Multiple Pesticides Detoxification Function of Maize (Zea mays

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Multiple Pesticides Detoxification Function of Maize (Zea mays) GST34 Dongzhi Li, Li Xu, Sen Pang, Zhiqian Liu, Weisong Zhao, and Chengju Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00057 • Publication Date (Web): 21 Feb 2017 Downloaded from http://pubs.acs.org on February 22, 2017

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Journal of Agricultural and Food Chemistry

Multiple Pesticides Detoxification Function of Maize (Zea mays) GST34

1 2

Dongzhi Lia, Li Xua, Sen Panga, Zhiqian Liub, Weisong Zhaoa, Chengju Wanga,*

3 4 5

a

6

road, Haidian District, Beijing 100193, People’s Republic of China.

7

b

8

Centre for AgriBioscience, 5 Ring Road, La Trobe University, Bundoora, Victoria

9

3083, Australia.

College of Science, China Agricultural University, No.2 of Yuan Ming Yuan west

Department of Economic Development, Jobs, Transport and Resources, AgriBio,

10 11

*Corresponding authors: Prof. Chengju Wang

12

E-mail: [email protected]

13

Tel: +86 (0)10 62733924

14

Fax: +86 (0)10 62734294

15 16 17 18 19 20 21 22 1

ACS Paragon Plus Environment


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Abstract: ZmGST34 is a maize Tau class GST gene and was found to be differently

24

expressed between two maize cultivars differing in tolerance to herbicide metolachlor.

25

To explore the possible role of ZmGST34 in maize development, the expression

26

pattern and substrate specificity of ZmGST34 were characterized by quantitative

27

RT-PCR and heterologous expression system, respectively. The results indicated that

28

the expression level of ZmGST34 was increased approximately 2 to 5-fold per day

29

during the second-leaf stage of maize seedling. Chloroacetanilide herbicides or

30

phytohormones treatments had no influence on the expression level of ZmGST34,

31

suggesting that ZmGST34 is a constitutively expressed gene in maize seedling.

32

Heterologous expression in Escherichia coli and in Arabidopsis thaliana proved that

33

ZmGST34 can metabolize most chloroacetanilide herbicides and increase tolerance to

34

these herbicides in transgenic Arabidopsis thaliana. The constitutive expression

35

pattern and broad substrate activity of ZmGST34 suggested that this gene may play an

36

important role in maize development in addition to the detoxification of pesticides.

37 38

Keywords: ZmGST34, Expression pattern, Substrate specificity, Chloroacetanilide

39

herbicides, Constitutive expression, Heterologous expression

40 41 42 43 44 2


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1 Introduction

46

Herbicide application is considered to be the most effective manner for weed

47

control due to the high activity, wide weed-control spectrum, and selectivity between

48

weeds and crops. The selectivity between crops and weeds is based on different rate

49

of herbicide penetration, translocation and metabolism. Glutathione S-transferases

50

(GSTs, EC 2.5.1.18) have been widely studied in relation to their role in herbicide

51

detoxification and tolerance.1-2

52

Plant GSTs are a superfamily with multiple functions, which play an important role

53

in detoxifying cellular xenobiotics and toxins.3 To date, 54 GST genes were identified

54

in Arabidopsis thaliana (A. thaliana), over 25 in soybean (Glycine max), 59 in rice

55

(Oryza sativa) and at least 42 in maize (Zea mays).4-6 Based on substrate recognition

56

and antibody cross reactivity, plant GSTs can be classified into six groups: Phi, Tau,

57

Theta, Zeta, Lambda and dehydroascorbate reductases (DHAR).7 The Phi and Tau

58

class GSTs are specific in plants and are the most numerous and abundant members of

59

the family. They are dimeric and can catalyze the conjugation of a diverse range of

60

xenobiotics. The Theta and Zeta class GSTs are conserved in animals and plants.

61

Theta GSTs have limited transferase activity toward xenobiotics but are highly active

62

glutathione-dependent peroxidases (GPOXs).8 The Zeta GSTs are acting as

63

glutathione-dependent maleylacetoacetate isomerases (MAAI) and could also catalyze

64

glutathione-dependent dechlorination reactions.9 The DHAR and Lambda class GSTs

65

differ from the other plant GSTs in being monomeric and act as glutathione-dependent

66

oxidoreductases rather than conjugating enzymes.10 Because of the large member and 3



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various functions, GST superfamily confers tolerance to a wide range of biotic and

68

abiotic stresses, such as organic pollutants, natural toxins and especially herbicides.2

69

The major mode of action of GSTs in detoxifying xenobiotics is by catalyzing the

70

nucleophilic addition of glutathione (GSH) to the electrophilic groups of a large

71

variety of hydrophobic toxic molecules forming water soluble, inactive conjugates.11

72

Heterologous expression is a useful method for functional characterization of genes.

73

Huang et al. (2016) found that rice laccases gene involved in the detoxification of

74

herbicides atrazine and isoproturon by the method of heterologous expression12. To

75

date the function of multiple GST genes in plants had been characterized by

76

expression in prokaryote and eukaryote system. To facilitate the synthesis of a foreign

77

protein in Escherichia coli (E. coli), many expression vectors had been constructed.

78

Among

79

isopropyl-β-D-thiogalactopyranoside (IPTG), is the most frequently employed vectors

80

for production of recombinant proteins.13 A. thaliana is commonly used for genes

81

function characterization in vivo. For example, Jo et al. (2011) and Lee et al. (2011)

82

expressed successfully OsGSTU4 and OsGSTU3 in E. coli using pET-26b(+) vector

83

and the recombinant enzymes exhibited high activity toward chloroacetanilide

84

herbicides, such as acetochlor, acifluorofen, alachlor and metolachlor.14-15 Johnson

85

and Dowd (2004) characterized the insect resistance function of a conserved MYB

86

transcription factor of phenylpropanoid biosynthesis by transgenic A. thaliana.16

them

pET,

which

is

based

on

promoter

inducible

with

87

Maize is one of the most widely planted crops around the world and its GSTs had

88

been extensively studied. Among the 42 GST genes in maize, 12 belong to phi class, 2 4


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zeta class and 28 tau class.4 Six GSTs in phi class had been functionally characterized

90

by heterologous expression, especially for GSTI, GSTIII and GSTIV, which were the

91

most abundant members and showed high activity toward herbicides, such as alachlor,

92

metolachor and atrazine.4 By contrast, GST17, a member of zeta class, showed limited

93

activity toward herbicide.4 For the majority of the 42 GST genes in maize, their

94

functions have not been fully characterized, especially those of tau class. In our

95

previous study, ZmGST34 (Locus name AF244699) was found to be expressed in the

96

leaves of a metolachlor tolerant maize cultivar Nongda86, but not in the metolachlor

97

susceptible maize cultivar Zhengdan958 (Figure S1). To date, there is no report

98

concerning the role of this gene product.

99

The objectives of this study are to (1) explore the expression pattern of ZmGST34

100

in maize leaves; (2) functionally characterize ZmGST34 in pesticides detoxification in

101

vitro and vivo.

102 103

2 Materials and Methods

104 105

2.1 Plant Material and Treatment

106

Maize kernels of cultivar Nongda86 were soaked in distilled water for 12 h at room

107

temperature and then were allowed to germinate on moist cheesecloth in an artificial

108

climate chamber RXZ-3808 (Jiangnan Instrument, Ningbo, China) at 28 °C, 75% RH

109

and 16/8 h day/night cycle. Plastic pots filled with sterile sand were used as a culture

110

medium. Water (60 mL) was applied to each pot before sowing of germinated kernels. 5



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The plastic pots were maintained in the same growth chamber with the same growing

112

conditions for the whole duration of the experiment.

113

To determine the intrinsic expression of ZmGST34 in maize seedlings, leaf samples

114

were collected at 60, 84, 108, 132 and 156 h after treatment and frozen in liquid

115

nitrogen.

116

To test the induction of ZmGST34 expression by chloroacetanilide herbicides

117

(including alachlor, acetochlor, pretilachlor, butachlor, propisochlor and metolachlor),

118

60 mL of herbicide (60 µmol L-1 dissolved in 1/1000 water mixture of acetone and

119

tween 80) was applied to each pot before sowing of germinated kernels. An equal

120

volume of water was applied for the control treatment. Leaves were harvested at 60 h

121

and 84 h after treatment and frozen in liquid nitrogen. Each treatment contained three

122

pots (6 plants per pot).

123

To test the induction of ZmGST34 expression by phytohormones and elicitors,

124

ethephon (ET, 700 µmol L-1), salicylic acid (SA, 500 µmol L-1), abscisic acid (ABA,

125

100 µmol L-1) and methyl jasmonate (MeJA, 100 µmol L-1) were applied to

126

second-leaf stage plants (96 h after sowing) using a moving-boom cabinet sprayer

127

delivering 1313.7 L ha-1 water at the pressure of 0.4 MPa by a flat fan nozzle

128

positioned 54 cm above the foliages level. Leaves were harvested at 8 h and 24 h after

129

treatment and frozen in liquid nitrogen.

130 131 132

2.2 Expression Analysis of ZmGST34 in Maize Leaves The harvested leaves were ground into fine powder in liquid nitrogen. Total RNA 6


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was isolated using RNAprep pure Plant Kit (Tiangen Biotech, Beijing, China)

134

according to the manufacturer’s protocols (Manual S-1, Supporting Information).

135

First strand cDNA was synthesized from 1µg total RNA using the Fast Quant RT Kit

136

(Tiangen Biotech, Beijing, China) in accordance with the manufacturer’s

137

recommendations (Manual S-2, Supporting Information). Quantitative real-time PCR

138

(qRT-PCR) was carried out with SYBR Green PCR Master Mix regent kits (Tiangen

139

Biotech, Beijing, China) by ABI Prism7500 Real-Time PCR System (Applied

140

Biosystems by Life Technologies, Foster, CA, USA). Reactions were run in a 20 µL

141

mixture containing 10 µL 2×SuperReal PreMix solution, 0.4 µL 50×ROX Reference

142

Dye, 0.6 µL of each of the forward and reverse primer (10 µmol L-1), 1 µL cDNA

143

template and 7.4 µL ddH2O. The amplification procedure was as follows: 95 °C for

144

15 min followed by 40 cycles of 95 °C for 10 s, 60 °C for 32 s. Primers for

145

amplification of 18S rRNA (used as reference gene) and ZmGST34 were the following:

146

18S rRNA (F: 5’-GCTCTTTCTTGATTCTATGGGTGG-3’, R: 5’-GTTAGCAGGC

147

TGAGGTCTCGTTC-3’) and ZmGST34 (F: 5’-GTGAAGGCGGTGGA GAA -3’, R:

148

5’-TTTGGGAGCATTGATAGGA-3’). Reference gene 18S rRNA was used as internal

149

control to normalize the amount of transcripts among different samples. The primer

150

specificity was checked by a melt curve analysis, and the amplification efficiency was

151

estimated using the equation E=10-1/slope, where the slope was derived from the plot of

152

amplification cycle time (Ct value) versus serially diluted template cDNA. The

153

relative expression levels of genes were calculated by 2-△△CT method.17 Three

154

biological replicates and three technical replicates were performed for each sample. 7



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2.3 Construction of pET-26b(+)/ZmGST34 and pCAMBIA1304/ZmGST34

157

Vectors

158

Primer pair F: 5’-GAATTCCATATGTCGGAGGCCGCCGTG-3’ with an Nde I

159

cloning site at 5’ end and R: 5’-GGATCCTTAAGAGAACGACCCATAGGT-3’ with

160

a BamH I cloning site at 5’ end, was used for full-length ZmGST34 gene amplification

161

to

162

CCATGGATATGTCGGAGGCCGCCGTG-3’ and R: 5’ CCATGGCAGAGAACG

163

ACCCATAGGTC-3’ with an Nco I cloning site at each 5’ end, was used for

164

full-length ZmGST34 gene amplification to construct pCAMBIA1304/ZmGST34

165

vector. The amplifications were initially cloned in the pLB vector (Tiangen Biotech,

166

Beijing, China) and then restriction digested to produce a fragment with staggered

167

terminal ends, which was finally ligated to the specific restriction sites of vectors

168

pET-26b(+)

169

pET-26b(+)/ZmGST34 and pCAMBIA1304/ZmGST34, respectively (Figure 1).

construct

pET-26b(+)/ZmGST34

and

pCAMBIA1304

vector.

to

form

Primer

the

pair

F:

expression

5’

vectors

170 171

(Figure 1 Insert here)

172 173

2.4 Functional Analysis of Recombinant ZmGST34 in E. coil

174 175 176

2.4.1 Expression and Extraction of Recombinant ZmGST34 in E. coil The pET-26b(+)/ZmGST34 construct was transformed into E. coil strain BL21 8


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(DE3) (Tiangen Biotech, Beijing, China) and then grown in Luria-Bertani (LB)

178

medium until the OD600 reached 0.4-1.0. Expression of recombinant protein

179

ZmGST34 was induced by the addition of IPTG at a final concentration of 1.0 mM.

180

After incubation for 3 h at 37 °C, the induced cells were harvested by centrifugation

181

at 10000 g for 10 min at 4°C, and then resuspended in 20 mM potassium phosphate

182

buffer (pH 7.0). The cell suspension was subjected to sonication for 10 min with an

183

ultrasonic processor (Sonics and Materials Inc., USA), followed by a centrifugation at

184

20000 g for 30 min.14 The supernatant was used for subsequent experiments. All

185

purification procedures were performed either at 4°C or on ice.

186 187

2.4.2 Protein Assay and SDS-PAGE

188

The concentration of protein preparations was determined by the Bradford

189

method18 and then diluted to identical concentration (1mg mL-1). Denaturing

190

SDS-Polyacrylamide gel (12.5%) electrophoresis (PAGE) of E. coli cell proteins was

191

carried out together with protein markers (Tiangen Biotech, Beijing, China). The gels

192

were then stained with Coomassie Blue R-250 (Tiangen Biotech, Beijing, China)

193


194 195

2.4.3 Enzyme Activity Assays

196

GST activity of crude protein extract of E. coli toward substrate 1-chloro-2,

197

4-dinitrobenzene (CDNB) was assayed spectrophotometrically by measuring change

198

of A340 according to our previous study.19 9



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GST activities toward herbicides were assayed by high performance liquid

200

chromatography (HPLC) according to Edwards et al. with following modifications.7

201

The enzyme preparation (120 µL, adjusted to 1 mg mL-1) was added to 50 µL (0.1

202

mol L-1) potassium phosphate buffer (pH 6.8). The mixture was then transferred to

203

water bath at 37℃ and 10 µL pesticide solution (10 mmol L-1 dissolved in acetone)

204

was added, immediately followed by addition of 20 µL freshly prepared GSH (100

205

mmol L-1) adjusted to pH 7.0 with 0.1 M NaOH. After incubation for 60 min, the

206

reaction was stopped by adding 10 µL HCl (3 mol L-1). After standing on ice for 30

207

min, the precipitated protein was removed by centrifugation (12000 g, 5 min). The

208

non-transformed pesticide in supernatant was analyzed by an Agilent 1260 HPLC

209

system equipped with a reversed-phase C18 analytical column (4.6 mm × 150 mm,

210

3.5 µm) kept at 25 °C. The mobile phase was acetonitrile/water (60:40, V/V)

211

containing 0.1% acetic acid at 1 mL min-1 and the injection volume was 20 µL.

212

Chloroacetanilide herbicides (including alachlor, acetochlor, pretilachlor, butachlor,

213

propisochlor

214

chlorotoluron were quantified by a UV detector at 230 nm20, 22221 nm, 24022 nm,

215

25523 nm and 24021 nm respectively. The relative detoxification rate was calculated

216

based on the reduction of added pesticide. The protein preparation of E. coli

217

transformed by empty vector was used as control to correct for non-target enzyme

218

reaction.

and

metolachlor),

atrazine,

nicosulfurion,

azoxystrobin

and

219 220

2.5 Functional Analysis of Recombinant ZmGST34 in A. thaliana 10


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2.5.1 Transformation of A. thaliana

223

The prepared over-expression construct pCAMBIA1304/ZmGST34 was introduced

224

into Agrobacterium tumefaciens strain GV3101 by a freeze-thaw method.24 The A.

225

thaliana cv. Columbia with abundant flowering buds were subjected to flora-dip

226

transformation of recombinant Agrobacterium cells.25 The transformed plants were

227

cultured in an artificial climate chamber (RXZ-3808) at 20°C, 75% RH and 16/8 h

228

day/night cycle. Seeds were harvested and positive transformants were screened by

229

sowing the resulting seeds on 1/2 MS plates (pH 5.8) containing 0.7% (w/v) agar and

230

1.5% (w/v) sucrose supplemented with 50 mg L-1 hygromycin B. Seeds, screened for

231

two generations, were used for subsequent experiments.

232 233

2.5.2 PCR and GUS Staining Analysis of Transgenic A. thaliana

234

To verify the successful transformation of ZmGST34 into A. thaliana, genomic

235

PCR (polymerase chain reaction) and GUS (β-glucuronidase) staining experiments

236

were performed. The screened positive transgenic seeds were cultured in 1/2 MS

237

plates with the same conditions as described in 2.5.1. Ten days old seedlings were

238

used for the extraction of genomic DNA and GUS staining analysis. Genomic DNA

239

was extracted using Wizard genomic DNA purification kit (Promega, Madison, USA)

240


241

PCR reactions were performed by Mastercycler Gradient 5331 (Eppendorf, Hamburg,

242

Germany) using the 2×HotStart Taq PCR MasterMix (Tiangen Biotech, Beijing, 11



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China) with the same primers described in 2.3. The 25 µLPCR mixture consisted of

244

100 ng of genomic DNA, 1 µL (10 µM) of each primer and 12.5 µL of 2×HotStart

245

Taq PCR MasterMix and 10 µL ddH2O. PCR was run with the following procedures:

246

denaturation at 94 °C for 3 min, 30 cycles of 94 °C for 30 s, 56 °C for 30 s and 72 °C

247

for 60 s, followed finally by an extension step of 5 min at 72 °C. PCR products were

248

separated in a 2.5% (w/v) agarose gel. GUS staining kit (Real-Times Biotechnology

249

Co., Ltd, Beijng, China) was used to examine the expression of ZmGST34 in different

250

tissues as described by Jefferson et al. (1987)26. Tissues of A. thaliana seedling were

251

immersed in GUS solution (10 mM EDTA, 0.1% Triton X-100, 100 mM NaH2PO4,

252

25 mg L-1 X-gluc (5-bromo-4-chloro-3-indolyl-β-glucuronide) and 50% ethanol) and

253

incubated at 37 °C for 16 h. The GUS-positive tissues (blue-coloured precipitate)

254

were photographed with a digital camera.

255 256

2.5.3 Enzyme Activity Analysis of Transgenic A. thaliana

257

The same seedlings described in 2.5.2 were used for the GST activity assay. The

258

extraction of crude enzyme and method for GST activity test were the same as our

259

previous study.19 Three biological replicates and three technical replicates were

260

performed for each line.

261 262

2.5.4 Herbicide Tolerance Assay of Transgenic A. thaliana

263

The screened seeds were washed with 75% ethanol and the surface sterilized with

264

4% sodium hypochlorite. The sterilant was removed from the seeds by washing three 12


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times with autoclaved distilled water. The sterilized seeds were placed on 1/2 MS

266

plate as described in 2.5.1, which contained a series gradient concentrations (0, 5, 10,

267

20, 40, 80, 160, 320 µmol L-1) of chloroacetanilide herbicides. The plates were kept in

268

an artificial climate chamber (RXZ-3808) at 20°C, 75% RH and 16/8 h day/night

269

cycle. The fresh weight of the seedlings was measured after 15 days. The assay was

270

performed using 30 seedlings per treatment and three independent biological

271

replicates.

272 273

2.6 Statistical Analysis

274

All statistical analyses were performed by SPSS 16.0 (SPSS, Chicago, IL, USA).

275

Results related to ZmGST34 expression levels and enzymatic activity toward CDNB

276

in E. coli were subjected to one-way analysis of variance (ANOVA) combined with

277

Duncan post-hoc comparison test. Student’s t-test was used for enzymatic activity

278

assay in A. thaliana. The criterion for statistical significance was p < 0.05.

279 280

3 Results

281 282 283

3.1 Expression Profile of ZmGST34 in the Leaves of Maize Seedlings The expression of ZmGST34 in the leaves of maize cultivar Nongda86 was

284

determined by qRT-PCR. The intrinsic expression level of ZmGST34 was increased

285

approximately 2-5 folds per day during the period of second-leaf stage and reached

286

the highest level at 132 h after sowing (Figure 2). 13



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287 288


289 290

Treatment by chloroacetanilide herbicides (alachlor, acetochlor, pretilachlor,

291

butachlor, propisochlor and metolachlor) for 60 h or 84 h did not significantly affect

292

the expression of ZmGST34 (Figure 3A). Similarly, treatment by phytohormones or

293

elicitors (including ET, SA, ABA and MeJA) had no significant influence on the

294

expression of this gene (Figure 3B).

295 296


297 298

3.2 SDS-PAGE Analysis of Recombinant ZmGST34 in E. coli

299

To investigate the function of ZmGST34 gene product, the recombinant protein

300

expressed in E. coli was characterized. SDS-PAGE was conducted to verify the

301

presence of ZmGST34 protein in the extract of transformed E. coli. Figures 4 shows

302

that compared with the protein profile of E. coli transformed by an empty vector (lane

303

1), an extra band with the molecular weight below 27 kDa was found in the extract of

304

E. coli containing ZmGST34 (lane 2, arrow), which is consistent with the theoretical

305

value 24.6 kDa of recombinant ZmGST34 protein.

306 307


308 14


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309

3.3 GST Activity Analysis of Recombinant ZmGST34 in E. coli

310

The model substrate CDNB was used to assay the GST activity of recombinant

311

protein ZmGST34. Without IPTG induction, a minimum activity was detected in E.

312

coli strains regardless of vectors; after IPTG induction, GST activity was about 10

313

fold higher in ZmGST34 transformed strain as compared to empty vector transformed

314

strain (Table 1), which further confirmed the successful transformation of ZmGST34

315

into E. coli.

316 317

(Table 1 Insert here)

318 319

3.4 Pesticides Metabolism Analysis of Recombinant ZmGST34 in E. coli

320

The substrate specificity of the recombinant protein ZmGST34 toward a range of

321

pesticides was determined. As presented in Table 2, the recombinant protein of

322

ZmGST34 is able to detoxify most chloroacetanilide herbicides except for alachlor. It

323

also shows comparable activity toward other types of herbicides such as atrazine and

324

nicosulfuron, and the highest activity of this protein was observed with germicide

325

azoxystrobin. By contrast, no activity toward chlorotoluron was observed.

326 327


328 329 330

3.5 Verification of Transgenic A. thaliana The PCR and GUS staining experiments were used to verify the successful 15



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331

transformation of ZmGST34 gene into A. thaliana. As presented in Figure 5A, the

332

target gene of ZmGST34 was amplified in transgenic A. thaliana line but did not in the

333

wild line, which suggested that ZmGST34 had been transformed into the A. thaliana

334

genome. The GUS staining result further confirmed that ZmGST34 was successfully

335

expressed in all the tissues of transgenic A. thaliana lines, such as leaf, flower and

336

silique (Figure 5B).

337 338


339 340

3.6 GST Activity Analysis of Transgenic A. thaliana

341

GST activity of transgenic A. thaliana was assayed using CDNB as a substrate.

342

Compared with the wild lines, transgenic A. thaliana showed significant higher GST

343

activity (Table 3), which further confirmed the successful transformation of ZmGST34

344

into A. thaliana.

345 346


347 348

3.7 Tolerance Assay of Transgenic A. thaliana to Chloroacetanilide Herbicides

349

To verify the detoxification role of ZmGST34 in vivo, transgenic A. thaliana

350

seedlings were used for chloroacetanilide herbicides bioassay. According to Figure 6,

351

transgenic A. thaliana showed a higher tolerance than the wild lines to all the tested

352

chloroacetanilide herbicides, including alachlor which could not be detoxified by the 16


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353

recombinant protein of ZmGST34 expressed in E. Coli.

354 355


356 357

Discussion

358

GSTs, a major group of detoxification enzymes, appear to be implicated in various

359

functions, including detoxification of xenobiotics and endobiotics, primary and

360

secondary metabolism, stress tolerance and cell signaling.27 The progress made in the

361

understanding of plant GSTs in previous decades was mainly in relation to their role

362

in the detoxification of herbicides. In our previous study, ZmGST34 was found to

363

express differently in two maize cultivars with significant difference in tolerance to

364

metolachlor.19 To further understanding the role of ZmGST34 in herbicide tolerance,

365

the detoxification function of ZmGST34 was characterized by means of heterlogous

366

expression system in E. coli and in A. thaliana followed by in vitro and in vivo

367

enzymatic activity assay. The results indicated that ZmGST34 had a wide substrate

368

spectrum and is able to detoxify most chloroacetanilide herbicides (including

369

metolachlor), which could contribute to the contrasting tolerance of two maize

370

cultivars to this herbicide observed in our previous study.

371

Individual GSTs often respond to diverse stimuli rather than single environmental

372

factor or exogenous treatments, but certain GSTs respond only to a given treatment.

373

Wagner et al. (2002) investigated the transcript abundance of ten GST genes in A.

374

thaliana subjected to phytohormones and metolachlor, and found that five of them 17



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375

remained unaffected by both of the treatments, one was up-regulated by both of the

376

treatments, whereas four were differentially induced by the treatments, indicating that

377

diverse mechanisms are responsible for the regulation of gene expression.28 SA plays

378

a protective role against pathogen infection by inducing the expression of several

379

pathogenesis-related genes during systemic acquired resistance against the pathogen.

380

The induction of GSTs after SA treatment suggests their involvement in mounting a

381

defense response against pathogens.29 Mang et al. (2004) reported that the AtGSTF2

382

of A. thaliana was up-regulated by ET treatment, which indicated that AtGSTF2 may

383

play a role during early root development.30 In our study, treatments by ABA, ET,

384

MeJA and SA did not significantly affect the expression of ZmGST34 in maize leaves,

385

nor by chloroacetanilide herbicides. These findings suggest ZmGST34 expression in

386

Nongda86 is not responsive to chemical stress, nor to defense-related signalling. The

387

fact that the expression of ZmGST34 increased sharply during the early seedling stage

388

suggests that ZmGST34 may play a role in the growth and development of maize

389

plants. In contrast to the well-established involvement of plant GSTs in pesticide

390

detoxification, more studies are clearly needed to explore other physiological

391

functions of these enzymes.

392

The substrate specificity of an individual GST can be quite broad and functional

393

overlap is found within the GST superfamily.4 It was found that ZmGSTI expresses

394

constitutively in maize roots and shoots and showed activity toward alachlor, atrazine,

395

chlorimuron, ethacrymic acid, trans-stilbene oxide, 1,2-epoxy-3-(p-nitrophenoxy)

396

propane and CDNB, and ZmGSTIII also showed activity toward these compounds.4 18


Page 19 of 35


397

However, ZmGSTIV was reported to show no activity to atrazine, trans-stilbene oxide

398

or 1,2-epoxy-3-(p-nitrophenoxy) propane.4 In the present study, ZmGST34 showed

399

lower specific activity toward atrazine than chloroacetanilide herbicides and also had

400

activity toward non-halogenated herbicides nicosulfuron and azoxystrobin, but no

401

active for alachlor or chlorotoluron was observed. According to McGonigle et al.

402

(2000), ZmGST8, ZmGST9 and ZmGST10 and ZmGST17 also showed no activity to

403

alachlor.4 The reason for the lack of activity of ZmGST34 to chlorotoluron could be

404

that chlorotoluron is detoxified in plants by the combined action of CYPs and UGTs.31

405

Several classes of herbicides containing halogen have been found undergo readily

406

conjugation through substitution or addition reactions with glutathione in plants,

407

including

408

aryloxyphenoxypropionate and diphenylether classes.11 However the detoxification

409

mechanism of chlorotriazine, chloroacetanilide and sulfonylurea is halide substitution

410

by GSH, but aryloxyphenoxypropionate and diphenylether is cleavage reaction.11 In

411

addition, compounds without halogen, such as 2-crotonyloxymethyl-2-cycloalkenone

412

and 1,2-epoxy-3-(p-nitrophenoxy) propane, were also able to conjugate with GSH.32,4

413

So it appears that GSTs can be active toward compounds with different structures,

414

which coincides with the variable topology of GST substrate binding site (H-site).2 In

415

addition to the function of exogenous detoxification, some GSTs show glutathione

416

peroxidases (GPOX) that counteract oxidative stress.33 However no GPOX activity

417

was observed in the case of ZmGST34 (Data not shown). The in vivo activity assay in

418

transgenic A. thaliana showed that ZmGST34 can increase tolerance to all the tested

members

of

the

chlorotriazine,

chloroacetanilide,

sulfonylurea,

19



Page 20 of 35

419

chloroacetanilide herbicides including alachlor, which can’t be metabolized by

420

ZmGST34 in vitro. In addition to the well known function in xenobiotics metabolism,

421

GSTs also play an important role in diverse metabolic activities of plant, such as

422

binding potentially reactive hydrophobic metabolites and plant hormone IAA, for the

423

purposes of stabilizing pathway intermediates, or acting as protein delivery shuttles

424

between sites of synthesis and use5. In addition, the localization of GSTs was found to

425

be associated with distinct organelles such as the plastid, mitochondrion, vacuole,

426

nucleus and peroxisome, indicating that GSTs should have important functions in

427

plant cell structure and function5. All these functions may contribute indirectly to the

428

increased tolerance to alachlor.

429

In conclusion, our study showed that ZmGST34 is a constitutively expressed gene

430

and its expression increased with the development of maize seedling during the

431

second-leaf stage, indicating that ZmGST34 may play an important role in the growth

432

and development of maize. In addition, ZmGST34 had a broad substrate spectrum

433

toward pesticides especially for chloroacetanilide herbicides. The detoxification

434

properties of GSTs have been extensively employed for the development of herbicide

435

tolerant crop varieties.34-35 Therefore, ZmGST34 could be a good candidate gene to

436

improve pesticide tolerance of plants.

437 438 439 440

Acknowledgements We thank Jiazheng Jiang, Mingqi Zheng for their kindly help in experiment technical support during this study. 20


Page 21 of 35


441 442

Supporting Information Available: [Expression of ZmGST34 between Nongda86

443

and Zhengdan958; The manufacturer's protocol of RNAprep pure Plant Kit; The

444

manufacturer's protocol of Fast Quant RT Kit; The manufacturer's protocol of

445

Coomassie Blue R-250; The manufacturer's protocol of Wizard genomic DNA

446

purification kit] This materials are available free of charge via the Internet at

447

http://pubs.acs.org.

448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 21



463

Page 22 of 35

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[2] Frova, C. The plant glutathione transferase gene family: Genomic structure,

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[3] Tsuchiya, T.; Takesawa, T.; Kanzaki, H.; Nakamura, I. Genomic structure and

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[4] McGonigle, B.; Keeler, S. J.; Lau, S. M. C.; Koeppe, M. K.; O'Keefe, D. P. A

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[12]Huang, T. H.; Lu, Y. C.; Zhang, S.; Luo, F.; Yang, H. Rice (Oryza sativa) Laccases

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[13] Zelena, K.; Eisele, N.; Berger, R. G. Escherichia coli as a production host for

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novel enzymes from basidiomycota. Biotechnol. Adv. 2014, 32, 1382–1395.

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[14] Jo, H. J.; Lee, J. J.; Kong, K. H. A plant-specific tau class glutathione

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1-chloro-2,4-dinitrobenzene and chloroacetanilide herbicides. Pestic. Biochem.

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Physiol. 2011, 101, 265–269.

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[15] Lee, J. J.; Jo, H. J.; Kong, K. H. A Plant-specific Tau Class Glutathione

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S-transferase from Oryza sativa Having Significant Detoxification Activity Towards

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Chloroacetanilide Herbicides. Bull. Korean Chem. Soc. 2011, 32, 3756–3759.

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[16] Johnson, E. T.; Dowd, P. F. Differentially Enhanced Insect Resistance, at a Cost,

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in Arabidopsis thaliana Constitutively Expressing a Transcription Factor of Defensive

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Metabolites. J. Agric. Food Chem. 2004, 52, 5135–5138.

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[17] Livak, K. J.; Schmittgen, T. D. Analysis of relative gene expression data using

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real-time quantitative PCR and the 2-△△CT Method. Methods 2001, 25, 402–408.

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[18] Bradford, M. M. A rapid and sensitive method for the quantitation of microgram 24


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Glutathione S-Transferases Are Responsible for the Differential Tolerance to

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Metolachlor between Maize (Zea mays) Shoots and Roots. J. Agric. Food Chem. 2017,

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[20] Zhang, J.; Zheng, J. W.; Liang, B.; Wang, C. H.; Cai, S.; Ni, Y. Y.; He, J.; Li, S.

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P. Biodegradation of Chloroacetamide Herbicides by Paracoccus sp. FLY-8 in Vitro.

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[21] Fingler, S.; Mendas, G.; Dvorscak, M.; Stipicevic, S.; Vasilic, Z.; Drevenkar, V.

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Herbicide micropollutants in surface, ground and drinking waters within and near the

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Chen, Y. H. Determination of Sulfonylurea Herbicides in Pears Using Hollow

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Fiber‑Protected Magnetized Solvent-Bar Liquid-Phase Microextraction HPLC.

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Chromatographia 2014, 77, 1283-1290.

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[23] Abdelraheem, E. M. H.; Hassan, S. M.; Arief, M. M. H.; Mohammad, S. G.

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beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.

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structure and functional role in xenome network and plant stress response. Curr. Opin.

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Biotechnol. 2015, 32, 186–194.

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[29] Sappl, P. G.; Sanchez, L.; Singh, K. B.; Millar, A. H. Proteomic analysis of

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acid-induced expression of plant-specific phi and tau classes. Plant Mol. Biol. 2004,

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Y. Y.; Kim, W. T. A proteomic analysis identifies glutathione S-transferase isoforms

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root epidermis in Arabidopsis seedlings. Biochim. Biophys. Acta, Gene Struct.

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Expression 2004, 1676, 231–239.

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[31] Van Eerd, L. L.; Hoagland, R. E.; Zablotowicz, R. M.; Hall, J. C. Pesticide

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metabolism in plants and microorganisms. Weed Sci. 2003, 51, 472– 495.

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[32] Sau, A.; Tregno, F. P.; Valentino, F.; Federici, G.; Caccuri, A. M. Glutathione

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transferases and development of new principles to overcome drug resistance. Arch.

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functioning as glutathione peroxidases in resistance to multiple herbicides in

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[34] Hu, T. A glutathione S-transferase confers herbicide tolerance in rice. Crop Breed.

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Appl. Biotechnol. 2014, 14, 76–81.

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[35] Govindarajan, S.; Mannervik, B.; Silverman, J. A.; Wright, K.; Regitsky, D.;

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Hegazy, U.; Purcell, T. J.; Welch, M.; Minshull, J.; Gustafsson, C. Mapping of amino

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602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 28


Page 29 of 35


617

Fig 1. Structure of constructed vectors pET-26b(+)/ZmGST34 (A) and

618

pCAMBIA1304/ZmGST34 (B) used for Escherichia coli and Arabidopsis thaliana

619

transformation, respectively. Kan, kanamycin resistance site; Ori, replication origin;

620

Lac I, the lac operon regulatory gene; T7, T7 promoter; HYG, hygromycin resistance

621

site; 35S, CaMV 35S promoter; Lac Z, the lac operon structure gene β-galactosidase;

622

GFP, green fluorescent protein gene; GUS, β-glucuronidase gene; Nde I, BamH I and

623

Nco I, restriction sites; RB, right border; LB, left border.

624 625

Fig 2. Relative expression level of ZmGST34 in the leaves of maize cultivar

626

Nongda86 during the second-leaf stage. Columns with different letters (a-e) are

627

significantly different (p< 0.05 by one-way ANOVA combined with Duncan’s

628

post-hoc comparison).

629 630

Fig 3. The effects of chloroacetanilide herbicides (A) and phytohormones/

631

elicitors (B) on the expression of ZmGST34 in the leaves of maize cultivar

632

Nongda86.

633 634

Fig 4. SDS-PAGE profile of putative ZmGST34 protein. Denaturing SDS–PAGE

635

was carried out in 12.5% gels stained with Coomassie Blue R-250. Marker, molecular

636

weight markers of protein standards; Lane 1, extract of vector only E. Coli strain;

637

Lane 2, extract of E. coli transformed by ZmGST34 gene; Arrow indicates the putative

638

recombinant protein ZmGST34. 29



Page 30 of 35

639 640

Fig 5. Verification of transgenic A. thaliana. (A) Genomic PCR of wild and

641

transgenic A. thaliana. Marker, molecular weight markers of DNA standards; Lane 1,

642

Genomic PCR of wild A. thaliana; Lane 2, Genomic PCR of transgenic A. thaliana.

643

(B) GUS staining of wild and transgenic A. thaliana.

644 645

Fig 6. Bioassay of wild and transgenic A. thaliana to chloroacetanilide herbicides.

646

Data expressed as mean ± standard deviation.

647 648 649 650 651 652 653 654 655 656 657 658 659 660 30


Page 31 of 35


661

Table 1. GST activity (mean ± SD) * of control and ZmGST34 transformed E. coli strains with and without IPTG induction

662

E. coil strain and treatment

Enzyme activity (nmol/min/mg)

Vector

17.8 ± 3.2 a

Vector + IPTG

19.2 ± 5.3 a

Vector + ZmGST34

15.1 ± 2.6 a

Vector + ZmGST34 + IPTG

177.1 ± 27.3 b

663

*

664

letter are significantly different (p< 0.05 by one-way ANOVA combined with

665

Duncan’s post-hoc comparison).

Mean values of three replicates ± standard deviation. Values followed by a different

666 667

Table 2. Pesticides metabolism activity of ZmGST34 Substrate Alachlor Acetochlor Pretilachlor Butachlor Propisochlor Metolachlor Atrazine Nicosulfuron Azoxystrobin Chlorotoluron

668

Specific activity (nmol/min/mg) ND 1.20 ± 0.15 0.86 ± 0.26 0.76± 0.17 0.94 ± 0.11 0.94 ± 0.29 0.72 ± 0.14 0.56 ± 0.12 1.44 ± 0.25 ND

ND: non-detectable

669 670

Table 3. GST activity (mean ± SD) * of wild and transgenic A. thaliana strains

Line 1 Line 2 Line 3

Enzyme activity (nmol/min/mg) Wild line Transformed line 50.3 ± 1.3a 85.4 ± 1.8b 57.2 ± 0.9a 80.8 ± 5.3b 61.4 ± 1.3a 73.9 ± 1.4b 31



671

*

672

different letter between the same line are significantly different (analyzed by

673

Student’s t-test, p < 0.05).

Page 32 of 35

Mean values of three technical replicates ± standard deviation. Values followed by a

674 675

Figure 1

676 677

Figure 2

678

679 680

32


Page 33 of 35


681

Figure 3

682 683 684

Figure 4

685 686

33



687

Page 34 of 35

Figure 5

688 689

Figure 6

34


Page 35 of 35


690

For table of contents only

691

35


Multiple Pesticides Detoxification Function of Maize (Zea mays

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