

800

600

a A

D

a

a a A A

40

b A

400

a B

a B

a a B b C D c c C D

b b C C

ab b b b A A AB B

b b B BC c C

a a B B

30

20

10

200

0

0

As0

600

350

AsA content (ug g-1 FW-1 )

SOD activity (U g-1 FW-1 )

A

GSH content (ug g-1 FW-1)

600

As1 As treatments (mg L-1)

As5

As0

As1

As5

As treatments (mg L-1)

601 602 603 604 24

605

Fig. 7

6

4

a B

b A

-1

MDA content(nmol g )

5

a A

Se 0 Se 0.1 Se 1 Se 5 a C a D

b B

3

c A

b D

a C

a c Ba B B

2 1 0 As0

606

As1

As5

As treatments (mg L-1)

607

25