Transcriptomic analysis of maize uncovers putative genes involved in metabolic detoxification under four safeners treatment.

Isoxadifen-ethyl (IDF) and cyprosulfamide (CSA) can effectively protect maize from nicosulfuron (NIC) injury, while mefenpyr-diethyl (MPR) and fenchlorazole-ethyl (FCO) did not. Their chemical diversity and requirement to use them in combination with the corresponding herbicides suggest that their elicitation of gene expression are complex and whether it is associated with the safening activity remains elusive. In this study, our first objective was to determine whether or not the ability of four safeners to enhance the metabolic rate of nicosulfuron. It was found that nicosulfuron degradation in maize was accelerated by IDF and CSA, but not by FCO and MPR. Transcriptomic analysis showed that the number of genes induced by IDF and CSA were larger than that induced by FCO and MPR. Overall, 34 genes associated with detoxification were identified, including glutathione S-transferase (GST), cytochrome P450 (CYP450), UDP-glucosyltransferase (UGT), transporter and serine. Moreover, 14 detoxification genes were screened for further verification by real-time PCR in two maize inbred lines. Two maize inbred lines exhibited high expression levels of four genes (GST31, GST39, AGXT2 and ADH) after IDF treatment. GST6, GST19, MATE, SCPL18 and UF3GT were specifically up-regulated in telerant maize inbred line under IDF and IDF + NIC treatments. Seven genes, namely GST31, GST6, GST19, UF3GT, MATE, ADH and SCPL18, are induced by IDF and CSA to play a vital role in regulating the detoxification process of NIC. Accordingly, the GST activity in maize was accelerated by IDF and CSA, but not by FCO and MPR. This result is consistent with transcriptome and metabolic data.These results indicate that the mitigation of NIC damage is associated with enhanced herbicide metabolism. IDF and CSA were more effective in protecting maize from NIC injury due to their ability to enhance the detoxification of specific types of herbicides, compared to FCO and MPR. The chemical specificity of four safeners is attributed to the up-regulated genes related to the detoxification pathway.

Investigation of resistance mechanisms to fomesafen in Ipomoea nil from China.

Recently, the herbicide fomesafen has frequently failed to control the troublesome weed Ipomoea nil in soybean fields in Liaoning Province, China. Hence, we collected 10 suspected resistant populations and evaluated their sensitivity to fomesafen. The results revealed various degrees of Ipomoea nil resistance to fomesafen, with a resistance index of 2.88 to 22.43; the highest value occurred in the LN3 population. Therefore, the mechanisms of the resistance in LN3 to fomesafen were explored. After fomesafen treatment, the expression levels of InPPX1 and InPPX2 genes were 4.19- and 9.29-fold higher, respectively, in LN3 than those in the susceptible (LN1) population. However, mutations and copy number variations were not detected between the two populations. Additionally, malathion pretreatment reduced the dose necessary to halve the growth rate of LN3 by 58%. Liquid chromatography with tandem mass spectrometry demonstrated that metabolism of fomesafen was significantly suppressed by malathion. Moreover, LN3 displayed increased reactive oxygen species scavenging capacity, which was represented by higher superoxide dismutase and peroxidase activities after fomesafen application than those in LN1. An orthogonal partial least squares-discriminant analysis revealed that the high resistance in LN3 could be attributed mainly to enhanced metabolism. Fortunately, the fomesafen-resistant I. nil remained sensitive to 2,4-D-ethylhexylester and bentazon, providing methods for its control.

Molecular and biochemical biomarkers in the American oyster Crassostrea virginica exposed to herbicide Roundup® at high temperature.

Aquatic organisms are frequently exposed to various environmental stressors. Thus, the effects of high temperatures and herbicides on aquatic organisms are a major subject of interest. In this study, we studied the effects of short-term exposure (1 week) to Roundup®, a glyphosate-based herbicide (concentrations: 0.5 and 5 µg/L), on the morphology of gills, digestive glands, and connective tissues, and the expression of heat shock protein-70 (HSP70, a chaperone protein), cytochrome P450 (CYP450, a biomarker of environmental contaminants), dinitrophenyl protein (DNP, a biomarker of protein oxidation), nitrotyrosine protein (NTP, a biomarker of protein nitration), antioxidant enzymes such as superoxidase dismutase (SOD) and catalase (CAT) in tissues of American oyster, Crassostrea virginica (Gmelin, 1791) maintained at high temperature (30 °C). Histological analyses showed an increase in mucous production in the gills and digestive glands, and in hemocyte aggregation in the connective tissues as well as a structural change of lumen in the digestive glands of oysters exposed to Roundup. Immunohistochemical and quantitative RT-PCR analyses showed significant (P < 0.05) increases in HSP70, CYP450, DNP, NTP, CAT, and SOD mRNA and protein expressions in the tissues of oysters exposed to Roundup. Taken together, these results suggest that exposure to Roundup at high temperature induces overproduction of reactive oxygen species/reactive nitrogen species which in turn leads to altered prooxidant-antioxidant activity in oyster tissues. Moreover, our results provide new information on protein oxidation/nitration and antioxidant-dependent mechanisms for HSP70 and CYP450 regulations in oysters exposed to Roundup at high temperature.

The effects of glyphosate-based herbicide on the hypothalamic-pituitary thyroid axis are tissue-specific and dependent on age exposure.

The significant increase in glyphosate-based herbicide (GBH) use raises concerns about residues in the environment and food, potentially jeopardizing human health. The involvement of GBHs in the increased incidence of thyroid disorders is speculated, since glyphosate has been linked to an increased risk of thyroid disease in farmers. In this sense, this study aims to investigate the potential effects of low levels of GBH exposure (0, 0.5 or 5 mg/kg) from weaning (postnatal day PND23) to adult life (PND60 and PND90) in male Wistar rats on hypothalamic-pituitary-thyroid (HPT) axis function. The serum levels of T4 were increased. The hypothalamus showed reduced expression of Dio2, Thra1, and Thra2. The pituitary showed reduced expression of Mct8 and Dio2 and increased expression of Thra1. The thyroid showed increased expression of Tshr and Thra1. The heart showed increased expression of Mct8 and Myh6. The liver showed reduced expression of Mct8 and Thra2 and increased expression of Thra1. In thyroid morphometry, a decrease in both follicular diameter and area and decreased follicular and colloid diameters and areas were observed. These results suggested that GBH may affect several steps of HPT axis regulation at the transcriptional level in an age-dependent manner and alter the morphometric parameters of the thyroid gland and TH synthesis, with potential repercussions in the TH-target organs.

Multiple Metabolism Pathways of Bentazone Potentially Regulated by Metabolic Enzymes in Rice.

Bentazone (BNTZ) is a selective and efficient herbicide used in crop production worldwide. However, the persistence of BNTZ residues in the environment has led to their increasing accumulation in farmland and crops, posing a high risk to human health. To evaluate its impact on crop growth and environmental safety, a comprehensive study was conducted on BNTZ toxicity, metabolic mechanism, and resultant pathways in rice. The rice growth was compromised to the treatment with BNTZ at 0.2-0.8 mg/L (529.95-1060.05 g a.i./ha), while the activities of enzymes including SOD, POD, CAT, GST, GT, and CYP450 were elevated under BNTZ stress. A genome-wide RNA-sequencing (RNA-Seq) was performed to dissect the variation of transcriptomes and metabolic mechanisms in rice exposed to BNTZ. The degradative pathways of BNTZ in rice are involved in glycosylation, hydrolysis, acetylation, and conjugation processes catalyzed by the enzymes. Our data provided evidence that helps understand the BNTZ metabolic and detoxic mechanisms.

Designing a cercosporin-bioinspired bifunctional algicide with flocculation and photocatalysis for efficiently controlling harmful cyanobacterial blooms.

Harmful cyanobacterial blooms (HCBs) are spreading in freshwater ecosystems worldwide, adversely affecting drinking water supplies, aquatic production, recreational and tourism activities. Therefore, the efficient and environmentally friendly method is still of interest to be developed to effectively control HCBs. Inspired by the excellent algicidal activity of cercosporin (CP), a novel metal-free algaecide SiO2@EDU@CP (EDU, N-ethyl-N'-(3-dimethylaminopropyl)urea) with flocculation and photoremoval functions, was successfully designed and prepared in one-step to simultaneously introduce CP and EDU on SiO2 nanoparticles. It could rapidly form algae flocs in 20 min with 97.1% flocculation rate, and remove Microcystis aeruginosa within 12 h with 91.0% algicidal rate under 23 W compact fluorescent light irradiation without any leaked CP detected. Additionally, odorant ß-cyclocitral and toxin microcystin-LR were both photodegraded after treatment of SiO2@EDU@CP. Further mechanistic studies showed that the introduction of EDU significantly reversed the zeta potential of SiO2-COOH to achieve the flocculation through neutral charge, and the photophysical characterization of SiO2@EDU@CP revealed the improved charge separation ability to generate reactive oxygen species. More importantly, the utility of SiO2@EDU@CP was well demonstrated by its effectiveness for algae from Taihu Lake under natural sunlight and inability to regrow after treatment. This study not only establishes a bifunctional algicide SiO2@EDU@CP to efficiently control HCBs, but also provides design possibilities to develop more novel and efficient algicides for the better control of practical HCBs.

Teratogenic effects of the dicamba herbicide in Zebrafish (Danio rerio) embryos.

Dicamba has been used worldwide for 60 years, but few studies have been conducted on its environmental safety and health effects. Therefore, this study aims to evaluate the acute toxicity, teratogenic effects, oxidative stress, and neurotoxicity of Dicamba in zebrafish embryos. Embryos were exposed to concentrations of 4.5, 18, 72, and 288 mg/L of Dicamba for 96 h. Among the teratogenic effects, yolk sac edema predominated, besides malabsorption of nutrients (grayish yolk sac). The presence of edema may indicate problems with circulation and water efflux from the embryos, which may be related to kidney and cardiovascular problems. Other effects such as hemorrhage, spinal and eye malformations, and dwarfism were also observed. The hatching rate was reduced in the highest concentration, and in the other concentrations, a decrease was noticeable indicating a delay in development. Neurotoxic effects were also observed. Oxidative stress analysis showed a significant decrease in SOD at all concentrations and an increase in GPx, GSH, and LPO at 288 mg/L of Dicamba. It was observed that the herbicide is capable of causing teratogenic effects, developmental delay, and oxidative stress. These results show that exposure to Dicamba, in a commercial formulation, can bring risks during embryonic development. In addition, it highlights the need for further studies on the effects of the herbicide and a reassessment of toxicity categorization.

Mycoremediation of pretilachlor and its metabolite by Aspergillus ficuum.

Pretilachlor is one of the widely used chloroacetamide herbicides in Asian countries to control weeds in the rice field. The extensive use of herbicides has caused major concern among scientists throughout the world. Therefore, it is essential to develop an efficient method for the remediation of pretilachlor and its harmful by-products from contaminated surfaces. Mycoremediation is known to play a key role in the removal of various environmental contaminants. Hence, in the present study, strain AJN2 Aspergillus ficuum was isolated from a paddy field that was in continuous exposure to pretilachlor for over a decade. The degradation studies showed that the strain was efficiently able to degrade 73% of pretilachlor in an aqueous medium within 15 days of incubation and 70% of its major metabolite PME (2-methyl-6-ethylalanine). The GC/MS profile revealed the formation of aldehyde as the end product of degradation which was confirmed through the infrared fingerprint of the degradation sample. The ligninolytic enzyme activity studies showed that the lignin peroxidase enzyme system could be responsible for the degradation of pretilachlor and its major metabolite. The results highlight that the strain AJN2 A. ficuum could be a potential strain for the bioremediation of pretilachlor from the contaminated areas.

A nitroreductase DnrA catalyzes the biotransformation of several diphenyl ether herbicides in Bacillus sp. Za.

Diphenyl ether herbicides, typical globally used herbicides, threaten the agricultural environment and the sensitive crops. The microbial degradation pathways of diphenyl ether herbicides are well studied, but the nitroreduction of diphenyl ether herbicides by purified enzymes is still unclear. Here, the gene dnrA, encoding a nitroreductase DnrA responsible for the reduction of nitro to amino groups, was identified from the strain Bacillus sp. Za. DnrA had a broad substrate spectrum, and the Km values of DnrA for different diphenyl ether herbicides were 20.67 µM (fomesafen), 23.64 µM (bifenox), 26.19 µM (fluoroglycofen), 28.24 µM (acifluorfen), and 36.32 µM (lactofen). DnrA also mitigated the growth inhibition effect on cucumber and sorghum through nitroreduction. Molecular docking revealed the mechanisms of the compounds fomesafen, bifenox, fluoroglycofen, lactofen, and acifluorfen with DnrA. Fomesafen showed higher affinities and lower binding energy values for DnrA, and residue Arg244 affected the affinity between diphenyl ether herbicides and DnrA. This research provides new genetic resources and insights into the microbial remediation of diphenyl ether herbicide-contaminated environments. KEY POINTS: • Nitroreductase DnrA transforms the nitro group of diphenyl ether herbicides. • Nitroreductase DnrA reduces the toxicity of diphenyl ether herbicides. • The distance between Arg244 and the herbicides is related to catalytic efficiency.

High doses of clethodim-based herbicide GrassOut Max poses reproductive hazard by affecting male reproductive function and early embryogenesis in Swiss albino mice.

Clethodim is a widely used and approved class II herbicide, with little information about its impact on the reproductive system. Herein, we investigated the male reproductive toxicity of clethodim using a mouse model. GrassOut Max (26% clethodim-equivalent) or analytical grade clethodim (≥90%) were given orally to male mice for 10 d in varying doses. All parameters were assessed at 35 d post-treatment. Significant decrease in testicular weight, decreased germ cell population, elevated DNA damage in testicular cells and lower serum testosterone level was observed post clethodim based herbicide exposure. Epididymal spermatozoa were characterized with significant decrease in motility, elevated DNA damage, abnormal morphology, chromatin immaturity and, decreased acetylated-lysine of sperm proteins. In the testicular cells of clethodim-based herbicide treated mice, the expression of Erß and Gper was significantly higher. Proteomic analysis revealed lower metabolic activity, poor sperm-oocyte binding potential and defective mitochondrial electron transport in spermatozoa of clethodim-based herbicide treated mice. Further, fertilizing ability of spermatozoa was compromised and resulted in defective preimplantation embryo development. Together, our data suggest that clethodim exposure risks male reproductive function and early embryogenesis in Swiss albino mice via endocrine disrupting function.

Atrazine decontamination by a newly screened psychrotroph Paenarthrobacter sp. KN0901 in an aquatic system: Metabolic pathway, kinetics, and hydroponics experiment.

Atrazine residues running off the fields and entering water resources are a major threat to food security and the ecosystem. In this study, a psychrotrophic functional strain named KN0901 to remove atrazine residues was screened. KN0901 could degrade 30 mg·L-1 atrazine in 4 days at 15ºC with 105 CFU·mL-1 incubation. The phylogenetic results showed KN0901 belonged to Paenarthrobacter sp. PCR results showed that the functional genes consist of trzN, atzB, and atzC, suggesting atrazine was transformed to cyanuric acid by KN0901. KN0901 could degrade atrazine without adding exogenous carbon and nitrogen sources. What's more, KN0901 could tolerate extreme low temperature (5ºC) and high atrazine concentration (100 mg·L-1). When growth and degradation curves were compared, the results indicated the length of lag time showed significant correlation to atrazine degradation rate. The hydroponic experiments showed that the toxicity of atrazine was significantly reduced with KN0901 treatment. The study provided an effective, economic, and eco-friendly bioremediation measure to address atrazine contamination.

Enhanced degradation of herbicides in groundwater using sulfur-containing reductants and spinel zinc ferrite activated persulfate.

A challenge to successfully implementing an injection-based remedial treatment in aquifers is to ensure that the oxidative reaction is efficient and lasts long enough to contact the contaminated plume. Our objective was to determine the efficacy of zinc ferrite nanocomposites (ZnFe2O4) and sulfur-containing reductants (SCR) (i.e., dithionite; DTN and bisulfite; BS) to co-activate persulfate (S2O82-; PS) and treat herbicide-contaminated water. We also evaluated the ecotoxicity of the treated water. While both SCRs delivered excellent PS activation in a 1:0.4 ratio (PS:SCR), the reaction was relatively short-lived. By including ZnFe2O4 in the PS/BS or PS/DTN activations, herbicide degradation rates dramatically increased by factors of 2.5 to 11.3. This was due to the SO4- and OH reactive radical species that formed. Radical scavenging experiments and ZnFe2O4 XPS spectra results revealed that SO4- was the dominant reactive species that originated from S(IV)/PS activation in solution and from the Fe(II)/PS activation that occurred on the ZnFe2O4 surface. Based on liquid chromatography mass spectrometry (LC-MS), atrazine and alachlor degradation pathways are proposed that involve both dehydration and hydroxylation. In 1-D column experiments, five different treatment scenarios were run using 14C-labeled and unlabeled atrazine, and 3H2O to quantify changes in breakthrough curves. Our results confirmed that ZnFe2O4 successfully prolonged the PS oxidative treatment despite the SCR being completely dissociated. Toxicity testing showed treated 14C-atrazine was more biodegradable than the parent compound in soil microcosms. Post-treatment water (25 %, v/v) also had less impact on both Zea Mays L. and Vigna radiata L. seedling growth, but more impact on root anatomies, while ≤4 % of the treated water started to exert cytotoxicity (<80 % viability) on ELT3 cell lines. Overall, the findings confirm that ZnFe2O4/SCR/PS reaction is efficient and relatively longer lasting in treating herbicide-contaminated groundwater.

Nontarget-site resistance due to rapid physiological response in 2,4-D resistant Conyza sumatrensis: reduced 2,4-D translocation and auxin-induced gene expression.

BACKGROUND: Resistance to 2,4-Dichlorophenoxyacetic acid (2,4-D) has been reported in several weed species since the 1950s; however, a biotype of Conyza sumatrensis showing a novel physiology of the rapid response minutes after herbicide application was reported in 2017. The objective of this research was to investigate the mechanisms of resistance and identify transcripts associated with the rapid physiological response of C. sumatrensis to 2,4-D herbicide. RESULTS: Differences were found in 2,4-D absorption between the resistant and susceptible biotypes. Herbicide translocation was reduced in the resistant biotype compared to the susceptible. In resistant plants 98.8% of [14 C] 2,4-D was found in the treated leaf, whereas ≈13% translocated to other plant parts in the susceptible biotype at 96 h after treatment. Resistant plants did not metabolize [14 C] 2,4-D and had only intact [14 C] 2,4-D at 96 h after application, whereas susceptible plants metabolized [14 C] 2,4-D into four detected metabolites, consistent with reversible conjugation metabolites found in other 2,4-D sensitive plant species. Pre-treatment with the cytochrome P450 inhibitor malathion did not enhance 2,4-D sensitivity in either biotype. Following treatment with 2,4-D, resistant plants showed increased expression of transcripts within plant defense response and hypersensitivity pathways, whereas both sensitive and resistant plants showed increased expression of auxin-response transcripts. CONCLUSION: Our results demonstrate that reduced 2,4-D translocation contributes to resistance in the C. sumatrensis biotype. The reduction in 2,4-D transport is likely to be a consequence of the rapid physiological response to 2,4-D in resistant C. sumatrensis. Resistant plants had increased expression of auxin-responsive transcripts, indicating that a target-site mechanism is unlikely. © 2023 Society of Chemical Industry.

Fitness assessment of Mytilus galloprovincialis Lamarck, 1819 after exposure to herbicide metabolite propachlor ESA.

The lack of data on the chronic effects of chloroacetanilide herbicide metabolites on non-target aquatic organisms creates a gap in knowledge about the comprehensive impacts of excessive and repeated pesticide use. Therefore, this study evaluates the long-term effects of propachlor ethanolic sulfonic acid (PROP-ESA) after 10 (T1) and 20 (T2) days at the environmental level of 3.5 µg.L-1 (E1) and its 10x fold multiply 35 µg.L-1 (E2) on a model organism Mytilus galloprovincialis. To this end, the effects of PROP-ESA usually showed a time- and dose-dependent trend, especially in its amount in soft mussel tissue. The bioconcentration factor increased from T1 to T2 in both exposure groups - from 2.12 to 5.30 in E1 and 2.32 to 5.48 in E2. Biochemical haemolymph profile and haemocyte viability were not affected by PROP-ESA exposure. In addition, the viability of digestive gland (DG) cells decreased only in E2 compared to control and E1 after T1. Moreover, malondialdehyde levels increased in E2 after T1 in gills, and DG, superoxidase dismutase activity and oxidatively modified proteins were not affected by PROP-ESA. Histopathological observation showed several damages to gills (e.g., increased vacuolation, over-production of mucus, loss of cilia) and DG (e.g., growing haemocyte trend infiltrations, alterations of tubules). This study revealed a potential risk of chloroacetanilide herbicide, propachlor, via its primary metabolite in the Bivalve bioindicator species M. galloprovincialis. Furthermore, considering the possibility of the biomagnification effect, the most prominent threat poses the ability of PROP-ESA to be accumulated in edible mussel tissues. Therefore, future research about the toxicity of pesticide metabolites alone or their mixtures is needed to gain comprehensive results about their impacts on living non-target organisms.

Herbicide atrazine impairs metabolic plasticity of mixotrophic organisms: Evidence in photochemistry, morphology, and gene expression.

Herbicide pollution is a main form of water pollution. As a result of additional harms to other non-target organisms, it threatens the function and structure of ecosystems. Previous researches mainly focused on the assessment of the toxicity and ecological effect of herbicides on monotrophic organisms. Responses of mixotrophs as an important component of functional groups are rarely understood in contaminated waters, although their metabolic plasticity and unique ecological functions in ecosystem stability are a major concern. This work aimed to investigate the trophic plasticity of mixotrophic organisms in atrazine-contaminated waters, and a primarily heterotrophic Ochromonas was used as the tested organism. Results showed that the herbicide atrazine significantly inhibited the photochemical activity and impaired the photosynthetic machine of Ochromonas, and photosynthesis activated by light was sensitive to atrazine. However, phagotrophy was unaffected by atrazine and closely correlated with growth rate, indicating that heterotrophy helped population maintenance during herbicide exposure. Mixotrophic Ochromonas upregulated the gene expression level involved in photosynthesis, energy synthesis, and antioxidation to adapt to increasing atrazine after long-term exposure. Compared with bacterivory, herbivory increased atrazine tolerance of photosynthesis under mixotrophic status. This study systematically illustrated the mechanism by which mixotrophic Ochromonas respond to the herbicide atrazine at population, photochemical activity, morphology, and gene expression levels and demonstrated the potential effect of atrazine on the metabolic flexibility and ecological niches of mixotrophs. These findings will provide important theoretical reference for governance and management decision-making in contaminated environments.

Elucidating the effects of acute and chronic exposure to 2,4-Dichlorophenoxyacetic acid on fathead minnow (Pimephales promelas) innate immunity.

Aquatic herbicides, such as 2,4-dichlorophenoxyacetic acid (2,4-D) formulations, are commonly used for invasive species management throughout the United States. Ecologically relevant concentrations of 2,4-D can impair essential behaviors, reduce survival, and act as an endocrine disruptor; however, there is limited knowledge of its effects on the health of non-target organisms. Here, we investigate the acute and chronic exposure impacts of 2,4-D on adult male and female fathead minnow (Pimephales promelas) innate immune function. We exposed both adult male and female fathead minnows to three different ecologically relevant concentrations of 2,4-D (0.00, 0.40, and 4.00 mg/L) and took blood samples at three acute time points (6, 24, and 96 h) and one chronic time point (30 days). We found that male fatheads had higher total white blood cell concentrations when exposed to 2,4-D at the acute time points. For the females, only proportions of specific cell types were altered when exposed to 2,4-D at the acute time points. However, we did not observe any significant impacts of chronic exposure to 2,4-D on any innate immune responses for either males or females. Overall, this study is the first step in answering an important question for game fisheries and management agencies while providing insight to future studies that investigate the impacts of herbicide exposure to freshwater fish health and immunity.

Potential Role of EPSPS Mutations in the Resistance of Eleusine indica to Glyphosate.

Gene mutation is a basic evolutionary mechanism in plants under selection pressure of herbicides. Such mutation has pleiotropic effects on plant growth. We systemically investigated the effects of Pro106Leu (P106L), Pro106Ser (P106S), and Thr102Ile + Pro106Ser (TIPS) mutations on EPSPS functionality and fitness traits in Eleusine indica at the biochemical and physiological levels. The affinity of natural EPSPS for glyphosate was 53.8 times higher than that for phosphoenolpyruvate (PEP), as revealed by the dissociation constant; the constant decreased in both the P106L (39.9-fold) and P106S (46.9-fold) mutants but increased in the TIPS (87.5-fold) mutant. The Km (PEP) values of the P106L, P106S, and TIPS mutants were 2.4-, 0.7-, and 4.1-fold higher than that of natural EPSPS, corresponding to resistance levels of 2.5, 1.9, and 11.4, respectively. The catalytic efficiency values (maximum reaction rates) were 0.89-, 0.94-, and 0.26-fold higher than that of natural EPSPS. The levels of metabolites related to amino acids and nucleotides were significantly reduced in the mutated plants. The fitness costs were substantial for the biomass, total leaf area, seed number, and seedling emergence throughout the growth period in the plants with P106L and TIPS mutations. These results provide insights into EPSPS kinetics and their effect on plant growth.

Improved Chromatography and MS-Based Detection of Glyphosate and Aminomethylphosphonic Acid Using iTrEnDi.

Glyphosate (GLY), a synthetic, nonselective systemic herbicide that is particularly effective against perennial weeds, is the most used weedkiller in the world. There are growing concerns over GLY accumulation in the environment and the attendant human health-associated risks, and despite increased attention in the media, GLY and its breakdown product aminomethylphosphonic acid (AMPA) remain elusive to many analytical strategies. Chemical derivatization coupled with high-performance liquid chromatography-mass spectrometry (HPLC-MS) addresses the challenge of quantifying low levels of GLY and AMPA in complex samples. Here we demonstrate the use of in situ trimethylation enhancement using diazomethane (iTrEnDi) to derivatize GLY and AMPA into permethylated products ([GLYTr]+ and [AMPATr]+, respectively) prior to analysis via HPLC-MS. iTrEnDi produced quantitative yields and resulted in a 12-340-fold increases in HPLC-MS-based sensitivity for [GLYTr]+ and [AMPATr]+, respectively, compared with underivatized counterparts. The limits of detection of derivatized compounds were found to be 0.99 ng/L for [GLYTr]+ and 1.30 ng/L for [AMPATr]+, demonstrating significant sensitivity improvements compared to previously established derivatization techniques. iTrEnDi is compatible with the direct derivatization of Roundup formulations. Finally, as proof of principle, a simple aqueous extraction followed by iTrEnDi enabled the detection of [GLYTr]+ and [AMPATr]+ on the exterior of field-grown soybeans that were sprayed with Roundup. Overall, iTrEnDi ameliorates issues relating to low proton affinity and chromatographic retention, boosting HPLC-MS-based sensitivity and enabling the elucidation of elusive analytes such as GLY and AMPA within agricultural systems.

Computational Study of the Fe(II) and α-Ketoglutarate-Dependent Aryloxyalkanoate Dioxygenase (AAD-1) in the Degradation of the Herbicide 2,4-Dichlorophenoxyacetic Acid.

The AAD-1 enzyme belongs to the Fe(II) and α-ketoglutarate (Fe/αKG)-dependent nonheme aryloxyalkanoate dioxygenase family (AADs), which catalyzes the breakdown of 2,4-dichlorophenoxyacetic acid (2,4-D, an active ingredient of thousands of commercial herbicides) by using the highly active Fe(IV)═O complex. Multiple species of bacteria degrade 2,4-D via a pathway initiated by AADs; however, the detail of how they promote the cleavage of the ether C-O bond to generate 2,4-dichlorophenol (2,4-DCP) and glyoxylate is still unclear, which is the prerequisite for the further degradation of these halogenated aromatics. In this work, based on the crystal structure of AAD-1, the computational models were constructed, and a series of QM/MM and QM-only calculations were performed to explore the cleavage of the ether bond in 2,4-D with the catalysis of AAD-1. Our calculations reveal that AAD-1 may be only responsible for the hydroxylation of the substrate to generate the intermediate hemiacetal, which corresponds to an overall energy barrier of 14.2 kcal/mol on the quintet state surface, and the decomposition of the hemiacetal in the active site center of AAD-1 was calculated to be rather slow, corresponding to an energy barrier of 24.5 kcal/mol. In contrast, the decomposition of the free hemiacetal molecule in a solvent was calculated to be quite easy. Whether the decomposition of the hemiacetal occurs inside or outside the activation site is still worthy of experimental verification.

Recurrent Selection of Echinochloa crus-galli with a Herbicide Mixture Reduces Progeny Sensitivity.

Herbicide mixtures are used to increase the spectrum of weed control and to manage weeds with target-site resistance to some herbicides. However, the effect of mixtures on the evolution of herbicide resistance caused by enhanced metabolism is unknown. This study evaluated the effect of a fenoxaprop-p-ethyl and imazethapyr mixture on the evolution of herbicide resistance in Echinochloa crus-galli using recurrent selection at sublethal doses. The progeny from second generations selected with the mixture had lower control than parental plants or the unselected progeny. GR50 increased 1.6- and 2.6-fold after two selection cycles with the mixture in susceptible (POP1-S) and imazethapyr-resistant (POP2-IR) biotypes, respectively. There was evidence that recurrent selection with this sublethal mixture had the potential to evolve cross-resistance to diclofop, cyhalofop, sethoxydim, and quinclorac. Mixture selection did not cause increased relative expression for a set of analyzed genes (CYP71AK2, CYP72A122, CYP72A258, CYP81A12, CYP81A14, CYP81A21, CYP81A22, and GST1). Fenoxaprop, rather than imazethapyr, is the main contributor to the decreased control in the progenies after recurrent selection with the mixture in low doses. This is the first study reporting the effect of a herbicide mixture at low doses on herbicide resistance evolution. The lack of control using the mixture may result in decreased herbicide sensitivity of the weed progenies. Using mixtures may select important detoxifying genes that have the potential to metabolize herbicides in patterns that cannot currently be predicted. The use of fully recommended herbicide rates in herbicide mixtures is recommended to reduce the risk of this type of resistance evolution.