Article pubs.acs.org/est
Enantioselective Damage of Diclofop Acid Mediated by Oxidative Stress and Acetyl-CoA Carboxylase in Nontarget Plant Arabidopsis thaliana Qiong Zhang,†,‡ Meirong Zhao,†,* Haifeng Qian,† Tao Lu,† Quan Zhang,† and Weiping Liu‡,* †
College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou 310032, China MOE Key Lab of Environmental Remediation and Ecosystem Health, Institute of Environmental Science, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, China
‡
S Supporting Information *
ABSTRACT: Diclofop-methyl (DM) is a widely used chiral herbicide, which rapidly hydrolyzes to its major metabolite diclofop acid (DC) after application. With a carbon chiral center, DC not only is an important ingredient of herbicidal activity, but also has a long half-life in soil. Studies so far have only considered the activity of racemic DM in target organisms, and the enantioselective toxicity in nontarget plants of DM and DC has yet to be explored. In this study, the enantioselective phytotoxicity of DC mediated by oxidative stress and the key enzyme ACCase in the fatty acid synthesis system on the model plant Arabidopsis thaliana was investigated. Significant differences between the two enantiomers were observed in phytotoxicity including growth inhibition, oxidative damage and alteration of key genes expression of ACCase, with R-DC showing greater toxicity to Arabidopsis thaliana than S-DC. The results of molecular docking showed that there was a stronger affinity between RDC and the target enzyme carboxyltransferase domain of ACCase, likely leading to the enantioselective phytotoxicity of DC. This study suggested that chirality of both parent compounds and metabolites should be considered to improve our understanding of the environmental fate and risks of chiral pesticides.
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INTRODUCTION Diclofop-methyl {2-[4-(2, 4-dichlorophenoxy)]-phenoxypropionate methyl ester (DM)} is one of the aryloxyphenoxy propionic acid (AOPP) postemergence herbicides and is widely used for annual grass control.1 The usage of DM was up to 5 million kilograms in China in 2006.2 When applying to crops and weeds, up to 73% of the active ingredient of DM may fall onto the soil surface.3 In soil, DM is known to hydrolyze to its corresponding acid, that is, diclofop acid {2-[4-(2, 4dichlorophenoxy)]-phenoxypropionic acid (DC)} within one day.4,5 Both the methyl and acid forms of the herbicide have been detected in runoff and irrigation return waters.6 For instance, Waite et al.7 detected DM at a 53% frequency in a watershed during the growing season over a four-year period. The frequency of detection of DC in water from nearby farm ponds and dugouts in the Canadian prairies from the fall of 1987 to the spring of 1989 was 46−95%.8 Both DM and DC are herbicidally active and each consists of a pair of enantiomers (Figure 1). © 2012 American Chemical Society
Figure 1. Enantiomers of chiral diclofop methyl (DM) and diclofop acid (DC). DM enantiomers are on the top with (S) and (R) indicating their optical difference.
An increasing number of studies on the toxicity and environmental fate of chiral herbicides have demonstrated the Received: Revised: Accepted: Published: 8405
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between chiral DC and CT domain of ACCase to explain the enantioselective phytotoxicity of DC.
occurrence of enantioselectivity in their interactions with a range of organisms.9−11 However, so far chiral selectivity of their metabolites has been largely ignored. It is known that metabolic intermediates of a pesticide may have enhanced activity or environmental stability. For example, 2,6-diethylaniline and 2-ethyl-6-methylaniline, the degradation products of alachlor and metolachlor, respectively, are more teratogenic than their parent compounds when evaluated with embryos of Xenopus laevis.12 In some other cases, although the parent compound itself is achiral, its metabolites may become chiral due to the structural alteration.5,13 Therefore, while chirality of parent compounds has gained gradual attention, similar emphasis has yet to be placed on chiral metabolites.14 To our knowledge, the toxicological studies of DC in plants were mainly concentrated on alga,1,10 little was known about high plants. Shimabukuro and Hoffer15 summarized two models to explain the action mechanisms of AOPP herbicides. Model 1 was called as a catabolic mechanism that has a single-site and multiple-effects involving the collapse of the transmembrane proton gradient and generation of highly destructive free radicals. Reactive oxygen species (ROS), like H2O2, O−2•, HO•, are necessary free radicals and signaling molecules in signaling transduction and other biochemical processes. Meanwhile, plants have evolved antioxidative strategies in which enzymatic and nonenzymatic systems scavenge the excess generation of free radicals. In enzymatic systems, cellular damages induced by oxidative stress conditions may be alleviated by protective metabolites involving antioxidative enzymes such as SOD, CAT, and POD. Qian et al.16 studied the enantioselective effects of herbicide imazethapyr (IM) on SOD, POD, and CAT activities in rice. They found that the maximum activities of SOD, POD, and CAT were all induced at the 0.5 mg/L concentration of R-IM treatment, and reached levels that were 1.21-, 4.6-, and 2.4- times over the control, respectively. However, once ROS accumulate under environmental stresses to a certain amount that the antioxidant systems in plants cannot scavenge efficiently, it may damage the cell irreversibly. Herbicide is also considered as one of environmental stresses that can lead to an oxidative damage in plants.17−20 Model 2, as an anabolic mechanism, is a single-site and single effect mechanism involving the inhibition of ACCase. In plants, acetyl-CoA carboxylase (ACCase) catalyzes acetyl-CoA carboxylation to produce two types of malonyl-CoA, one in plastids that is an important substrate of fatty acid synthesis and the other in the cytosol that is the precursor for fatty acid elongation and secondary metabolites.21,22 ACCase has been considered as the target site of inhibition by AOPP herbicides such as DM. During the recent decades, a mount of research focused on the DM toxicity on susceptible species. The target enzyme site or membrane potentials mechanisms of DM action had also been discussed extensively.23−25 However, the toxicity mechanism of DC on nontarget plant, the stereoselective toxicity of DC enantiomers are rarely referred to. In this study, we obtained enantiomers of DC and characterized their selective phytotoxicity on a model plant Arabidopsis thaliana. Oxidative damage was explored by antioxidative enzymes and lipid peroxidation. Meanwhile, the target enzyme ACCase related genes of Arabidopsis thaliana, which is a nontarget plant with a fully sequenced genome, were selected to study the selectivity mechanisms. Furthermore, molecular docking was employed to calculate the affinity
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MATERIALS AND METHODS Reagents and Plant Materials. Diclofop-methyl was provided by Iprochem in Shenzhen, China. The racemic DC was prepared from DM according to Smith26 and identified by high-performance liquid chromatography (HPLC). The enantiomers R- and S-DC (purity >99.0%) were prepared using a previously reported procedure.27 Arabidopsis thaliana (ecotype Columbia [Col]) seeds were obtained from the National Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences (Beijing, China). All organic reagents used in this study were of analytical purity. Plant Cultivation and Herbicide Treatment. The A. thaliana seeds were screened by soaking in water and the seeds that floated to the surface were discarded. The selected seeds were sterilized with HClO (4%) and 75% ethanol, followed by rinsing with distilled water. The sterilized seeds were vernalized at 4 °C for 2−3 d. The mineral salt medium was sterilized at 121 °C for 20 min and spiked with the rac-DC, R-DC or S-DC, at 1 mg/L each. After the medium was solidified, the vernalized seeds were sowed in the solid medium. Herbicide-free plates were similarly prepared as the control. The plates were placed in a constant temperature room (25 ± 0.5 °C) equipped with cool-white fluorescence lights of 300 μmol/m2/s fluorescence intensity and a 12 h light/12 h dark cycle. Substructure Detection by Transmission Electron Microscopy. Leaf samples of control and DC-treated plantlets were cut to 1 cm2 pieces and fixed for over 2 h in a cacodylate buffer solution containing 2.5% glutaraldehyde. Samples were then treated with 1.0% OsO4 for 1.5 h and dehydrated in acetone several times. After that, samples were embedded in epoxy resin. Ultrathin sections (70−90 nm) were obtained using a Reichert Ultracuts ultramicrotome (Tokyo, Japan), and stained with uranyl acetate by lead citrate. Finally, the leaf samples were observed with a JEM-1230 microscope (JEOL Ltd., Tokyo, Japan). Antioxidation Enzyme Extraction and Analysis. A. thaliana plantlets were ground in a mortar with 2 mL PBS buffer (pH 7.4) in an ice bath. Every treatment was done with four replicates. After centrifugation at 2500 rpm for 10 min, the supernatant was collected to assay the antioxidant enzyme activity and malondialdehyde (MDA) level. The activity of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and lipid peroxidation level reflected by MDA was determined by using a commercial kit (Jiancheng Bioengineering Institute, Nanjing, China). The principles of measurements can be found in the literature.28−31 CAC1, CAC2, CAC3, aCCD, and ACCaseI Gene Analysis. The whole A. thaliana plantlets were collected and ground to powder in liquid nitrogen. Trizol (0.5 mL per sample) was added to extract RNA. Reverse transcription and PCR were performed with a reverse transcription and PCR kit (Toyobo, Tokyo, Japan). The housekeeping gene was Actin 2 for plants. A two-step PCR protocol was used: denaturation process at 95 °C for 1 min and 40 cycles for 15 s, followed by 60 °C for 1 min. A real-time PCR was carried out by employing the Eppendorf MasterCycler ep RealPlex4 (Wesseling-Berzdorf, Hamburg, Germany). Statistical Analysis. Data were presented as mean ± standard error of the mean (SD) and statistical significance was 8406
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The substructure of Arabidopsis thaliana leaves was examined to survey the changes of cell and chloroplast configuration after treatment for 4 weeks. Treatments resulted in irregular cell shapes with disappearing cell membranes, and chloroplasts wandered in cytosol, especially with the S-DC treatment (Figure 3A). After exposed to DC, chloroplasts appeared swollen and the numbers of starch granules increased significantly as compared with the control (Figure 3B). After exposure to R-DC, the starch granules occupied almost the whole chloroplast space. As the major sites of photosynthesis, chloroplasts produce substances such as glucose during photosynthesis, which can be used to make other molecules such as cellulose and starch needed for their structure and functions.32 Chloroplast injuries can inhibit the flow of energy and conversion of organic matters. Therefore, the observed increase of starch granules suggested that the intracellular glycometabolism was significantly inhibited. Furthermore, both the pigment of photosynthesis and the electron transfer systems are located on the thylakoid membrane; the changes of thylakoid structure such as thinning or loss of integrity would inevitably affect the normal photosynthesis processes. The thylakoid structure in A. thaliana chloroplast is shown in Figure 3C. Compared to the control treatment, the granal thylakoid was thinner in the DC treatments. The connection between the granal thylakoid and stromal thylakoid was lessened and in some places the links disappeared. This phenomenon was more obvious in the R-DC treatment because of the extrusion by starch granules. Therefore, all DC treatments in this study led to damages to the plant in both morphology and organelles like chloroplast, and the injury caused by R-DC was more pronounced. Activities of Oxidative Stress Related Enzymes. The activity of CAT was stimulated by DC treatments (Figure 4A), and the stimulation was the most pronounced after treating for three weeks. CAT activity of S-DC treatment was inhibited at two weeks, while enhanced obviously at three and four weeks. However, the activity of R-DC treatment was stimulated after
analyzed by Orign 6.0 (Microcal Software, Northampton, MA). Values were considered to be significantly different when the probability (P) was less than 0.05.
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RESULTS AND DISCUSSION Growth Macrographs and Substructure of Arabidopsis thaliana Plants. Compared to the control, the whole plants were smaller when treated with rac-DC or R-DC, with the root length decreased. Plant leaves were partially yellow and displayed pigment precipitation, especially for plants receiving R-DC treatment. The root length of plants treated with S-DC was similar to that of the control, but the leaves were generally smaller than those of the control treatment (Figure 2).Ye et
Figure 2. Photographs of Arabidopsis thaliana plants treated by DC for three weeks.
al.27 studied the 72 h acute toxicity of DC enantiomers toward rice Xiushui 63 seedlings and also observed that the Renantiomer was more toxic to roots than its S counterpart.
Figure 3. The electron microscopic photos of Arabidopsis thaliana leaves treated with DC for four weeks. (A) Mesophyll cell structure; (B) Chloroplast; (C) Thylakoid structure. 8407
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Figure 4. The activity of antioxidative enzyme and Malondialdehyde content in Arabidopsis thaliana after different exposure times. (A) catalase (CAT); (B) superoxide dismutase (SOD); (C) peroxidase (POD); (D) malondialdehyde (MDA) content. * and ** depict a statistically significant difference as compared to control (p < 0.05 or 0.01 respectively, ANOVA); and represent a statistically significance between R-DC treatment and SDC treatment (p < 0.05 or 0.01 respectively, ANOVA). Similarly hereinafter.
two weeks and increased at three weeks, and then the activity decreased to a level similar to that of the control after treating for four weeks. The results of CAT activity showed that plants treated by R-DC were more susceptible than S-DC treatments. The activity of SOD indicates the capacity that an organism eliminates the free radicals in cells. In this study, as shown in Figure 4B, SOD activity in plants was stimulated by R-DC significantly after treating for 3 or 4 weeks. The increase of POD activity is considered as a physiological index of aging tissues. In this study, POD activity was increased substantially after 3 weeks of exposure to DC. The POD activity following R-DC treatment was greater than that after S-DC treatment except for the two week interval (Figure 4C). Overall, the enzymatic systems responsible for scavenging excess free radicals were greatly affected by DC as reflected by the high level of CAT, SOD and POD activity following exposure. Meanwhile, as shown in Figure 4D, oxygen free radicals attacked polyunsaturated fatty acid (PUFA) in organism membranes and initiated lipid peroxidation. MDA is one of the important products and MDA level is an index of lipid peroxidation.33,34 Compared to the control, MDA level increased after two weeks of exposure to R-DC or S-DC. A statistically significant difference was observed for 4 weeks of RDC treatment. Changes in antioxidant enzymes and plant substructures together showed that plants treated by DC likely underwent transmembrane potential changes when DC molecules were bonded at the putative membrane binding sites, and that the
antioxidant system in the plant was not able to deal with the excess ROS effectively, leading to consequent disruptions to cellular functions exhibited through the important organelle chloroplast. As a result, the normal photosynthesis was affected and the growth was inhibited. Furthermore, the changes of gene transcription level related with ROS may supply a more thorough understanding of oxidation stress caused by herbicides, as described by other researchers.35,36 Gene Expression of Heteromeric and Homomeric Acetyl-CoA Carboxylase (ACCase I and ACCase II) in Arabidopsis thaliana. The gene expression of four subunits of heteromeric ACCase is shown in Figure 5. Compared to the control, the gene expression of BCC domain was stimulated after four weeks of exposure to R and S-DC treatments. In contrast, the expression of the other genes was inhibited except for α-CT domain after two weeks of exposure. The gene expression of β-CT domain was inhibited significantly in all DC treatments. For the gene expression of BC domain and α-CT domain, there was a time lag in S-DC treatment as compared with R-DC treatment, that is, although the inhibition of BC domain was stronger in R-DC treatment than in S-DC treatment two weeks later, the gene expression was inhibited more by S-DC treatment than with the R-DC treatment after four weeks of exposure. The gene expression of α-CT domain was stimulated by S-DC treatment in the early two weeks and then inhibited more than other treatments as time elapsed. In contrast to the heteromeric ACCase, in which the functional domains of the enzyme are separated on distinct subunits, these 8408
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Figure 5. Gene expression of four subunits of heteromeric acetyl-CoA carboxylase (ACCase) in Arabidopsis thaliana. (A) Biotin carboxyl carrier (BCC) domain; (B) Biotin carboxylase (BC) domain; (C) α-carboxyltransferase (CT) domain; and (D) β- carboxyltransferase (CT) domain.
Figure 6. The gene expression of homomeric acetyl-CoA carboxylase (ACCase) in Arabidopsis thaliana.. 8409
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thaliana CAT appeared to be more lasting. According to Diao et al.,5 S-DC was found to readily undergo enantiomerization, and the enantiomer conversion may be used to explain the observed time lag in activity inhibition by the S-enantiomer. Molecular Docking to Simulate the Affinity between DC and Target Enzyme. In order to give a structural explanation for the experimental results, molecular docking was performed to further explore the mechanisms of enantioselective inhibition on ACCase by DC. Zhang et al.44 simulated a possible binding mode between DM and CT domain of ACCase from yeast. They found that DM was bound at the interface of the dimer of the CT domain that was considered as the active site. Conformational changes of several amino acid residues in this interface created a highly conserved hydrophobic pocket. After placing chiral DC micromolecules (molecules with a small molecular weight, for example, micromolecule peptides) into the same hydrophobic pocket that was the active site for DM, the total free energy of binding and inhibition constant (Ki) were estimated. Chiral DC micromolecules and amino acid residues within 6 Å radius are shown in Figure 7. The binding pocket started from a Pyr-
domains are fused into a single polypeptide in the homomeric ACCase. In our study, after treating for two weeks, the gene expression of homomeric ACCase was significantly inhibited by R-DC while slightly stimulated by S-DC. When treated by DC for four weeks, the gene expression of homomeric ACCase in all plants treated by DC was inhibited and the inhibition in SDC treatment was more pronounced than in R-DC treatment (Figure 6). Model 2 is a single-site mechanism involving the inhibition of ACCase. ACCase, which catalyzes acetyl-CoA carboxylation to produce malonyl-CoA, is an important intermediate metabolite in fatty acid synthesis and metabolism. Fatty acid synthesis is the basic course in biomembrane and lipin synthesis.18 Heteromeric ACCase (ACCase II) is composed of four distinct subunits. The α- and β-CT subunits constitute the CT catalytic domain, and the other two subunits constitute the BC and BCC domains of this enzyme. The BCC domain carries the biotin prosthetic group, which is thought to swing between the BC and CT active sites. At the BC active site biotin is carboxylated, and at the CT active site the carboxyl group is transferred to the final product of the reaction. Homomeric ACCase (ACCase I) is a 500-kDa enzyme that is composed of two identical subunits. Homomeric ACCase is also the target site of action for the AOPP herbicides, and to be exact, its carboxyltransferase (CT) domain of homomeric ACCase is the main target binding site in the plastid of susceptible biotypes.23,37 Although the heteromeric ACCase was insusceptible to AOPP,38 the subunits of BC and CT domain genes expression of nontarget plant A. thaliana were still inhibited significantly in all DC treatments after four weeks of exposure in this study. Previous studies showed that the target site which AOPP herbicides interact with is located in the single isoleucine/leucine residue in the CT domain of homomeric ACCase in plastid.39,40 However, our results of gene expression indicated that most gene expression of heteromeric and homomeric ACCase except for BCC domain in heteromeric ACCase was inhibited, rather than one single site as reported before.41 Just because the presence of both ACCase I and ACCase II in Arabidopsis thalina, it provided us an opportunity for thoroughly clarifying the toxicology effect of DC mediated by target enzyme (ACCase) system. Menéndez and De Prado42 purified and separated two kinds of ACCase in both resistant and susceptible biotypes of A. myosuroides and also found that DM resistance in the resistant biotype was not due to a mutation at the target site. Therefore the target of DC action should be clarified in future studies. Besides, the two weeks exposure results in this study indicated that R-DC treatment was more sensitive than S-DC treatment in ACCase gene expression in almost every gene. The stereoselectivty of DC on isolated maize chloroplasts was studied by Hoppe and Zacher,43 where the authors also found that the R-(+)-stereoisome was >40 times more sensitive than the S-(−)- stereoisomer. Diao et al.5 found that S-DC was preferentially degraded under aerobic and anaerobic conditions in soil, and therefore, the enantioselective degradation may further contribute to the relatively greater phytotoxicity by R-DC because of its longer residue time. Interestingly, although the gene expression of S-DC treatment was not strongly inhibited or even stimulated in αCT domain when compared to R-DC treatment, S-DC could induce a more forceful gene expression inhibition in A. thaliana after time has elapsed. The phenomenon was analogous to the CAT result discussed above, or the action of S-DC to A.
Figure 7. The schematic drawing of the interactions between diclofop acid and the CT domain of ACCase from yeast.
1738 amino acid, the distance between O atom in R-DC micromolecule and H atom in Pyr-1738 was measured and a hydrogen bond was likely formed there. The value of free energy of binding in R-DC action (−5.09 kcal/mol) was lower than for S-DC action (−4.99 kcal/mol). Compared to S-DC action (221.06 μM), the inhibition constant of R-DC action (186.84 μM) was also smaller. The smaller of the two values, the stronger the binding capacity. Compared with S-DC, the toxicity of R-DC was stronger because of the stronger binding capacity between the R-DC and the possible target site. Therefore, our simulation results agreed with the experimental results, or R-DC was more toxic because of its stronger affinity to the target enzyme. Zhou et al.45 also employed computational molecular docking to evaluate the molecular interactions between herbicide imazethapyr (IM) enantiomers and targeting acetolactate synthase (ALS). Results showed that the IM enantiomers enantioselectively suppressed the in vitro and in vivo ALS activity of maize leaves and R-(−)-IM was more active than S-(+)-IM. Therefore, the molecular docking was useful in providing further insight into the possible toxicity mechanism of chemicals at enantiomeric level. Results from this study indicated that the enantioselective damage of DC in the model plant A. thaliana involved both models, that is, the oxidative damage and inhibition of ACCase. Plants, as the producers and the bottom of food chain in the ecosystem, their physiological properties, as well as their ability of absorption and transformation of chiral chemicals in their body, may affect the whole food chain in the ecosystem. Therefore, the study of toxic effects and residues of herbicides in plants can give a more comprehensive understanding of herbicide safety. In addition, the risk assessment of parent 8410
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chemicals should extend to its main metabolites, especially for the parent chemicals that have shorter residence time or lower toxicity compared to their metabolites. A. thaliana, as a model plant with a clear physiological characteristic and genome, is a good model in studying the action mechanism of herbicide and other deleterious environmental pollutants.
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ASSOCIATED CONTENT
S Supporting Information *
A table for the primers of acetyl-CoA carboxylase genes is listed in the Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
* Fax: +86-571-88982341. E-mail:
[email protected] (W.P.L.);
[email protected] (M.R.Z.). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This study was supported by the National Basic Research Program of China (2009CB421603 and 2010CB126101), and the National Natural Science Foundations of China (20837002 and 30771255).
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ABBREVIATIONS AOPP aryloxyphenoxy propionic acids DM diclofop methyl {2-[4-(2, 4-dichlorophenoxy)]-phenoxypropionate methyl ester} DC diclofop acid {2-[4-(2, 4-dichlorophenoxy)]-phenoxypropionic acid} ROS reactive oxygen species CAT catalase SOD superoxide dismutase POD peroxidase MDA malondialdehyde ACCase acetyl-CoA carboxylase BCC biotin carboxyl carrier domain of acetyl-CoA carboxylase BC biotin carboxylase domain of acetyl-CoA carboxylase CT carboxyltransferase domain of acetyl-CoA carboxylase
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dx.doi.org/10.1021/es300049q | Environ. Sci. Technol. 2012, 46, 8405−8412
