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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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Enantioselective Phytotoxicity of Dichlorprop to Arabidopsis

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thaliana: Effect of Cytochrome P450 enzymes and Role of Fe

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

5 6 7



MOE Key Laboratory of Environmental Remediation & Ecosystem Health, College

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

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China

10 11



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

Station, Texas 77843, United States

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*

Corresponding author:

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

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ABSTRACT:

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The ecotoxicology effects of chiral herbicides have long been recognized and

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

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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

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

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

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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.

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

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

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herbicides in the environment.

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Graphic Abstract

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INTRODUCTION

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

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

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through the food chain.1,

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

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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

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

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

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

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

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

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

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

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

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

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

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

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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

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

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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

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

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

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stress

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

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

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

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

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

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

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

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that CYP450s may mediate the enantioselective phytotoxicity of chiral AOPP

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

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herbicides has still been minimally studied.

may

not

only

reveals

situation

of element itself,

but also the

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

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

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mechanisms.

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

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experiments,22,

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

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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

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

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

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

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

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which will provide opportunities for better understandings herbicide security and

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

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environment.

88 89

MATERIALS AND METHODS

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

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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

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

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

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

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

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

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

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

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and our previous research,

respectively. The cultivation process and herbicide

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

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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

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

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

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was defined as the control group.

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Measurements of Reactive Oxygen Species (ROS) Production. The

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

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

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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,

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

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

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

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

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

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

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Bethesda, Maryland, USA) according to previous26.

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

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

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

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

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

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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

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performed in an ice bath and in triplicate.

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

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

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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

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

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

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

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

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provided in Text S2 of the Supporting Information.

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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

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

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

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

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

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

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

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

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

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

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Determination of DCPP Concentration in Arabidopsis thaliana. Fresh

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

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

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

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

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The mixtures were centrifuged and the supernatants were purified by using

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

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

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

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

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

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

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

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provided in Text S4 of the Supporting Information.

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

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

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

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

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

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

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

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than 0.05. 9

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RESULTS AND DISCUSSION

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

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

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

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

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

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

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

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

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

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

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

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

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also became smaller.

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ABT is a nonselective substrate inhibitor of cytochromes P450 enzymes

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(CYP450s) in vitro and in vivo.28,

29

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

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

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

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

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

The regulation effect of ABT shown above

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

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

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

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

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

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explore the potential mechanism.

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

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

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

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

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

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

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

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

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

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

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

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

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homeostasis.37,

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

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

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

38

However, the ROS level in organisms can increase dramatically

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

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

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

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

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

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that the CYP450s were responsible for the enantioselectivity exhibited in ROS

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production. Furthermore, considering that ROS production contributes to DCPP

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

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

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enantioselective toxicity.

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

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

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

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

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

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

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

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

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exhibited the opposite trend and SOD activities gradually increased as the

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

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

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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)

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

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

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the gap between the effects of DCPP enantiomers.

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

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

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

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

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

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the experimental results of ROS production revealed above and confirms that

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

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

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

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

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

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

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

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

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

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

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

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

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

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enantioselective differences between DCPP enantiomers by adjusting the effects in a

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

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

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

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

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DCPP enantiomers also became smaller.

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

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

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blocks of the membranes that physically divide all subcellular and cellular

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compartments,

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

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

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

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

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

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

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

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

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

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

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thoroughly understanding how CYP450s mediated the enantioselective phytotoxicity

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

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of DCPP to A. thaliana.

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

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

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

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

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

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

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

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

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

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(Figure S3).

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

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

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

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

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

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

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

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

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

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which were corresponding to the Fe aggregation phenomenon discussed above.

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

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

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

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

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

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

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

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

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

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

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

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

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Effects of CYP450s on the Concentration and Enantioselective Toxicity of

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

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

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

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

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

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

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

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

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

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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.

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

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

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

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

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

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

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

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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

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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.

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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

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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