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Enantioselective Phytotoxicity of Dichlorprop to Arabidopsis thaliana: Effect of Cytochrome P450 enzymes and Role of Fe Zunwei Chen, Jia Wang, Hui Chen, Yuezhong Wen, and Weiping Liu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b04252 • Publication Date (Web): 14 Sep 2017 Downloaded from http://pubs.acs.org on September 17, 2017

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Environmental Science & Technology

1

Enantioselective Phytotoxicity of Dichlorprop to Arabidopsis

2

thaliana: Effect of Cytochrome P450 enzymes and Role of Fe

3 4

Zunwei Chen†，‡, Jia Wang†, Hui Chen†, Yuezhong Wen†,* Weiping Liu†

5 6 7

†

MOE Key Laboratory of Environmental Remediation & Ecosystem Health, College

8

of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058,

9

China

10 11

‡

Department of Veterinary Integrative Bioscience, Texas A&M University, College

Station, Texas 77843, United States

12

*

Corresponding author:

Tel.: +86-571-8898-2421. Fax: +86-571-8898-2421. E-mail address: [email protected] (Y.W.) 1

ACS Paragon Plus Environment


13

ABSTRACT:

14

The ecotoxicology effects of chiral herbicides have long been recognized and

15

have drawn increasing attention. The toxic mechanisms of herbicides in plants are

16

involved in production of reactive oxygen species (ROS) and cause damage to target

17

enzyme, but the relationship between these two factors in the enantioselectivity of

18

chiral herbicides has rarely been investigated. Furthermore, even though cytochromes

19

P450 enzymes (CYP450s) have been related to the phytotoxicity of herbicides, their

20

roles in the enantioselectivity of chiral herbicides have yet to be explored. To solve

21

this puzzle, the CYP450s suicide inhibitor 1-aminobenzotriazole (ABT) was added to

22

an exposure system made from dichlorprop (DCPP) enantiomers in the model plant

23

Arabidopsis thaliana. The results indicated that different phytotoxicities of DCPP

24

enantiomers by causing oxidative stress and ACCase damage, were observed in the

25

presence/absence of ABT. The addition of ABT decreased the toxicity of (R)-DCPP

26

but not significantly affected that of (S)-DCPP, resulting in smaller differences

27

between enantiomers. Furthermore, profound differences were also observed in Fe

28

uptake and distribution, exhibiting different distribution patterns in A. thaliana leaves

29

exposed to DCPP and ABT, which helped to bridge the relationship between ROS

30

production and target enzyme ACCase damage through the function of CYP450s.

31

These results offer an opportunity for a more comprehensive understanding of chiral

32

herbicide action mechanism and provide basic evidence for risk assessments of chiral

33

herbicides in the environment.

34 2


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Graphic Abstract

35 36

3



37

INTRODUCTION

38

Herbicides have garnered special attention due to the fact that the detrimental

39

effects on non-target plants will eventually influence the safety of human beings

40

through the food chain.1,

41

currently belong to the chiral family, and this proportion is expected to increase as

42

more complex compounds are introduced into use.3, 4 Therefore, enormous efforts

43

have been made to reveal the enantioselective effects of chiral pesticides on a wide

44

range of organisms.5-7 For instance, dichlorprop (DCPP) is a widely used

45

broad-spectrum chiral aryloxyphenoxy propionic acid (AOPP) herbicides and its

46

enantioselective action mechanisms have been increasingly explored. On the one hand,

47

the enzyme acetyl-CoA carboxylase (ACCase) has long been recognized as the target

48

site of AOPP herbicides.8-10 An anabolic mechanism has been unveiled involving the

49

inhibition of ACCase, which is necessary for fatty acid synthesis and secondary

50

metabolites.11 On the other hand, evidence has also been found that AOPP herbicides

51

could stimulate the production of reactive oxygen species (ROS), which play essential

52

roles in signaling transduction and other biochemical process.12 Once ROS

53

accumulates to a certain degree and exceeds the scavenging ability, then occurs the

54

irreversibly oxidant damage and eventually the death of plants.12 However, the

55

intrinsic relevance between ROS and ACCase in the enantioselective effects of chiral

56

herbicides remains elusive.

2

In particular, as many as 30% of the herbicides used

57

It should also be noted that the cross talk between ROS production and

58

microelement stress has been investigated.5 Among the elements, iron (Fe) is the most 4


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crucial mineral, not only for cellular functions and plant growth,13 but more

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importantly, because the quantification and distribution of Fe exhibit an abnormal

61

effect under abiotic stress,5, 14 making it a promising marker of the phytotoxicity

62

caused by contaminants. Furthermore, the abnormal behavior of Fe in plants under

63

stress

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bio-macromolecules consisting of Fe such as enzymes those are probably involved.

65

For instance, the cytochromes P450 enzymes (CYP450s), which have a Fe atom in the

66

structural center, were intensively reported to mediate the homeostasis of Fe.15-17

67

Because the superfamily includes a large group of enzymes in organisms, CYP450s

68

have also been found to be related to the phytotoxicity of herbicides.18-20 Considering

69

that CYP450s can enantioselectively catalyze the oxidation of plant fatty acids,21

70

which are also involved in the action mechanism of AOPP herbicides, it is reasonable

71

that CYP450s may mediate the enantioselective phytotoxicity of chiral AOPP

72

herbicides. However, the role of CYP450s in the enantioselective effects of chiral

73

herbicides has still been minimally studied.

may

not

only

reveals

situation

of element itself,

but also the

74

In this study, we selected A. thaliana as a model plant for the clear physiological

75

characteristics and genome, making it a good model for the study of herbicide action

76

mechanisms.

77

1-aminobenzotriazole (ABT), which has been successfully used in plant

78

experiments,22,

79

enantioselectivity of the chiral AOPP herbicide DCPP for the first time to date; this

80

was conducted in two ways, namely by measuring ACCase damage and oxidative

Moreover,

23

the

nonselective

substrate

inhibitor

of

CYP450s,

was added to explore the potential role of CYP450s in the

5



81

stress as well as Fe behavior in Arabidopsis. And DCPP levels in plants were also

82

determined. All of the experiments were performed at an enantiomeric level. The

83

results from this study will provide insights into the roles of CYP450s in the

84

enantioselective phytotoxicity of chiral herbicides as well as the mechanisms involved,

85

which will provide opportunities for better understandings herbicide security and

86

provide basic evidence for the risk assessments of chiral herbicides in the

87

environment.

88 89

MATERIALS AND METHODS

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Chemical Reagents. Dichlorprop enantiomers ((R)-DCPP and (S)-DCPP) with

91

99% purity were synthesized according to a previous study.24 The reagents

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1-aminobenzotriazole (ABT, 98% purity), 2’, 7-dicholordihydrofluorescein diacetate

93

(H2DCFDA) and 5, 5’-dithiobis (2-nitrobenzoic acid) (DTNB) were purchased from

94

Sigma-Aldrich (St. Louis, MO, USA). All other reagents were analytically pure, and

95

the glassware used in the experiments was sterilized in an autoclave.

96

Plant Growth Inhibition. Seeds of Arabidopsis thaliana (ecotype Columbia)

97

were first sterilized and then planted in 24-well culture plate (six seeds per cell),

98

containing 1 mL Murashige and Skoog medium mixed with ABT and DCPP in each

99

well; the exposure concentration (0.2 µM) was set based on preliminary experiments 25

100

and our previous research,

respectively. The cultivation process and herbicide

101

treatments are explained in detail in Text S1 of the Supporting Information. The plants

102

were cultivated for 20 days until analysis. The effects of DCPP combined with ABT 6


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on the growth of A. thaliana in terms of fresh weight, root length and chlorophyll

104

content were determined based on a previous study.5 Experiments were all performed

105

in triplicate. The group where plants grown in MS medium without ABT or DCPP

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was defined as the control group.

107

Measurements of Reactive Oxygen Species (ROS) Production. The

108

measurements of reactive oxygen species (ROS) in A. thaliana leaves and roots were

109

analyzed according to a previous study with slight modification.25 Briefly, the fresh

110

prepared leaves and roots of A. thaliana were and immersed in 25 µM H2DCFDA for

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30 min under darkness then washed by phosphate buffered saline (PBS, 0.05 mol·L-1,

112

pH=7.0). The fluorescence was visualized using a laser-scanning confocal microscope

113

(Carl Zeiss LSM 780, Jena, Germany) with excitation and emission wavelength at 488

114

and 515 nm, respectively. Confocal images were automatically acquired with ZEN

115

2010 software (Carl Zeiss MicroImaging, Inc., Jena, Germany). For each treatment, at

116

least 15 stomata were randomly chosen for the measurement of fluorescence intensity,

117

which were further analyzed with ImageJ software (National Institute of Health,

118

Bethesda, Maryland, USA) according to previous26.

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Antioxidant System Response Analysis. A certain number of A. thaliana was

120

ground with 2 mL of PBS. After centrifugation at 13,000 rpm for 20 min, the

121

supernatant was collected to perform further assays. The activity of superoxide

122

dismutase (SOD) and catalase (CAT) as well as the content of malondialdehyde

123

(MDA) and glutathione (GSH) were determined with a spectrophotometric method

124

using a Cary 100 UV-Vis spectrophotometer (Agilent Technology, Tokyo, Japan) and 7



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the detailed process has been described by Chen et al.25 All of the process were

126

performed in an ice bath and in triplicate.

127

Detection of Fe Concentration and Distribution. For the quantification of Fe,

128

plants were successively washed three times using distilled water and then oven-dried

129

at 60°C for 12 h. Then, 80- to 100-mg samples for each treatment were digested using

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6 mL of HNO3 and 200 µL of H2O2. The concentrations of Fe were determined with

131

ICP-MS (PerkinElmer, MA, USA). To measurer the distribution of Fe in plant tissues,

132

we used synchrotron X-ray microfluorescence (µ-XRF) with the beamline BL15U1 at

133

the Shanghai Synchrotron Radiation Facility (SSRF) of the Chinese Academy of

134

Science. The process was performed refer to our previous study,5 and details are

135

provided in Text S2 of the Supporting Information.

136

Preparation of Total RNA and Real-time PCR Analysis. Total RNA of A.

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thaliana was extracted using Trizol reagent (Invitrogen) according to the

138

manufacturer’s instructions. The RNA was then reverse-transcribed to cDNA using a

139

reverse transcriptase kit (Toyobo, Tokyo, Japan); Actin 2 was selected as the

140

housekeeping gene to normalize the expression changes. A three-step PCR protocol

141

was used as follows: a denaturation process at 95°C for 1 min and 40 cycles for 15 s,

142

followed by 55°C for 15 s and 72°C for 45 s. Real-time PCR was carried out by

143

employing the Stratagene Mx3000P (Agilent Technologies, Tokyo, Japan). Genes

144

related to Fe transportation and aggregation as well as with the acetyl-CoA

145

carboxylase (ACCase) were selected according to previous studies.5,

146

gene information is provided in Table S1 of the Supporting Information. 8


9, 27

Detailed

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Determination of DCPP Concentration in Arabidopsis thaliana. Fresh

148

Arabidopsis thaliana seedlings were harvested after 3-week cultivation for the

149

determination of DCPP concentrations. At first, 1-2 g seedlings were accurately

150

weighted and washed with ddH2O. Then 10 mL acetonitrile was added to mortar and

151

plants were thoroughly ground. The mortar was washed by another 15 mL acetonitrile.

152

The mixtures were centrifuged and the supernatants were purified by using

153

Carb-GCB/NH2 double-layer solid phase extraction (SPE) columns, which was

154

activated by 10mL of acetonitrile-toluene (3:1, v/v) in advanced. The eluents were

155

concentrated by rotary evaporation (40 °C) and dried under nitrogen gas (40 °C).

156

Then the samples (n=3 per treatment) were re-dissolved in 1 mL of methanol and

157

analysized by UPLC (Waters Corp., Milford, MA, USA) combined with quadrupole

158

time-of-flight tandem mass spectrometer (TripleTOF 5600+ System, AB SCIEX Corp.,

159

Framingham, USA). The detailed measurement process and parameters have been

160

provided in Text S4 of the Supporting Information.

161

Data Analysis. The data are presented as the mean ± standard deviation and

162

analyzed using Origin 9.0 software (OriginLab, Northampton, MA, USA). The

163

enantioselective difference (ED) value was calculated as the percentage of the

164

differences between the (R)-DCPP and (S)-DCPP treatments compare to the control

165

(i.e. (R-S)/control*100%). Comparisons were made with one-way analyses of

166

variance (ANOVA) followed by a multiple-comparison test of means (Tukey test).

167

The differences were considered statistically significant when the P value was less

168

than 0.05. 9



169 170

RESULTS AND DISCUSSION

171

Effect on the Growth of A. thaliana. In the preliminary experiment, a

172

concentration range of 1-aminobenzotriazole (ABT) was set to detect the potential

173

effect on the growth of A. thaliana (Text S1 of Supporting Information). As shown in

174

Figure S1, ABT was safe for the test plant at a wide range of concentrations.

175

Thereafter, we selected 20 and 40 µM for further experiments. As for the effect on 0.2

176

µM DCPP, the whole plants were much smaller when exposed to (R)-DCPP than those

177

of (S)-DCPP and the control (Figure 1A). However, the addition of ABT slightly

178

reduced the toxicity of (R)-DCPP in terms of fresh weight (Figure 1B), root length

179

(Figure 1C) and chlorophyll content (Table S2). Surprisingly and interestingly, the

180

toxicity of (S)-DCPP was mildly intensified by ABT, where the fresh weight

181

decreased by 12.27% and 24.48% with 20 and 40 µM ABT, respectively. And gap of

182

effects caused by DCPP enantiomers in aspects of root length and chlorophyll content

183

also became smaller.

184

ABT is a nonselective substrate inhibitor of cytochromes P450 enzymes

185

(CYP450s) in vitro and in vivo.28,

29

186

suggests that CYP450s may play an important role in the toxicity of DCPP. It should

187

also be noted that DCPP is a chiral herbicide, whose enantiomers exhibit

188

enantioselectivity in their interaction with organisms.30-33 In this study, DCPP

189

inhibited the growth of A. thaliana in an enantioselective manner, which was

190

consistent with our previous study.25 Additionally and more importantly, the presence

The regulation effect of ABT shown above

10


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of ABT reduced the toxicity of (R)-DCPP but didn’t significantly change that of

192

(S)-DCPP. This indicated that the inactivation of CYP450s attributed to the change in

193

the enantioselective toxicities of DCPP enantiomers to A. thaliana, which has not

194

been reported so far. These findings provide evidence of the role that CYP450s play

195

in the enantioselectivity of chiral herbicides. Therefore, further attempts were made to

196

explore the potential mechanism.

197

Production of Reactive Oxygen Species (ROS). Previous investigations have

198

revealed that reactive oxygen species (ROS) play multiple roles in plants under

199

abiotic stress.34-36 As for the leaves of A. thaliana shown in Figure 2A, significant

200

differences in ROS production were observed by comparing the (R)-DCPP treatment

201

(b) with that of the control (a) and (S)-DCPP (c). After adding 20 µM ABT, the

202

production of ROS in the (R)-DCPP-treated group exhibited a slightly decrease (e),

203

companied by an increase in that of (S)-DCPP (f). As the concentration of ABT

204

increased to 40 µM, ROS production of (R)-DCPP decreased by 36.51% but

205

(S)-DCPP increased by 24.33% compared to the groups without ABT (Figure 2B). A

206

similar phenomenon also occurred in the roots of A. thaliana (Figure S2).

207

ROS are chemically reactive molecules formed as a natural byproduct of the

208

normal metabolism of oxygen and play important roles in cell signaling and

209

homeostasis.37,

210

when under environmental stress, making it triggered by the number of external

211

contaminants and their harmful effects have long been recognized.37, 38 Furthermore, it

212

has been reported that interaction of ROS with contaminants can tune the

38

However, the ROS level in organisms can increase dramatically

11



213

enantioselectivity of chiral herbicides.6 As described above, even though DCPP still

214

profoundly induced the production of ROS in A. thaliana compared to the control

215

under high concentration of ABT, a significant difference between the enantiomers of

216

DCPP was disappeared, where the production of ROS in the (R)-DCPP groups

217

decreased and enhancement occurred in the (S)-DCPP groups. These results indicate

218

that the CYP450s were responsible for the enantioselectivity exhibited in ROS

219

production. Furthermore, considering that ROS production contributes to DCPP

220

phytotoxicity as previously reported,25 we further proposed that CYP450s mediated

221

the ROS production in A. thaliana induced by DCPP, which then caused the

222

enantioselective toxicity.

223

Response of Antioxidant System. The antioxidant response of A. thaliana

224

exposed to DCPP enantiomers and ABT was detected. The results are depicted in

225

Figure 3. In detail, as for superoxide dismutase (SOD) activities, (R)-DCPP stimulated

226

a 77.28% enhancement comparing to the control, but no significant change was

227

observed in the (S)-DCPP-treated groups, which is consistent with the production of

228

ROS production described above. With the introduction of 20 and 40 µM ABT, the

229

SOD activity of the (R)-DCPP groups decreased by 8.68% and 24.71%, respectively,

230

compared to under the absence of ABT. However, groups under (S)-DCPP stress

231

exhibited the opposite trend and SOD activities gradually increased as the

232

concentration of ABT increased, indicating that the oxidative stress was slightly

233

aggravated. In particular, it should also be noted that under high concentrations of

234

ABT, significant differences between DCPP enantiomers disappeared as also occurred 12


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in the comparison with control. Moreover, the catalase activities (CAT) (Figure 3B)

236

and the contents of malondiadehyde (MDA) (Figure 3C) as well as glutathione (GSH)

237

(Figure 3D) also exhibited similar results, where the addition of ABT help to narrow

238

the gap between the effects of DCPP enantiomers.

239

Changes in antioxidant enzymes showed that A. thaliana treated by DCPP

240

enantiomers underwent an oxidant stress. The damaged antioxidant system in the

241

plant was not able to scavenge the excess ROS effectively, which could further disrupt

242

to cellular functions.9 In this study, the addition of ABT stimulated a different change

243

trend of antioxidant response for (R)-DCPP and (S)-DCPP, which is consistent with

244

the experimental results of ROS production revealed above and confirms that

245

CYP450s play critical roles in the oxidative damage of DCPP in plants.

246

Gene Expression of Acetyl-CoA Carboxylase (ACCase). There are two types

247

ACCase in plants, homomeric and heteromeric ACCase (Types I and II). ACCase II

248

consists of four distinct subunits, in which the α- and β-carboxyltransferase domain

249

(α- and β-CT) subunits constitute the CT catalytic domain and the other two subunits

250

constitute the biotin carboxylase (BC) and biotin carboxyl carrier (BCC) domain of

251

this enzyme.9 Gene expression of ACCase was then detected. As shown in Figure 4A,

252

(R)-DCPP significantly downgraded the expression of CAC1, which encodes the BCC

253

domain but (S)-DCPP remained stable. With the addition of ABT, the expression of

254

CAC1 in the (R)-DCPP groups gradually increased. However, the expression of CAC1

255

was suppressed in (S)-DCPP treatment. Similarly, for gene CAC3, which encode the

256

α-CT domain, DCPP enantiomers still induced significant suppression in their 13



257

expression levels (Figure 4C). The addition of ABT helped to narrow the

258

enantioselective differences between DCPP enantiomers by adjusting the effects in a

259

different direction, especially for gene CAC2 and (R)-DCPP exhibited almost the

260

same inhibition effect to (S)-DCPP. In contrast to ACCase II, the domains were fused

261

into a single polypeptide in ACCase I, and the gene expression of ase I (Figure 4D)

262

also exhibited a similar change pattern to that of ACCase II, where the gap between

263

DCPP enantiomers also became smaller.

264

In plants, ACCase catalyzes the first and committed biosynthesis reaction of fatty

265

acids, whose multiple functions in plant biology, such as serving as the building

266

blocks of the membranes that physically divide all subcellular and cellular

267

compartments,

268

site of DCPP, and its CT domain is the main target binding sit in the plastid of

269

susceptible biotypes.41 Although the ACCase II was insusceptible to DCPP, the

270

expression of genes in the BC and CT domain in this study was still profoundly

271

inhibited (Figure 4A, 4B and 4C), indicating that most of gene expression of ACCase

272

I and ACCase II was affected, rather than only one single site as reported before,42

273

which is consistent with a previous study of another AOPP herbicide,

274

diclofop-methyl.9 Furthermore, the gene expression evidence also revealed that with

275

the addition of ABT, the damage to ACCase caused by DCPP enantiomers was

276

adjusted; to be exact, the BC domain of ACCase was probably the first part to be

277

disturbed (Figure 4B). All of these results provide us with an opportunity for

278

thoroughly understanding how CYP450s mediated the enantioselective phytotoxicity

have long been recognized.39, 40 The ACCase I is the action target

14


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of DCPP to A. thaliana.

280

Fe Uptake and Distribution in A. thaliana. Cross talk between enantioselective

281

phytotoxicity and element stress in plants exposed to chiral herbicides has been

282

previously investigated.5 In this study, as shown in Table 1, the Fe concentration in A.

283

thaliana seedlings exposed to (R)-DCPP was significantly lower than in the control or

284

(S)-DCPP treatments. With the addition of ABT, the inhibition effect on Fe uptake

285

gradually weakened in the (R)-DCPP groups, but Fe concentrations became lower

286

comparing to that without ABT in the (S)-DCPP groups. Furthermore, evidence about

287

the gene related to Fe transportation (IRT1, IRT2, FRO2, FRO3, NRAMP1, NRAMP3

288

and NRAMP4)16,43 in A. thaliana also supported the change in Fe concentration

289

(Figure S3).

290

In addition to quantification, the distribution of Fe also exhibited in an

291

enantioselective manner (Figure 5A), where the (R)-DCPP stimulated significant

292

aggregation around the edges of leaves. However, Fe was distributed uniformly in the

293

control and (S)-DCPP groups. When 40 µM ABT was added, the distribution of Fe in

294

the (R)-DCPP groups became more uniform, but only part of the aggregation was

295

observed compared to without ABT. In the (S)-DCPP groups, however, Fe began to

296

aggregate around the leaf stalk. Gene expression related to Fe aggregation in A.

297

thaliana is depicted in Figures 5B and 5C. The addition of ABT regulated the

298

expression of ISU 1 and ISU 3 induced by DCPP enantiomers in similar patterns,

299

which were corresponding to the Fe aggregation phenomenon discussed above.

300

As mentioned before, the abnormal behavior of Fe in plants revealed more than 15



301

the Fe, as molecules such as enzymes containing Fe may also be involved. In this

302

study, gene ISU1 and ISU3 were both negatively correlated with the expression of

303

four genes related to ACCase (Figure S4 and Table S3), indicating that the damage to

304

ACCase caused by DCPP was more severe as Fe in leaves aggregated to a greater

305

extent. A similar effect also occurred in another chiral herbicide imazethapyr as

306

previously reported.5 The addition of ABT adjusted the distribution of Fe in

307

Arabidopsis leaves exposed to DCPP enantiomers in an opposite direction, further

308

confirming that the enantioselective phytotoxicity of DCPP on plants was mediated by

309

CYP450s. Moreover, ROS were also correlated with Fe behavior. Therefore, with the

310

help of ABT, the role of Fe in the intrinsic relevance of ROS and ACCase damage,

311

which contribute the toxicity of DCPP, was revealed for the first time in this study.

312

Effects of CYP450s on the Concentration and Enantioselective Toxicity of

313

DCPP. Except for discussing the two action mechanism of DCPP, we further

314

determined the concentration of DCPP in A. thaliana by using UPLC-QTOF-MS.

315

Negative ion mode was selected due the higher intensity comparing to positive ion

316

mode (Figure S3 A&B). The primary and secondary structure (Figure 6 A&B) of

317

DCPP standard (5 mg/L) as well as the extraction of ion current (XIC) (Figure S3B)

318

have been obtained. Then DCPP concentration in the plants of control group was

319

determined and there was no significant high concentration of DCPP in A. thaliana

320

(Figure S3G). As for the DCPP levels in plants treated to ABT and DCPP depicted in

321

Figure 6C, different change trend occurred in both DCPP enantiomers. To be exact,

322

concentration of (R)-DCPP decreased by 69.45% with the presence of 40 µM ABT in 16


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0.2 µM DCPP; however, (S)-DCPP increased by 1.43–fold.

324

As one typical types of chiral herbicides, both DCPP enantiomers exhibited

325

toxicity by different mechanism. As the herbicidal active ingredient, (R)-DCPP can

326

attack target enzyme such as acetyl-CoA carboxylase in plants;4,

327

non-herbicidal active (S)-DCPP only exhibited its toxicity only when the

328

concentration raised to relative high. In the present study, the decrease of (R)-DCPP

329

concentration were consistent with the toxicity decrease; however, enhancement of

330

(S)-DCPP may also leading it to start exhibiting toxic effects comparing to the control.

331

The concentration and enantioselective toxicity of DCPP enantiomers changed by the

332

addition of ABT, which did not exhibit a significant effect on the growth of A.

333

thaliana by itself (Figure S1). Therefore, the concentration data indicated that

334

CYP450s mediate metabolism influences the level of DCPP enantiomers in A.

335

thaliana, and as a result, these differences in DCPP levels finally affect toxic

336

outcomes. On the other hand, interestingly, the enantioselective difference (ED)

337

between DCPP enantiomers was decreased remarkably in aspects ranging from basic

338

growth effects to gene expression patterns (Figure 7). The detailed ED data are shown

339

in Table S4 of the Supporting Information. Taking ROS production in leaves as an

340

example, the ED value was calculated to be as large as 291.88% without ABT. With

341

the addition of ABT, the ED between DCPP enantiomers decreased to 114.59% at 20

342

µM and to 26.68% at 40 µM.

44

but the

343

As for CYP450s, they have been reported to be able to catalyze the oxidation of

344

plant fatty acids in an enantioselective manner.21 Furthermore, enantioselective 17



345

investigation was also performed for CYP450s in the interactions between plants and

346

pathogens.45 In this study, considering that ABT is one of the nonselective substrate

347

inhibitors of CYP450s, the addition of ABT helped to classify the responsible roles of

348

CYP450s for the enantioselective effects of the chiral herbicide DCPP. In addition, the

349

abnormal uptake and distribution of Fe in A. thaliana were correlated with the

350

phytotoxicity of DCPP, which in turn revealed that Fe played an important role in

351

elucidating the intrinsic relevance between ROS and ACCase in enantioselective

352

damage. An insightful understanding of the roles of Fe and CYP450s in the

353

enantioselective phytotoxicity of chiral herbicides is of great importance. It not only

354

makes the mechanism of herbicides much brighter but also beneficial for the whole

355

ecosystem. Plants are at the bottom of the food chain in an ecosystem, and they may

356

adsorb and transfer the chiral herbicides into their body, which may impact the health

357

of the whole ecosystem through the food chain. Therefore, investigations into the

358

toxic effects of chiral herbicides on plants would offer an opportunity for a more

359

comprehensive understanding of herbicide security and could provide basic evidence

360

for risk assessments of chiral herbicides in the environment.

361

18


Page 18 of 34

Page 19 of 34


362

ASSOCIATED CONTENT

363

Supporting Information. Texts S1-S4, Table S1-S4 and Figure S1-S5 are

364

referenced in this paper. This information is available free of charge via the Internet at

365

http://pubs.acs.org.

366 367

AUTHOR INFORMATION

368

*Corresponding Author: Phone: (86)-88982421. E-mail: [email protected]

369

MOE Key Laboratory of Environmental Remediation & Ecosystem Health, College

370

of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058,

371

China

372 373

ACKNOWLEGMENTS

374

We thank profoundly to Mr. Zhiwei Ge of Core Facilities for Agriculture, Life

375

and Environment Sciences of Zhejiang University for the UPLC-QTOF-MS data

376

analysis. This work was supported by the National Natural Science Foundation of

377

China (NSFC, No. 21377111, 21677124, Key Program Grant No. 21427815 and

378

International Cooperation Grant No. 21320102007).

379

19



380

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Table 1. Fe content in Arabidopsis thaliana seedlings treated with dichlorprop (DCPP) and 1-aminobenzotriazole (ABT) a Treatment Fe Content (mg/g plant) a

Control

(R)-DCPP

(S)-DCPP

Without ABT 0.85 ± 0.01a

0.30 ± 0.02f

0.83 ± 0.04a

20 µM ABT

0.81 ± 0.02a

0.41 ± 0.07e

0.67 ± 0.03b

40 µM ABT

0.82 ± 0.02a

0.52 ± 0.01d

0.61 ± 0.01c

The data presented consist of average values ± standard deviation of three independent batches.

Different letters in the column represent statistically significant differences (p

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