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Thymol Ameliorates Cadmium-Induced Phytotoxicity in the Root of Rice (Oryza sativa) Seedling by Decreasing Endogenous Nitric Oxide Generation Ting-Ting Wang, Zhi Qi Shi, Liang-Bin Hu, Xiao-Feng Xu, Fengxiang X Han, Ligang Zhou, and Jian Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02950 • Publication Date (Web): 03 Aug 2017 Downloaded from http://pubs.acs.org on August 3, 2017

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Journal of Agricultural and Food Chemistry

1

Thymol Ameliorates Cadmium-Induced Phytotoxicity in the

2

Root of Rice (Oryza sativa) Seedling by Decreasing

3

Endogenous Nitric Oxide Generation

4

Ting-Ting Wang,†,§ Zhi Qi Shi,†,§ Liang-Bin Hu,# Xiao-Feng Xu,§ Fengxiang X. Han,∆

5

Li-Gang Zhou,▲ and Jian Chen*,#,†,‡

6 7

†

8

Nanjing 210014, China

9

§

College of Life Sciences, Nanjing Normal University, Nanjing 210064, China

10

#

Department of Food Science, Henan Institute of Science and Technology, Xinxiang

11

453003, China

12

∆

13

39217, USA

14

▲

15

China

16

‡

17

Laboratory Breeding Base, Jiangsu Provincial Department of Agriculture and Forestry,

18

Nanjing 210014, China

Institute of Food Quality and Safety, Jiangsu Academy of Agricultural Sciences,

Department of Chemistry and Biochemistry, Jackson State University, Jackson, MS

Department of Plant Pathology, China Agricultural University, Beijing 100193,

Key Laboratory of Food Quality and Safety of Jiangsu Province-State Key

19 20 21

Corresponding Author

22

*(J.C.) Phone: +86-25-84391863; Fax: +86-25-84390422

23

E-mail: [email protected]

24 1

ACS Paragon Plus Environment


25

ABSTRACT

26

Thymol has been developed as medicine and food preservative due to its

27

immune-regulatory effect and anti-microbial activity, respectively. However, little is

28

currently known about the role of thymol in the modulation of plant physiology. In the

29

present study, we applied biochemical and histochemical approaches to investigate

30

thymol-induced tolerance in rice (Oryza sativa) seedlings against Cd (cadmium) stress.

31

Thymol at 20 µM recovered root growth completely upon CdCl2 exposure. Thymol

32

pronouncedly decreased Cd-induced ROS accumulation, oxidative injury, cell death,

33

and Cd2+ accumulation in roots. Pharmaceutical experiments suggested that

34

endogenous NO mediated Cd-induced phytotoxicity. Thymol decreased Cd-induced

35

NO accumulation by suppressing the activity of NOS (nitric oxide synthase) and NR

36

(nitrate reductase) in root. The application of NO donor (SNP, sodium nitroprusside)

37

resulted in the increase in endogenous NO level, which in turn compromised the

38

alleviating effects of thymol on Cd toxicity. Such findings may helpful to illustrate the

39

novel role of thymol in the modulation of plant physiology, which maybe applicable

40

to improve crop stress tolerance.

41 42

KEYWORDS: thymol, Oryza sativa, cadmium, nitric oxide, phytotoxicity

43 44 45 46 2


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INTRODUCTION

48

Cd (Cadmium) pollution is one of the most important environmental problems

49

worldwide.1 In agricultural environment, Cd poses thereat to crop growth and food

50

safety. Growth inhibition has been characterized as the most frequently-occurred

51

symptom of the phytotoxicity induced by Cd. Cd exposure always induces ROS

52

(reactive oxygen species) accumulation in plants. The over-generated ROS further

53

attacks macromolecules (e.g. membrane lipids, nucleic acids, and proteins), resulting in

54

oxidative stress, cell death, and growth inhibition in plants upon Cd exposure.2 It is well

55

documented that Cd-induced phytotoxicity involves a complex regulatory network with

56

multiple interactions among plant signaling molecules.3 Cd-triggered ROS signaling

57

and cell death can be regulated by various plant signaling modulators, such as NO

58

(nitric oxide),4 Ca2+/calmodulin,5 and protein kinases,6 etc. NO seems to be a

59

regulatory node during cadmium sensing.4

60

NO is not only a hazardous gaseous molecule but also a multifunctional regulator in

61

both mammals and plants. Plant endogenous NO is mainly generated through NOS

62

(nitric oxide synthase) and NR (nitrate reductase).7 Deoxygenated hemeproteins and

63

polyamines have been associated with endogenous NO generation as well, but the

64

manipulating mechanisms remain unclear.7 It has been reported that exogenous

65

application of NO donor SNP (sodium nitroprusside) is capable of protecting plant

66

from Cd stress.8-10 However, the physiological roles of endogenous NO in the

67

modulation of plant tolerance against Cd stress still remain controversial. In contrast to

68

the above reports, several studies suggest that endogenous NO can contribute to Cd

69

toxicity in Arabidopsis,11 barley,12 and tobacco.13 Therefore, it is important to focus on

70

endogenous NO level to understand the heterogeneous function of NO in plants upon

71

Cd exposure.4 3



72

Basically, it is hard to remove Cd permanently from the environment. Alternatively,

73

plant resistant physiology can be regulated exogenously to combat Cd stress.14

74

Thymol is an important component of essential oil extracted from Thymus vulgaris.15

75

Thymol has been developed as medicine due to its medicinal properties, such as

76

anti-inflammatory activity,16 anti-oxidative activity,17 and anti-microbial activity.18

77

The regulation of immune pathways by thymol has been closely linked to the

78

modulation of endogenous NO and protein kinase signaling in mammalian cells.16

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Compared to the extensive study on medicinal and anti-microbial properties of thymol,

80

the knowledge for thymol-regulated plant physiology and its manipulating mechanism

81

are still very limited. Thymol is probably a new candidate for the development of

82

environmental-friendly agrochemical based on the risk evaluation by U.S.EPA

83

(United States Environmental Protection Agency) Office of Pesticide Programs.19 Our

84

previous study have found that thymol has the ability to confer plant tolerance against

85

Cd toxicity,20 but its signaling regulatory mechanism needs to be revealed.

86

In this work, we performed detailed analysis of thymol on the alleviation of

87

CdCl2-induced phytotoxicity in the roots of rice seedling. The possible role of

88

endogenous NO in thymol-facilitated Cd tolerance was investigated by detecting

89

endogenous NO production. Then endogenous NO level was altered by using NO

90

donor, NO scavenger, or inhibitors of NO production, in order to investigate the

91

relationship between NO and other biochemical parameters (e.g. ROS, cell death, and

92

root growth) under the treatment of CdCl2 or thymol. Finally, the manipulating

93

mechanisms for thymol modulating the above physiological processes are discussed,

94

which would advocate a positive role for thymol in helping plants against metal toxicity

95

by decreasing endogenous NO.

96

MATERIALS AND METHODS 4


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Plant Culture, Treatment, and Chemicals. Seeds of commercial rice (Oryza

98

sativa) (Nangeng 9108) were obtained from Institute of Food Crops, Jiangsu

99

Academy of Agricultural Sciences. The seeds were placed on a floating net for

100

germination. After germination for two days, thirty selected identical seedlings were

101

transferred to another floating plastic net in a new container with modified Kimura B

102

nutrient solution [2 mM KNO3, 1 mM KCl, 0.36 mM CaCl2, 0.18 mM KH2PO4, 0.54

103

mM MgSO4, 40 µM Fe(II)-EDTA (ethylenediaminetetraacetic acid), 18.8 µM H3BO3

104

(boric acid), 0.03 µM Na2MoO4 (sodium molybdate), 0.32 µM CuSO4, 13.4 µM

105

MnCl2, 0.3 µM ZnSO4, and 1.6 mM Na2SiO3, pH 6.0].21 The chamber for seedling

106

growth were set up with the condition of temperature at 28°C, photoperiod of 12 h,

107

and photosynthetic active radiation of 200 µmol/m2/s.

108

The roots of seedlings were treated with CdCl2 (0-8 µM) and thymol (0-40 µM),

109

alone or their combination. The NO-related reagents were selected according to our

110

previous study.22 Exogenous NO donor: SNP (20 µM); NO scavenger: 20 µM of

111

cPTIO (2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide); NOS

112

activity inhibitor: 30 µM of L-NMMA (NG-monomethyl-L-arginine); NR activity

113

inhibitor: Na2WO4 (Sodium Tungstate) at 30 µM. All the chemicals were purchased

114

from Sigma-Aldrich (St. Louis, MO, USA) at analytical purity. Various combinations

115

of the above chemicals were designated to treat seedling roots according to specific

116

experimental setup. After that, root samples were harvested and prepared immediately

117

for further analysis.

118

Fluorescence-Based Histochemical Analysis. Root samples used for fluorescent

119

detection were washed with distilled water followed by incubation with specific

120

fluorescent probe. Then the excessive probes attached on root surface were washed

121

away with distilled water. Afterwards, the roots were visualized and captured using a 5



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fluorescence microscope (ECLIPSE, TE2000-S, Nikon, Melville, NY, USA) at

123

specific wavelength according to different fluorescent probes. Image-Pro Plus 6.0

124

(Media Cybernetics, Inc., Rockville, MD, USA) was applied to calculate relative

125

fluorescent density.

126

Total ROS in roots was detected in situ using DCFH-DA (2′,7′-dichlorofluorescein

127

diacetate) (Beyotime Biotechnology Institute, Haimen, China).23 The harvested roots

128

were

129

(2′,7′-dichlorofluorescein) fluorescence was detected under excitation 488 nm and

130

emission 525 nm.

loaded

with

DCFH-DA

(10

µM)

for

10

min

at

25°C.

DCF

131

Intracellular H2O2 (hydrogen peroxide) was detected in situ using HPF

132

[3′-(p-hydroxyphenyl) fluorescein] (Invitrogen, Ltd. Paisley, UK).24 The harvested

133

roots were loaded with HPF (5 µM) for 15 min at 25°C. HPF fluorescence was

134

detected under excitation 490 nm and emission 515 nm.

135

Intracellular O2•¯ (superoxide radical) was detected in situ using DHE

136

(dihydroethidium) (Beyotime Biotechnology Institute, Haimen, China).25 The

137

harvested roots were loaded with DHE (15 µM) for 15 min at 25°C. DHE

138

fluorescence was detected under excitation 535 nm and emission 610 nm.

139

Intracellular NO was detected in situ using DAF-FM DA (3-amino,

140

4-aminomethyl-2′,7′-difluorescein, diacetate) (Beyotime Biotechnology Institute,

141

Haimen, China).26 The harvested roots were loaded with DAF-FM DA (15 µM)

142

dissolved

143

acid-potassium hydroxide] (20 mM, pH 7.5) for 15 min at 25°C. DAF (3-amino,

144

4-aminomethyl-2′,7′-difluorescein) fluorescence was detected under excitation 490

145

nm and emission 525 nm.

146

in

HEPES-KOH

[4-(2-Hydroxyethyl)-1-piperazineethanesulfonic

Root cell death was detected in situ using PI (propidium iodide) (Beyotime 6


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Biotechnology Institute, Haimen, China).20 The harvested roots were loaded with PI

148

(20 µM) for 20 min at 25°C. PI fluorescence was detected under excitation 535 nm

149

and emission 615 nm.

150

Free Cd2+ inside of root cells was detected in situ using LeadmiumTM Green AM

151

(Invitrogen, Ltd. Paisley, UK).27 The harvested roots were washed in 1 mM of EDTA

152

(ethylenediamine tetra-acetic acid) for 3 min followed by rinsing with distilled water

153

for three times to remove Cd2+ attached on root surface. Then roots were loaded with

154

LeadmiumTM Green AM (1 µg/mL) for 60 min at 25°C. LeadmiumTM Green

155

fluorescence was detected under excitation 488 nm and emission 525 nm.

156

Non-Fluorescent

Histochemical

Detection.

Root

samples

used

for

157

non-fluorescent detection were washed with distilled water followed by incubation

158

with specific staining solution. Then the excessive dye attached on root surface were

159

washed away with distilled water. Afterwards, the roots were visualized and captured

160

using a stereoscopic microscope (SteREO Discovery.V8, ZEISS, Oberkochen,

161

Germany).

162

Intracellular H2O2 was also visualized using DAB (3,3-diaminobenzidine)

163

staining.20 The harvested roots were incubated in DAB-HCl (hydrogen chloride)

164

solution (0.1%, w/v, pH 3.8) for 20 min. DAB reacted with H2O2 to produce brown

165

product in roots, which were further observed and captured.

166

Intracellular O2•¯ was also visualized using NBT (nitro-blue tetrazolium)

167

(Sigma-Aldrich St. Louis, MO, USA) staining.20 The harvested roots were incubated

168

in NBT (6 mM) dissolved in Na-citrate buffer (10 mM, pH 6.0) under light for 20 min

169

at 25°C. NBT reacted with O2•¯ to produce dark blue product in roots, which were

170

further observed and captured.

171

Lipid peroxidation of root cells was visualized using Schiff′s reagent staining.28 7



172

The harvested roots were stained with Schiff′s reagent for 20 min. Then roots were

173

washed with K2S2O5 (0.5%, w/v) dissolved in HCl (0.05 M) to exhibit light red,

174

which were further observed and captured.

175

Loss of plasma membrane integrity in roots was visualized using Evans blue

176

(Sigma-Aldrich St. Louis, MO, USA) staining.29 The harvested roots were stained

177

with Evans blue (0.025%, w/v) for 20 min to exhibit blue, which were further

178

observed and captured.

179

Root cell death was also visualized using trypan blue (Sigma-Aldrich St. Louis,

180

MO, USA) staining.20 The harvested roots were stained with trypan blue (10 mg/mL)

181

for 20 min to exhibit blue, which were further observed and captured.

182

Determination of H2O2 and O2•¯ content in Root. For the measurement of H2O2

183

content, hydroxylamine (1 mM) dissolved in 1.5 mL of phosphate buffer (50 mM, pH

184

6.5) was applied to homogenize fresh root samples (0.1 g). Then the mixture was

185

centrifuged for 10 min at 10,000 g. Supernatant (0.5 mL) was collected and mixed

186

thoroughly with TiCl4 (0.1% v/v) dissolved in 1.5 mL of H2SO4 (20%, v/v). After

187

centrifugation for 10 min at 10,000 g, supernatant was collected for the measurement

188

of absorbance at 410 nM. The extinction coefficient of 0.28 /µM/cm was used to

189

calculate H2O2 concentration.30

190

For the measurement of O2•¯ content, 1.5 mL of phosphate buffer (50 mM, pH 7.8)

191

was applied to homogenize fresh root samples (0.1 g). Then the mixture was

192

centrifuged at 4°C for 10 min at 5,000 g. Supernatant (0.5 mL) was collected for the

193

addition of hydroxylamine hydrochlorides (1 mM, 1 mL) and phosphate buffer

194

(50mM, pH 7.8, 0.5 mL). After reaction at 25°C for 1 h, the above mixture was added

195

with p-aminobenzene sulfonic acid (17 mM, 1 mL) and phosphate buffer (50mM, pH

196

7.8, 0.5 mL). Another reaction at 25°C for 25 min was allowed for the final 8


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measurement of absorbance at 530 nM. NaNO2 was applied to prepare a standard

198

curve for the calculation of O2•¯ content base on fresh weight.31

199

Determination of Enzymatic Activity of NR and NOS in Root. Ice-cooled

200

phosphate buffer (50 mM, pH 7.0, containing 1 mM EDTA and 1% w/v insoluble

201

polyvinylpyrrolidone) was applied to homogenize fresh root samples (0.05 g). Then

202

the mixture was centrifuged 4°C for 10 min at 15,000 g. Then supernatant was

203

collected as crude extract for the measurement of enzymatic activity.

204

NR activity was measured according to the method of Xiong et al.32 Enzymatic

205

crude extract was mixed with pre-warmed HEPES-KOH buffer (100 mM, pH 7.5)

206

containing KNO3 (5 mM) and NADH (nicotinamide adenine dinucleotide hydrogen)

207

(0.25 mM). After reacting for 60 min at 30°C, Zn-acetate was added to terminate the

208

reaction. Then the mixture was added with sulfanilamide (1 mg/L) dissolved in HCl

209

(3 M) and N-(1-naphthyl) ethylenediamine (1 mg/L) to generate nitrite, which was

210

further measured the absorbance at 540 nM. NaNO2 was applied to prepare a standard

211

curve for the calculation of the relative NR activity based on total protein content.

212

A commercial NOS colorimetric kit (A014-2; Nanjing Jiancheng Bioengineering

213

Institute, Nanjing, China) was selected to determine NOS activity according to

214

manufacturer’s

215

spectrophotometrically by quantifying the capacity to catalyze the generation of NO

216

from L-arginine, while NO generation was determined by the oxidation process of

217

oxyhaemoglobin to methaemoglobin. Relative NOS activity with 1 unit was

218

designated as the generation of NO (1 nmol) at 37°C per minute per mg protein.

219 220 221

instruction.33

The

activity

of

NOS

was

measured

Total protein content in crude extract was determined based on standard bovine serum albumin as described by Bradford.34 Measurement of Root TBARS Concentration. TBARS (thiobarbituric acid 9



222

reactive substances) concentration is always used to evaluate lipid peroxidation in

223

cells. A commercial TBARS kit (A003; Nanjing Jiancheng Bioengineering Institute,

224

Nanjing, China) was applied to determine TBARS content in roots according to

225

manufacturer’s instruction. TBA (1,3-diethyl-2-thiobarbituric acid) was allowed to

226

react with TBARS in the presence of TCA (trichloroacetic acid), which was further

227

spectrophotometrically measured to calculated TBARS content.35

228

Determination of Cd Content in Plant Tissue. Roots and shoots were harvested,

229

respectively. Roots were rinsed with of EDTA (1 mM) for 3 mins. Afterwards, plant

230

samples were oven-dried followed by digestion with HNO3 using a Microwave

231

Digestion System MARS 6 (CEM, Matthews, NC, USA). Then ICP-MS (Inductively

232

Coupled Plasma-Mass Spectrometer) (iCAPTM Q, Thermo Scientific, Waltham, MA,

233

USA) was applied to determine total Cd content in plant tissue.36

234

Cluster Analysis. Selected data under specific treatment was prepared as relative

235

fold change (log2) respect to control, which was further performed for hierarchical

236

cluster analysis using Cluster 3.0. Then Java Treeview was applied to display tree

237

figure generated from Cluster 3.0.37

238

Statistical Analysis. The result was shown as the average of at least three

239

replicates with standard deviation (SD). One-way analysis of variance (ANOVA) was

240

calculated to evaluate the difference significantly using SPSS 14.0 (Statistical

241

Package for the Social Science, SPSS Inc., Chicago, IL). To evaluate the significant

242

difference between two specific treatments, ANOVA was compared significantly

243

followed by F-test at p < 0.05. To evaluate the significant difference among multiple

244

treatments, least significant difference test (LSD) was applied to compare ANOVA

245

results significantly at p < 0.05.

246

RESULTS 10


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Thymol Alleviated Cd-Inhibited Rice Root Growth. Treatment with CdCl2 for

248

72 h significantly inhibited root growth in a dose-dependent manner. Root length

249

significantly decreased by 17.60%, 38.55%, 61.73%, and 72.91% at 2, 4, 6, and 8 µM

250

of CdCl2 level, respectively, as compared to control (Figure S1). CdCl2 at 4 µM was

251

selected for further experiments because it resulted in moderate root inhibition.

252

Simultaneous treatment with Cd (4 µM) and thymol at 5, 10, 20, and 40 µM led to the

253

significant increase in root length by 52.97%, 69.64%, 81.29%, and 29.45%,

254

respectively, as compared to Cd treatment alone (Figure 1A). Thymol also blocked the

255

decrease in root fresh weight under Cd stress (Figure 1B). In Cd-treated roots, the

256

fresh weight remarkably increased by 16.87%, 23.04%, 39.30%, and 15.23% under

257

the treatment of thymol at 5, 10, 20, and 40 µM, respectively, as compared to Cd

258

treatment alone (Figure 1B). In Cd-free solution, thymol at 5-20 µM did not

259

significantly impact root growth. However, treatment with thymol at 40 µM alone

260

significantly inhibited root growth (Figure 1A and 1B). In time-course experiments

261

(up to 72 h), root growth was significantly inhibited after Cd (4 µM) treatment for

262

24-72 h, whereas neither root length nor root fresh weight showed significant

263

difference between the control group and Cd+thymol treatment (Figure 1C and 1D).

264

Since 20 µM of thymol had the greatest ability for the recovery of root growth upon

265

Cd stress (Figure 1E), thymol at 20 µM was used to evaluate its effect on biochemical

266

parameters in Cd-treated root in the following experiments.

267

To avoid the in vivo interaction between thymol and Cd, we studied the effect of

268

thymol pretreatment with on root growth upon Cd exposure. Roots were treated with 11



269

of seedlings were treated with 20 µM thymol for 12 h, followed by transferring to

270

CdCl2 solution (4 µM) for another 72 h. As expected, pretreatment with thymol still

271

significantly alleviated Cd-inhibited root growth (Figure S2).

272

Thymol Inhibited Cd-Induced ROS Accumulation in Rice Root. Fluorescent

273

probe DCFH-DA reacted with total ROS in situ to exhibit green fluorescence in root.

274

Roots treated with Cd alone exhibited stronger DCF fluorescence compared to control.

275

However, thymol has the ability to weaken DCF fluorescence in Cd-treated roots

276

(Figure 2A). The relative total ROS content was indicated by the quantification of

277

DCF fluorescent density. Compared to the control group, Cd treatment induced

278

significant increase in relative total ROS content by 170.75% (Figure 2B). Thymol

279

supplement led to the remarkable decrease in total ROS back to control level in

280

Cd-treated root (Figure 2B). In addition, compared to Cd treatment alone, roots

281

pretreated with thymol followed by Cd treatment (Thymol→Cd) showed significant

282

decrease in the content of total ROS (Figure S3).

283

O2•¯and H2O2 are two representatives of ROS in plant cells. Both fluorescent

284

detection (Figure 3A and 3B) and chemical staining (Figure 3C) indicated that Cd

285

exposure induced remarkable accumulation of H2O2 in root, which was inhibited by

286

the addition of thymol. The in-tube assay of H2O2 content was performed by

287

spectrophotometrical measurement. Compared to control, H2O2 content in Cd-treated

288

roots remarkably increased by 62.75% (Figure 3D). However, H2O2 content in root

289

treated with Cd+thymol increased by only 29.41% as compared to control (Figure 3D).

290

Histochemical detection of O2•¯ indicated that thymol pronouncedly blocked 12


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291

Cd-induced O2•¯ accumulation in root (Figure 3E-G). Determination of O2•¯ content

292

indicated that O2•¯ concentration increased by 56.91% in Cd-treated roots than control

293

group (Figure 3H). However, O2•¯ content in root treated with Cd+thymol increased

294

by only 14.16% as compared to control (Figure 3H).

295

Thymol Inhibited Cell Death and Oxidative Damage in Rice Root under Cd

296

Stress. Cd-treated roots showed extensive staining of Shiff’s reagent indicating lipid

297

peroxidation, while only light staining was observed in control or roots under

298

Cd+thymol treatment (Figure 4A). Histochemical detection of loss of membrane

299

integrity with Evans blue also showed similar staining patterns with Shiff’s reagent

300

upon different treatments (Figure 4B). Lipid peroxidation can be indicated by the

301

concentration of TBARS. The content of TBARS pronouncedly increased by 96.43%

302

in Cd-treated roots than in control group (Figure 4C). However, Cd+thymol treatment

303

led to remarkable decrease in root TBARS content by 43.64% than Cd treatment

304

alone (Figure 4C). In addition, the addition of thymol effectively inhibited Cd-induced

305

cell death indicated in situ by trypan blue staining (Figure 4D).

306

Thymol Decreased Free Cd2+ Level in Rice Root. First, total Cd content in plant

307

tissues was determined by ICP-MS. The results showed that treatment with thymol

308

didn’t significantly affect total Cd concentration in both roots shoots and under CdCl2

309

treatment (Figure 5A). Then root free Cd2+ was labeled in situ with LeadmiumTM

310

Green AM to emit green fluorescence (Figure 5B). The fluorescence was not detected

311

in control and thymol-treated roots (Figure 5B). Cd-treated roots showed strong

312

fluorescence of LeadmiumTM Green. Compared to Cd treatment alone, thymol 13



313

addition resulted significantly decreased the fluorescent density of LeadmiumTM

314

Green in roots by 74.68% as (Figure 5C).

315

Thymol Inhibited NOS- and NR-Dependent NO Accumulation Induced by Cd

316

in Rice Root. Endogenous NO in root was detected in situ by specific probe DAF-FM

317

DA to emit green fluorescence (Figure 6A). Relative NO content can be indicated by

318

DAF fluorescent density. Relative NO content in roots treated with Cd was 9.75 times

319

higher than in control roots (Figure 6B). However, compared to Cd treatment alone,

320

Cd+thymol treatment resulted in remarkable decrease in NO content in roots (Figure

321

6B).

322

The increase in endogenous NO level induce by Cd could be inhibited by the

323

cPTIO (a NO scavenger), tungstate (NR inhibitor), and L-NMMA (a potent NOS

324

inhibitor) (Figure 6B). SNP (NO donor) application significantly enhanced the level

325

of endogenous NO in roots under either Cd or Cd+thymol treatment (Figure 6B).

326

Compared to Cd treatment alone, pretreatment with thymol followed by Cd treatment

327

(Thymol→Cd) resulted in significant decrease in endogenous NO level in roots

328

(Figure S4). To further ascertain the NO-producing pathways in roots upon Cd

329

exposure, the activity of NOS and NR in roots was determined, respectively. As

330

expected, treatment with Cd alone led to remarkable increase in the activity of NOS

331

and NR, both of which were inhibited by the addition of thymol (Figure 6C and 6D).

332

Endogenous NO was Involved Thymol-ameliorated Rice Root Growth

333

Induced by Cd Stress. Root length was measured under the conditions of changing

334

the level of endogenous NO level in roots treated with Cd or thymol. Addition of 14


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335

L-NMMA,

tungstate, or cPTIO was capable of alleviate Cd-induced inhibition of root

336

growth, showing similar action with that of thymol (Figure 7). The alleviating effect

337

of thymol on Cd-inhibited root growth was compromised by the addition of SNP

338

(Figure 7). In addition, SNP aggravated Cd-induced inhibition of root growth (Figure

339

7).

340

Endogenous NO was Involved in Thymol-Inhibited Cell Death and ROS

341

Accumulation in Cd-Treated Rice Root. Cell death in root was indicated in situ by

342

specific probe PI to emit red fluorescence (Figure 8A). Quantitative analysis of PI

343

fluorescent density indicated that Cd stress induced cell death in roots. Thymol

344

treatment showed obvious effect on the inhibition of Cd-induced cell death (Figure

345

8B). However, SNP application enhanced PI fluorescent density in roots treated with

346

Cd+thymol (Figure 8B). In Cd-treated roots, the addition of cPTIO, L-NMMA, or

347

tungstate significantly reduced PI fluorescent density, whereas the addition of SNP

348

remarkably enhanced PI fluorescent density (Figure 8B). Total ROS indicated by DCF

349

fluorescence showed very similar changing pattern with PI in roots upon different

350

treatments (Figure 9).

351

Hierarchical Cluster Analysis for the regulation of NO by Thymol in

352

Cd-Treated Rice Root. The data for NO, PI, total ROS, and RL (root length) (Figure

353

6-9) were selected to perform hierarchical clustering (Figure 10). Each parameter

354

under a specific treatment was calculated as the fold change of the corresponding

355

control. Black indicates that there was not significant change between treatment and

356

control. Red indicates the increase of designated treatment as compared to control 15



357

while green indicates the decrease of designated treatment as compared to control.

358

Hierarchical cluster analysis was performed based on the folder change of each value.

359

PI and NO and were grouped together (Figure 10A), suggesting that NO was

360

closely related to cell death. In addition, NO, PI, and ROS were clustered together,

361

which was related to RL (root length) negatively (Figure 10B). The relationship of

362

different treatments was analyzed as well. Cd and Cd+thymol+SNP were clustered

363

together (Figure 10C), indicating that thymol-facilitated changes of all the parameters

364

in Cd-treated roots could be compromised by SNP. Cd+cPTIO, Cd+L-NMMA, and

365

Cd+Tungstate were grouped together (Figure 10D), suggesting that blockade of

366

endogenous NO reversed all the toxic effects of Cd on root.

367

DISCUSSION

368

Thymol shows great potential to protect mammalian cells from stress conditions

369

including metal toxicity.38 In addition, thymol has been applied to improve fruits

370

quality,39 implicating the possible regulation of plant physiology by thymol. However,

371

how thymol regulates plant resistant physiology remains elusive. Our current study

372

provided important evidences to indicate that thymol conferred Cd tolerance in rice

373

root by repressing NO-dependent ROS accumulation and cell death. First, thymol

374

remarkably ameliorated Cd-induced toxicity, including the recovery root growth,

375

inhibition of ROS accumulation and cell death, and alleviation of oxidative injury.

376

Secondly, Cd exposure induced considerable NOS- and NR-dependent NO production

377

in roots. However, application of NO scavenger, NOS inhibitor, or NR inhibitor

378

effectively suppressed Cd-induced increase in endogenous NO, coinciding with Cd

379

detoxification. Thirdly, thymol was able to block Cd-induced over-generation of NO 16


Page 16 of 46

Page 17 of 46


380

by inhibiting NOS and NR activity. Fourthly, SNP-facilitated enhancement of

381

endogenous NO level in root compromised all the alleviating effects of thymol on

382

Cd-induced phytotoxicity. Finally, pretreatment with thymol was able to recover root

383

growth and to suppress the accumulation of NO and ROS under the subsequent

384

exposure of Cd.

385

Determination of root growth has been suggested as a reliable approach for the

386

evaluation of phytotoxicity induced by pollutants including heavy metals.40 Our

387

present results indicated that thymol remarkably recovered root growth of rice

388

seedlings upon Cd exposure, suggesting that thymol was able to confer plant tolerance

389

against Cd stress. Thymol at 5-20 µM stimulated root growth under Cd exposure in a

390

dose-dependent manner. However, thymol at high concentration (40 µM) showed

391

decreased capability to restore root growth under Cd exposure. The phytotoxic and

392

cytotoxic effect of thymol has been reported in plant bioassays.41 Here we also found

393

that thymol at 40 µM alone significantly inhibited root growth in Cd-free conditions.

394

Therefore, the phytotoxicity induced by thymol at high concentration may partially

395

compromise its protective effect against Cd stress.

396

NO plays dual functions during immune responses in mammalian cells. NO plays

397

important roles in cell protection, but endogenous NO has been considered as an

398

important signaling molecule triggering cell death in mammals.42 The similar role of

399

NO has been found in plants as well.11-13 In the present study, NO-deprived root

400

showed enhanced tolerance to Cd, suggesting that endogenous NO in rice root

401

mediated Cd-induced phytotoxicity. Thymol-conferred Cd tolerance was probably

402

ascribed to the suppression of NR- and NOS-dependent NO generation in rice root. In

403

mammalian cells, anti-inflammatory activity of thymol has been associated to its

404

capability to suppress NOS expression and NO production.16 Therefore, NO seems to 17



405

be a common signal in responses to thymol in both plants and mammals when thymol

406

helps cells combat stress conditions.

407

Cd-induced plant growth inhibition has been largely attributed to ROS-facilitated

408

oxidative injury (e.g. plasma membrane damage andlipid peroxidation) followed by

409

cell death.2 In the present study, thymol rescued Cd-triggered oxidative damage and

410

cell death in root, which may result from the decline in ROS accumulation (e.g. O2•¯

411

and H2O2). In vitro test suggests that thymol can quench ROS directly,43 but our

412

current results suggested that thymol-inhibited endogenous NO generation was also

413

important for the suppression of Cd-induced ROS accumulation in rice roots.

414

Compared to NO scavenger or NO-biosynthesis inhibitors, thymol exhibited similar

415

abilities to decrease endogenous NO level and ROS accumulation in Cd-treated roots.

416

NO-facilitated ROS generation is one of the most important signaling events to trigger

417

cell death in plants and mammals. In Arabidopsis suspension cultures, endogenous

418

NO-mediated H2O2 accumulation is required for Cd-induced cell death.44 Therefore, it

419

can be speculated that thymol may prevents cell death by suppressing the interaction

420

between NO and H2O2 in rice root under Cd stress.

421

NADPH (nicotinamide adenine dinucleotide phosphate) oxidase has been

422

recognized as an important enzymatic origin for ROS generation in mammals and

423

plants under stress conditions.45 NADPH oxidase-derived ROS production contributes

424

to cell death in tobacco under Cd stress.46 Intriguingly, NO is able to regulate cell

425

death through NADPH oxidase modification (S-nitrosylation) during plant immune

426

responses.47 In mammalian study, the inhibition of NADPH oxidase activity and ROS

427

production

428

macrophages.48 Total of nine genes encoding for NADPH oxidase homologues have

429

been characterized in rice.49 Therefore, it is of interest to further identify the NDAPH

by

thymol

has

been

found

in

lipopolysaccharide-stimulated

18


Page 18 of 46

Page 19 of 46


430

oxidase homologue possibly regulated by thymol during the inhibition of

431

NO-dependent ROS accumulation in rice root upon Cd exposure.

432

Root elongation is mainly dominated by the activity of meristem, which is

433

regulated by auxin.50 High level of endogenous NO inhibited meristem activity by

434

reducing auxin transport, which further inhibits the elongation of root.51 Intriguingly,

435

Cd inhibits the growth of root meristem through NO-repressed auxin signaling.52

436

Therefore, whether thymol rescues root growth under Cd stress by abolishing

437

NO-repressed meristem activity needs to be illuminated further.

438

In the present study, there was not significant difference for the total Cd content in

439

plant tissues between Cd and thymol+Cd treatment. This suggests that thymol cannot

440

impact the accumulation of total Cd in rice seedling. However, thymol significantly

441

decreased free Cd2+ level in rice root, which may contribute to the detoxification of

442

Cd by thymol via regulating NO. Endogenous NO has been evidenced to facilitate

443

Cd2+ transportation in plants. NO promotes Cd2+ influxes into tobacco suspension

444

cells.13 The increase in endogenous NO regulates the expression of IRT1

445

(IRON-REGULATED TRANSPORTER 1), leading to the further accumulation of Cd2+

446

in the roots of Arabidopsis.11 Glutathione is an important molecule protecting plant

447

from Cd toxicity by scavenging ROS or chelating free Cd2+.53 Our previous study

448

demonstrated that thymol enhanced glutathione level in tobacco roots to combat Cd

449

stress.20 In hepatic cells, NO plays negative roles in the regulation of glutathione

450

bisosynthesis during endotoxemia.54 Thus, whether and how thymol regulates the

451

interaction between NO and glutathione in the detoxification of Cd in plants need to

452

be studied further.

453

In sum, the above results advocate the protective role of thymol against

454

Cd-induced phytotoxicity, which provides important evidences for the novel function 19



Page 20 of 46

455

of thymol in the regulation of plant physiology. Thymol at proper concentrations

456

confers plant tolerance against Cd toxicity by suppressing a list of NO-mediated

457

downstream events, such as oxidative injury, cell death, and Cd2+ accumulation. The

458

results from pretreatment with thymol also suggest that thymol has the capability of

459

regulate plant NO signaling pathway to combat the subsequent Cd stress. In mammals

460

and plants, thymol seems to modulate a similar repertoire of NO signals to combat

461

stress

462

thymol-facilitated metal tolerance in plants would extend our knowledge of thymol in

463

physiological modulation.

464

ASSOCIATED CONTENT

465

Supporting Information

466

The Supporting Information is available free of charge on the ACS Publications

467

website.

468

Effect of Cd at different concentrations on root growth of rice seedlings (Figure S1);

469

Pretreatment with thymol attenuated the subsequent root growth inhibition induced by

470

Cd stress (Figure S2); Pretreatment with thymol repressed the subsequent ROS

471

accumulation induced by Cd stress (Figure S3); Pretreatment with thymol blocked the

472

subsequent accumulation of endogenous NO induced by Cd stress (Figure S4).

473

AUTHOR INFORMATION

474

Corresponding Author

475

*E-mail:

476

+86-25-84390422.

477

Notes

conditions.

Further

investigation

[email protected].

of

the

Phone:

detailed

mechanism

+86-25-84391863.

20


for

Fax:

Page 21 of 46


478

The authors declare no competing financial interest.

479

ACKNOWLEDGEMENTS

480

This work was supported by Jiangsu Agriculture Science and Technology Innovation

481

Fund (CX(14)2096), National Key Research and Development Program of China

482

(2017YFD0201105), The Program for Science and Technology Innovation Team in

483

Universities of Henan Province (16IRTSTHN007), and Natural Science Foundation of

484

Jiangsu Province, China (BK20140745).

485

ABBREVIATIONS USED

486

Cd,

chloride;

cPTIO,

487

2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide;

DAB,

488

3,3-diaminobenzidine; DAF, 3-amino, 4-aminomethyl-2′,7′-difluorescein; DAF-FM

489

DA,

490

2′,7′-dichlorofluorescein; DCFH-DA,

491

dihydroethidium; EDTA, ethylenediaminetetraacetic acid; H3BO3, boric acid; HCl,

492

hydrochloric acid; HEPES-KOH, 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic

493

acid-potassium hydroxide; H2O2, hydrogen peroxide; HPF, 3′-(p-hydroxyphenyl);

494

H2SO4, sulfuric acid; ICP-MS, Inductively Coupled Plasma-Mass Spectrometer;

495

K2S2O5,

496

nicotinamide adenine dinucleotide hydrogen; NADPH, nicotinamide adenine

497

dinucleotide phosphate; Na2MoO4, sodium molybdate; NaNO2, sodium nitrite; NBT,

498

nitro-blue tetrazolium; Na2WO4, sodium Tungstate; NO, nitric oxide; NOS, nitric

499

oxide synthase; NR, nitrate reductase; O2•¯, superoxide radical; PI, propidium iodide;

cadmium;

3-amino,

potassium

CdCl2,

cadmium

4-aminomethyl-2′,7′-difluorescein,

sulfite;

diacetate;

DCF,

2′,7′-dichlorofluorescein diacetate; DHE,

L-NMMA,

NG-monomethyl-L-arginine;

21


NADH,


500

RL, root length; ROS, reactive oxygen species; SNP, sodium nitroprusside; TBA,

501

1,3-diethyl-2-thiobarbituric acid; TBARS, thiobarbituric acid reactive substances;

502

TCA, trichloroacetic acid; TiCl4, titanium tetrachloride; U.S.EPA, United States

503

Environmental Protection Agency.

504 505

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(53) Hossain, M. A.; Piyatida, P.; da Silva, J. A. T.; Fujita, M. Molecular mechanism of

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heavy metal toxicity and tolerance in plants: central role of glutathione in

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detoxification of reactive oxygen species and methylglyoxal and in heavy metal

672

chelation. J. Bot. 2012, 2012, 872-875.

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(54) Payabvash, S.; Ghahremani, M. H.; Goliaei, A.; Mandegary, A.; Shafaroodi, H.;

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Amanlou, M.; Dehpour, A. R. Nitric oxide modulates glutathione synthesis during

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endotoxemia. Free Radic. Biol. Med. 2006, 41, 1817-1828.

1 (PIN1)-dependent acropetal auxin transport. Proc. Natl. Acad. Sci.

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676 677 678

FIGURE CAPTIONS

679

Figure 1. Effect of thymol on root growth of rice seedlings under Cd stress. In the

680

presence of CdCl2 at 4 µM (+Cd) or not (-Cd), the roots of seedlings were treated with

681

thymol at different concentrations (0-40 µM) for 72 h. Then root length (A) and root

682

fresh weight (B) were determined, respectively. The roots were treated with distilled

683

water (control), CdCl2 (4 µM), and CdCl2 (4 µM) + thymol (20 µM) for 6, 12, 24, 48,

684

and 72 h, respectively, for the measurement of root length (C) and root fresh weight

685

(D). The roots of seedlings were treated with water, 4 µM of CdCl2, 20 µM of thymol,

686

alone or their combinations for 72 h. Then the images of seedlings were captured (E).

687

Bar = 1 cm. The asterisk (*) in (A,B) indicated that the mean value of four replicates

688

was significantly different between -Cd and +Cd under 0 µM of thymol. Different

689

letters in (A,B) indicated that the mean values of four replicates were significantly

690

different among different thymol treatments in the presence of Cd (p < 0.05, ANOVA,

691

LSD). Different letters in (C,D) indicated that the mean values of four replicates were

692

significantly different among the treatments at each time point (p < 0.05, ANOVA,

693

LSD).

694 695

Figure 2. Effect of thymol on total ROS accumulation in the root of rice seedlings

696

under Cd stress. The roots of seedlings were treated with distilled water (control),

697

CdCl2 (4 µM), CdCl2 (4 µM) + thymol (20 µM), and thymol (20 µM) for 72 h. (A) 30


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698

The roots were loaded with DCFH-DA for the observation of total ROS fluorescence

699

with a fluorescent microscope; (B) The DCF fluorescent density was quantified to

700

indicate the relative total ROS level in roots. Bar = 1 mm. Different letters in (B)

701

indicated that the mean values of four replicates were significantly different between

702

the treatments (p < 0.05, ANOVA, LSD).

703 704

Figure 3. Effect of thymol on the accumulation of H2O2 and O2•¯ in the root of rice

705

seedlings under Cd stress. The roots of seedlings were treated with distilled water

706

(control), CdCl2 (4 µM), CdCl2 (4 µM) + thymol (20 µM), and thymol (20 µM) for 72

707

h. (A) The roots were loaded with HPF for the observation of H2O2 fluorescence with

708

a fluorescent microscope; (B) The HPF fluorescent density was quantified to indicate

709

the relative H2O2 level in roots; (C) The roots were stained with DAB to indicate

710

H2O2 accumulation; (D) Measurement of H2O2 content in roots; (E) The roots were

711

loaded with DHE for the observation of O2•¯ fluorescence with a fluorescent

712

microscope; (F) The DHE fluorescent density was quantified to indicate the relative

713

O2•¯ level in roots; (G) The roots were stained with NBT to indicate O2•¯

714

accumulation; (H) Measurement of O2•¯ content in roots. Bar = 1 mm. Different

715

letters in (B,D,F,H) indicated that the mean values of three replicates were

716

significantly different between the treatments (p < 0.05, ANOVA, LSD).

717 718

Figure 4. Effect of thymol on lipid peroxidation, loss of membrane integrity, TBARS

719

content, and cell death in the root of rice seedlings under Cd stress. The roots of 31



720

seedlings were treated with distilled water (control), CdCl2 (4 µM), CdCl2 (4 µM) +

721

thymol (20 µM), and thymol (20 µM) for 72 h. Then the roots were stained with

722

Shiff’s reagent (A), Evans blue (B), and Trypan blue (D), respectively, followed by

723

photographing with a stereoscopic microscope; (C) Measurement of TBARS content

724

in roots. Bar = 1 mm. Different letters in (C) indicated that the mean values of three

725

replicates were significantly different between the treatments (p < 0.05, ANOVA,

726

LSD).

727 728

Figure 5. Effect of thymol on Cd accumulation in rice seedlings under Cd stress. The

729

roots of seedlings were treated with distilled water (control), CdCl2 (4 µM), CdCl2 (4

730

µM) + thymol (20 µM), and thymol (20 µM) for 72 h. (A) The roots and shoots were

731

harvested, respectively, for the determination of total Cd content by using ICP-MS. (B)

732

The roots were loaded with LeadmiumTM Green AM and photographed with a

733

fluorescent microscope; (C) The LeadmiumTM Green fluorescent density was

734

quantified to indicate the relative Cd2+ level in roots. Bar = 1 mm. The asterisk (*) in

735

(B) indicated that the mean value of three replicates was significantly different

736

between Cd+thymol treatment and Cd treatment alone (p < 0.05, ANOVA).

737 738

Figure 6. Effect of thymol on endogenous NO level, NOS activity, and NR activity in

739

the root of rice seedlings under Cd stress. The roots of seedlings were treated with

740

distilled water (control), CdCl2 (4 µM), thymol (20 µM), SNP (20 µM), cPTIO (20

741

µM), L-NMMA (30 µM), tungstate (30 µM), alone or their combinations for 72 h. (A) 32


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742

The roots were loaded with DAF-FM DA and photographed with a fluorescent

743

microscope; (B) The DAF fluorescent density was quantified to indicate the relative

744

NO level in roots. Bar = 1 mm. The roots of seedlings were treated with distilled

745

water (control), CdCl2 (4 µM), CdCl2 (4 µM) + thymol (20 µM), and thymol (20 µM)

746

for 72 h. Then roots were harvested for the determination of NOS activity (C) and NR

747

activity (D). Different letters in (B,C,D) indicated that mean values of three replicates

748

were significantly different between the treatments (p < 0.05, ANOVA, LSD).

749 750

Figure 7. Effect of thymol, SNP, cPTIO, L-NMMA, and tungstate on root growth of

751

rice seedlings under Cd stress. The roots of seedlings were treated with distilled water

752

(control), CdCl2 (4 µM), thymol (20 µM), SNP (20 µM), cPTIO (20 µM), L-NMMA

753

(30 µM), tungstate (30 µM), alone or their combinations for 72 h. The root length (A)

754

and seedling images (B) were obtained, respectively. Different letters in (A) indicated

755

that the mean values of three replicates were significantly different between the

756

treatments (p < 0.05, ANOVA, LSD).

757 758

Figure 8. Effect of thymol, SNP, cPTIO, L-NMMA, and tungstate on cell death in the

759

roots of rice seedlings under Cd stress. The roots of seedlings were treated with

760

distilled water (control), CdCl2 (4 µM), thymol (20 µM), SNP (20 µM), cPTIO (20

761

µM), L-NMMA (30 µM), tungstate (30 µM), alone or their combinations for 72 h. (A)

762

The roots were loaded with PI and photographed with a fluorescent microscope; (B)

763

The quantification of PI fluorescent density in roots. Bar = 1 mm. Different letters in 33



764

(B) indicated that the mean values of three replicates were significantly different

765

between the treatments (p < 0.05, ANOVA, LSD).

766 767

Figure 9. Effect of thymol, SNP, cPTIO, L-NMMA, and tungstate on total ROS

768

accumulation in the roots of rice seedlings under Cd stress. The roots of seedlings

769

were treated with distilled water (control), CdCl2 (4 µM), thymol (20 µM), SNP (20

770

µM), cPTIO (20 µM),

771

combinations for 72 h. (A) The roots were loaded with DCFH-DA and photographed

772

with a fluorescent microscope; (B) The DCF fluorescent density was quantified to

773

indicate relative total ROS level in roots. Bar = 1 mm. Different letters in (B)

774

indicated that the mean values of three replicates were significantly different between

775

the treatments (p < 0.05, ANOVA, LSD).

L-NMMA

(30 µM), tungstate (30 µM), alone or their

776 777

Figure 10. Hierarchical cluster analysis of interaction between thymol and NO on

778

physiological responses of rice seedling roots under Cd stress. (A) and (B) indicated

779

the hierarchical groups among physiological parameters. (C) and (D) indicated the

780

hierarchical groups among treatments. The relative data of RL (root length), ROS

781

content (indicated by DCF fluorescent density), NO content (indicated by DAF

782

fluorescent density), and cell death (indicated by PI fluorescent density) in roots with

783

different treatment, were selected for cluster analysis. All the data were presented as

784

relative fold change respect to control. The cluster color bar was shown as log2 fold

785

change as compared to control (black). For each parameter, red indicates the increase 34


Page 34 of 46

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786

of designated treatment as compared to control, while green indicates the decrease of

787

designated treatment as compared to control.

35



Figure 1. Effect of thymol on root growth of rice seedlings under Cd stress. In the presence of CdCl2 at 4 µM (+Cd) or not (-Cd), the roots of seedlings were treated with thymol at different concentrations (0-40 µM) for 72 h. Then root length (A) and root fresh weight (B) were determined, respectively. The roots were treated with distilled water (control), CdCl2 (4 µM), and CdCl2 (4 µM) + thymol (20 µM) for 6, 12, 24, 48, and 72 h, respectively, for the measurement of root length (C) and root fresh weight (D). The roots of seedlings were treated with water, 4 µM of CdCl2, 20 µM of thymol, alone or their combinations for 72 h. Then the images of seedlings were captured (E). Bar = 1 cm. The asterisk (*) in (A,B) indicated that the mean value of four replicates was significantly different between -Cd and +Cd under 0 µM of thymol. Different letters in (A,B) indicated that the mean values of four replicates were significantly different among different thymol treatments in the presence of Cd (p < 0.05, ANOVA, LSD). Different letters in (C,D) indicated that the mean values of four replicates were significantly different among the treatments at each time point (p < 0.05, ANOVA, LSD).


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174x229mm (300 x 300 DPI)



Figure 2. Effect of thymol on total ROS accumulation in the root of rice seedlings under Cd stress. The roots of seedlings were treated with distilled water (control), CdCl2 (4 µM), CdCl2 (4 µM) + thymol (20 µM), and thymol (20 µM) for 72 h. (A) The roots were loaded with DCFH-DA for the observation of total ROS fluorescence with a fluorescent microscope; (B) The DCF fluorescent density was quantified to indicate the relative total ROS level in roots. Bar = 1 mm. Different letters in (B) indicated that the mean values of four replicates were significantly different between the treatments (p < 0.05, ANOVA, LSD). 114x208mm (300 x 300 DPI)


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Figure 3. Effect of thymol on the accumulation of H2O2 and O2•¯ in the root of rice seedlings under Cd stress. The roots of seedlings were treated with distilled water (control), CdCl2 (4 µM), CdCl2 (4 µM) + thymol (20 µM), and thymol (20 µM) for 72 h. (A) The roots were loaded with HPF for the observation of H2O2 fluorescence with a fluorescent microscope; (B) The HPF fluorescent density was quantified to indicate the relative H2O2 level in roots; (C) The roots were stained with DAB to indicate H2O2 accumulation; (D) Measurement of H2O2 content in roots; (E) The roots were loaded with DHE for the observation of O2•¯ fluorescence with a fluorescent microscope; (F) The DHE fluorescent density was quantified to indicate the relative O2•¯ level in roots; (G) The roots were stained with NBT to indicate O2•¯ accumulation; (H) Measurement of O2•¯ content in roots. Bar = 1 mm. Different letters in (B,D,F,H) indicated that the mean values of three replicates were significantly different between the treatments (p < 0.05, ANOVA, LSD). 115x51mm (300 x 300 DPI)



Figure 4. Effect of thymol on lipid peroxidation, loss of membrane integrity, TBARS content, and cell death in the root of rice seedlings under Cd stress. The roots of seedlings were treated with distilled water (control), CdCl2 (4 µM), CdCl2 (4 µM) + thymol (20 µM), and thymol (20 µM) for 72 h. Then the roots were stained with Shiff’s reagent (A), Evans blue (B), and Trypan blue (D), respectively, followed by photographing with a stereoscopic microscope; (C) Measurement of TBARS content in roots. Bar = 1 mm. Different letters in (C) indicated that the mean values of three replicates were significantly different between the treatments (p < 0.05, ANOVA, LSD). 111x100mm (300 x 300 DPI)


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Figure 5. Effect of thymol on Cd accumulation in rice seedlings under Cd stress. The roots of seedlings were treated with distilled water (control), CdCl2 (4 µM), CdCl2 (4 µM) + thymol (20 µM), and thymol (20 µM) for 72 h. (A) The roots and shoots were harvested, respectively, for the determination of total Cd content by using ICP-MS. (B) The roots were loaded with LeadmiumTM Green AM and photographed with a fluorescent microscope; (C) The LeadmiumTM Green fluorescent density was quantified to indicate the relative Cd2+ level in roots. Bar = 1 mm. The asterisk (*) in (B) indicated that the mean value of three replicates was significantly different between Cd + thymol treatment and Cd treatment alone (p < 0.05, ANOVA). 178x404mm (300 x 300 DPI)



Figure 6. Effect of thymol on endogenous NO level, NOS activity, and NR activity in the root of rice seedlings under Cd stress. The roots of seedlings were treated with distilled water (control), CdCl2 (4 µM), thymol (20 µM), SNP (20 µM), cPTIO (20 µM), L-NMMA (30 µM), tungstate (30 µM), alone or their combinations for 72 h. (A) The roots were loaded with DAF-FM DA and photographed with a fluorescent microscope; (B) The DAF fluorescent density was quantified to indicate the relative NO level in roots. Bar = 1 mm. The roots of seedlings were treated with distilled water (control), CdCl2 (4 µM), CdCl2 (4 µM) + thymol (20 µM), and thymol (20 µM) for 72 h. Then roots were harvested for the determination of NOS activity (C) and NR activity (D). Different letters in (B,C,D) indicated that mean values of three replicates were significantly different between the treatments (p < 0.05, ANOVA, LSD). 172x217mm (300 x 300 DPI)


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Figure 7. Effect of thymol, SNP, cPTIO, L-NMMA, and tungstate on root growth of rice seedlings under Cd stress. The roots of seedlings were treated with distilled water (control), CdCl2 (4 µM), thymol (20 µM), SNP (20 µM), cPTIO (20 µM), L-NMMA (30 µM), tungstate (30 µM), alone or their combinations for 72 h. The root length (A) and seedling images (B) were obtained, respectively. Different letters in (A) indicated that the mean values of three replicates were significantly different between the treatments (p < 0.05, ANOVA, LSD). 115x120mm (300 x 300 DPI)



Figure 8. Effect of thymol, SNP, cPTIO, L-NMMA, and tungstate on cell death in the roots of rice seedlings under Cd stress. The roots of seedlings were treated with distilled water (control), CdCl2 (4 µM), thymol (20 µM), SNP (20 µM), cPTIO (20 µM), L-NMMA (30 µM), tungstate (30 µM), alone or their combinations for 72 h. (A) The roots were loaded with PI and photographed with a fluorescent microscope; (B) The quantification of PI fluorescent density in roots. Bar = 1 mm. Different letters in (B) indicated that the mean values of three replicates were significantly different between the treatments (p < 0.05, ANOVA, LSD). 118x125mm (300 x 300 DPI)


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Figure 9. Effect of thymol, SNP, cPTIO, L-NMMA, and tungstate on total ROS accumulation in the roots of rice seedlings under Cd stress. The roots of seedlings were treated with distilled water (control), CdCl2 (4 µM), thymol (20 µM), SNP (20 µM), cPTIO (20 µM), L-NMMA (30 µM), tungstate (30 µM), alone or their combinations for 72 h. (A) The roots were loaded with DCFH-DA and photographed with a fluorescent microscope; (B) The DCF fluorescent density was quantified to indicate relative total ROS level in roots. Bar = 1 mm. Different letters in (B) indicated that the mean values of three replicates were significantly different between the treatments (p < 0.05, ANOVA, LSD). 118x125mm (300 x 300 DPI)



Figure 10. Hierarchical cluster analysis of interaction between thymol and NO on physiological responses of rice seedling roots under Cd stress. (A) and (B) indicated the hierarchical groups among physiological parameters. (C) and (D) indicated the hierarchical groups among treatments. The relative data of RL (root length), ROS content (indicated by DCF fluorescent density), NO content (indicated by DAF fluorescent density), and cell death (indicated by PI fluorescent density) in roots with different treatment, were selected for cluster analysis. All the data were presented as relative fold change respect to control. The cluster color bar was shown as log2 fold change as compared to control (black). For each parameter, red indicates the increase of designated treatment as compared to control, while green indicates the decrease of designated treatment as compared to control. 131x152mm (300 x 300 DPI)


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