Article pubs.acs.org/JAFC
Are Nutrient Stresses Associated with Enantioselectivity of the Chiral Herbicide Imazethapyr in Arabidopsis thaliana? Zunwei Chen,† Hui Chen,† Yuqin Zou,† Jiguo Qiu,† Yuezhong Wen,*,† and Dongmei Xu§ †
Institute of Environmental Science, Zhejiang University, Hangzhou 310058, China College of Biological and Environmental Engineering, Zhejiang Shuren University, Hangzhou 310015, China
§
S Supporting Information *
ABSTRACT: Plant growth can be inhibited by herbicides and is strongly limited by the availability of nutrients, which can influence human health through the food chain. Until now, however, cross talk between the enantioselectivity of herbicides and nutrient stresses has been poorly understood. We analyzed trace element and macroelement contents in shoots of Arabidopsis thaliana treated by the chiral herbicide imazethapyr (IM) and observed that multiple-nutrient stress (trace elements Mn, Cu, and Fe and macroelements P, K, Ca, and Mg) was enantioselective. The (R)-IM treatments resulted in Mn 23.37%, Cu 63.53%, P 30.61%, K 63.70%, Ca 34.32%, and Mg 36.14% decreases compared with the control. Interestingly, it was also found that herbicidally active (R)-IM induced notable aggregation of nutrient elements in leaves and roots compared with the control and (S)-IM. Through gene expression analyses, it was found that herbicidally active (R)-IM induced the up- or down-regulation of genes involved in the transport of nutrient elements. We propose that (R)-IM affected the uptake and translocation of nutrient elements in A. thaliana, which destroyed the balance of nutrient elements in the plant. This finding reminds us to reconsider the effect of nutrient stresses in risk assessment of herbicides. KEYWORDS: nutrient, stresses, enantioselectivity, chiral herbicide, food quality
■
INTRODUCTION The benefits of global pesticide use come at the cost of their widespread occurrence in the environment, which represents an important source of diffuse chemical pollution that is difficult to control.1 In view of irreplaceable pesticides, environmental protection, and food safety, there has been an increased use of “green” chiral pesticides, which target organisms for higher performance while being safer and friendlier for nontarget organisms and humans.2 Chiral pesticides constitute up to 30% of all pesticides used, and this ratio is increasing.3 The enantiomers of a chiral chemical generally undergo the same physical and chemical processes in the environment; however, their ecotoxic effects are typically enantioselective.4−8 Therefore, a comprehensive understanding of the significance of enantioselective behavior is imperative for improving risk assessment and regulation of these pesticides.9,10 It is commonly recognized that the enantioselectivity of a chiral pesticide is likely related to chemical structures, biological processes, and the environment.11−13 Among these factors, chemical structures and biological processes have been studied extensively for a long time. For example, from studies of biological factors, enantioselective toxicity has been found to be related to different microorganisms, enzyme systems, and the activation of signaling pathways in cells.11,14−18 Although these findings have explained some phenomena related to the enantioselectivity of chiral pesticides, they have relatively broad biological specificity and cannot easily be used to understand the effects of environmental factors on the enantioselectivity of pesticides.19,20 Due to the complexity of various environments, one of the major challenges in ecotoxicology is to trace the effects and side effects of chiral pesticides from their cellular targets in the environment. © 2015 American Chemical Society
Therefore, it is necessary to elucidate the relationship between the enantioselectivity of chiral pesticides and the environment to provide insights into how individual transformation processes contribute to observed bulk toxicity. Plants are intimately connected with the natural environment, and their growth is strongly limited by the availability of light and nutrients.21 Under conditions with sufficient light, plant growth is controlled by the supply of nutrients, particularly essential trace elements (hereafter referred to as trace elements), which can influence human health through the food chain. While trace elements are essential, they are required at very small concentrations.21 Numerous studies have shown that a relative balance of nutrient elements is necessary for plant growth and development; a lack or surplus of an element will cause an imbalance between nutrients and may result in toxic effects. In the meantime, pesticides can inhibit plant growth by adversely affecting important physiological processes. It is widely known that considerable differences between enantiomers of chiral pesticides are frequently observed in terms of their toxicity to plants.8 Therefore, the question arises as to whether chiral pesticides result in an imbalance of nutrient elements in plants and therefore enantioselectively toxic effects. Furthermore, it is crucial to understand the effects of chiral pesticides on the accumulation and distribution of nutrient elements in plants for comprehensive scientific assessment of the environmental safety and ecological risk of chiral herbicides. Received: Revised: Accepted: Published: 10209
September 14, 2015 October 18, 2015 November 13, 2015 November 13, 2015 DOI: 10.1021/acs.jafc.5b04495 J. Agric. Food Chem. 2015, 63, 10209−10217
Article
Journal of Agricultural and Food Chemistry C b = 22.9 × OD645 − 4.86 × OD663
Until now, however, cross talk between the enantioselectivity of herbicides and nutrient stresses has been poorly understood. Imazethapyr (IM) is an imidazolinone herbicide that is widely used on wheat, corn, sorghum, and olive trees to control broad-leaf weeds. In addition, IM is a chiral herbicide with one pair of enantiomers, that is, (R)-IM and (S)-IM, and the (R)enantiomer is known to be the active herbicide (shown in Figure S1 in the Supporting Information). However, IM is still commercially sold as a racemate and is released into the environment as a 1:1 mixture of its two enantiomers because the mechanisms of enantioselective toxicity of enantiomers in the environment have not been clearly elucidated. Thus, to evaluate the possible effects of chiral pesticides on nutrient stresses in plants, we used IM as a representative chiral herbicide and Arabidopsis thaliana as a biomarker. A. thaliana was selected as a test plant species for various reasons. This species has a rapid germination rate and short lifespan, which facilitate life-cycle toxicity screening. In addition, its small seed size results in a relatively large surface area to volume ratio, which is conducive to higher sensitivity to toxicants. The objectives of this study were (1) to determine the distribution of nutrient elements in the roots and leaves of A. thaliana; (2) to evaluate the relationship between the changes in selected nutrient elements and toxicities of the enantiomers of the herbicide IM; and (3) to explore the relationships between the expression of genes related to nutrient elements and toxicities of the enantiomers of IM. The findings of this study may be helpful in understanding the enantioselective toxicity of chiral pesticides and possibly improve risk assessment and regulation of chiral pesticides.
■
Determination of Nutrient Element Content in Shoots. After 3 weeks of cultivation, plants were separated into roots and shoots (including stems). The shoots from each treatment were successively washed three times using distilled water and oven-dried at 60 °C for 12 h. The concentrations of nutrient elements were determined from 80 to 100 mg of dried plant samples that were digested using 6 mL of HNO3/200 μL of H2O2. Elemental concentrations were determined using an inductively coupled plasma−mass spectrometer (ICP-MS, PerkinElmer, Boston, MA, USA). All reagents used were of analytical reagent grade or better. Statistical analysis was performed using SPSS software. Distribution of Nutrient Elements in Leaves and Roots. We used a high-resolution chemical imaging method, that is, synchrotron X-ray microfluorescence (μ-XRF) with a beamline 15U1 at the Shanghai Institute of Applied Physics, Chinese Academy of Science, to examine the distribution of several key elements in the leaves and roots of A. thaliana. A synchrotron source is required because it provides a sub-micrometer spot size with very high photon flux. The sample is raster-scanned through a focused X-ray beam, and the XRF signal emitted from each point is collected and analyzed. The areas of interest for XRF were selected using an in-chamber video microscope. Before analysis, plants were separated into leaves and roots and washed three times using distilled water. The samples were fixed on the tape for analysis, and a beam size of 2 × 2 μm2 was used. The majority of maps were focused on root tips and the entire leaf blade. Color-coded composite chemical maps of the target elements (K, Ca, Mn, Fe, Cu, and Zn) in the leaves and roots of A. thaliana were performed by Image-pro plus 6.0 (Media Cybernetics, Rockville, MD, USA). Distribution of Reactive Oxygen Species (ROS) in Leaves and Roots. After 3 weeks cultivation, plants were washed three times using distilled water and then separated into roots and leaves. The samples were placed into 25 μM 2′,7′-dichlorofluorescin diacetate for 30 min in the dark. Then, samples were washed three times using phosphate-buffered saline (PBS, 0.5 mM, pH 7.0), and the distributions of ROS in leaves and roots of A. thaliana were observed using confocal laser scanning microscopy (Carl Zeiss LSCM 780, Germany).25 Preparation of Total RNA and Real-Time PCR Analysis. Total leaf RNA was extracted using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. RNA was then reverse-transcribed to cDNA using a reverse transcriptase kit (Toyobo, Tokyo, Japan); the analysis was conducted using a SYBRGreen Realtime PCR Master Mix (Toyobo Co., Ltd.). Actin2 was used as a housekeeping gene to normalize the expression changes. The relative gene expression among the treatment groups was quantified using the 2 −ΔΔCt method.26 Six ZIP genes (IRT1 At4g19690; IRT2 At4g19680; ZIP2 At5g59520; ZIP4 At1g10970; ZIP5 At1g05300; ZIP9 At4g33020), two FRO genes (FRO2 At1g01580; FRO3 At1g23020), three NRAMP genes (NRAMP1 At1g80830; NRAMP3 At2g23150; NRAMP4 At5g67330), two COPT genes (COPT1 At5g59030; COPT2 At1g46900), and MTP11 (At2g39450) and ECA3 (At1g10130), which code for metal transporters in the leaves and roots of A. thaliana, were analyzed in our work. Detailed gene information is shown in Table S4 in the Supporting Information. Three replicates were used in each treatment, and every replicate contained 12 seedlings. Statistical Analysis. The data were analyzed using Origin 8.0 software (OriginLab, Northampton, MA, USA) according to the methods provided by the manufacturer of the test kit. Comparisons were made using one-way analyses of variance (ANOVA) followed by a multiple-comparison test of means (Tukey test). The differences were considered statistically significant when the p value was
