| The enantioselectivety of the chiral agricultural organic compounds widespread in the environment has received more and more extensive understanding and became one of the focus researches. Imidazolinones, as a pillar of China's herbicide products, are a class of pesticides with typical chiral structure. It has been reported that the imidazolinones enantioselectively inhibited the acetolactate synthase (ALS), however, the mechanism of the enantioselective inhibition on anzyme has not been revealed; and the enantioselective phytotoxicity has not yet reported. In this study, imazethapyr (IM), one of the imidazolinones, which is chiral and is number one among all of the imidazolinones in sales, was chosed in this article as a model herbicide. Enantiomeric pure IM was eperated by High Performance Liquid Chromatography (HPLC). On this basis, enantioselective phytotoxicity of IM on plant roots growth were determined by measuring changes in the root morphology, subcellular structure and root hair growth of tender maize and Arabidopsis thaliana seedlings. As IM is a soil-applied herbicide, the radicle is the first organ to contact the herbicide in the soil. Exploring the relationship between plant growth regulators responses and root hair development and the mechanism of enantioselective inhibition of IM on acetolactate synthase activity so as to explore and reveal the mechanism of enantioselective phytotoxicities. The main findings of the paper are as follows:The IM enantiomers were baseline separated by HPLC on chiral column Chiralpak OJ with the mobile phase of hexane/ethanol/acetic acid solution (75/25/0.5 by volume). Circular dichroism (CD) detectors were used to determine the elution order and CD spectra of the enantiomers. The absolute configuration of IM enantiomers was identified as S-(+)-IM and R-(-)-IM by the octant rule from force-field calculations and CD spectra.Plant growth was enantioselectively inhibited by IM enantiomers at the concentration of 100,200,400 and 800μg L-1. Visible crop injury symptoms were observed, including chlorosis of leaves, reduced plant growth, and putrescence on the root tips.R-(-)-IM was the most effective inhibitor in the damage of maize and Arabidopsis thaliana growth in comparison to the S-(+)-IM and racemate mixtures at equal concentrations. The inhibition ability of racemate was between the R and S enantiomer.The maize root morphology was enantioselectively affected by the IM at the concentration of 100,200,400 and 800μg L-1. Root diameter was increased; the number of root tips, the root volume and surface area were significantly decreased. IM also enantioselectively decreased root activity.IM Enantioselectively inhibited the growth of root hairs of maize and Arabidopsis at 200 and 400μg L-1, respectively. Root hair length was shortened, the number of root hair significantly was reduced, and cell membranes were damaged. The observation of hormone levels in transgenic Arabidopsis DR5::GFP showed that the auxin content was involved in root hair growth inhibition rate. Exogenous auxin treatment alleviated the growth inhibition of root hair.Ultrastructural studies revealed that IM at the concentration of 400μgL-1 had adverse effects on cell organelles in maize root caps:cell nucleuses were irregular, and nucleoluses were disappeared; other cell organelles were damaged by a reduction in number and size; mitochondrial membranes were disintegrated, mitochondrial cristae became blurred; endoplasmic reticulum membranes were ruptured; the dictyosomers were severely swollen, small vacuoles were disappeared; membranes and cell walls became thicker and showed the phenomenon of fibrosis, and lacked flexibility. The ultrastructure of the statocyte seriously damaged, releasing the starch grains into the cytoplasm, so that the root cap would no longer be able to respond correctly to gravity, resulting in a transverse growth of the root.Enantioselective toxicity of herbicide on the plants after applied into the soil first reflects in the rapid inhibition of root. The changing of root morphology, the destruction of the internal structure of root cells, root hair growth suppression and physical activity inhibition ultimately lead to plant toxicity.IM enantioselectively suppressed the in vitro and in vivo ALS activity of maize leaves. R-(-)-IM was more active than S-(+)-IM. The in vivo ALS activity study showed only a 2-fold difference between R-(-)-IM and S-(+)-IM at the concentration of 100,200,400 and 800μg L-1 which is accordance with the previous established difference in retarding the plant growth of maize. Quite different from the in vivo study, the in vitro study showed that the difference in inhibition between the enantiomers fell sharply as concentration increased. At the lowest concentration of 40μg L-1,R-(-)-IM appeared 25 times more active than S-(+)-IM, but only 7 times at 200μg L-1. At the highest concentration of 25 mg L-1, in vitro ALS activity was almost completely inhibited by S-(+)-IM, there was only 1.1times differences between S-(+)-IM and R-(-)-IM. In the study of molecular docking, the combination of the IM with ALS shows chiral discrimination. R-(-)-IM could bind to ALS in its preferred orientation. The different interaction modes of the R-(-)-and the S-(+)-IM with ALS obtained from molecular docking provide a structural explanation for the more potent activity of the R-(-)-IM in contrast to the S-(+)-IM.Because of the very different biological properties that selectively interacting with biological systems, enantiomers of chiral coumpounds can be regarded as different substances. The enantioselectivity in efficacy, environmental safety and other aspects can not be ignored. Realization of the major breakthrough of chiral pesticides researches and production of a single optically active isomer are the inevitable requirements for reducing environmental pollution and the implementation of green agriculture. The study provides indicative values for the objective evaluation of imida.zolinone herbicides in environmental effects, for the prevention of the succeeding crop from the poisoning of residual herbicides, for directing research into the manufacture of sole optically active herbicides with high efficiency and low side-effects.
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