Glutathione Transferase Photoaffinity Labeling Displays GST Induction by Safeners and Pathogen Infection.

Glutathione transferases (GSTs) represent a large and diverse enzyme family involved in the detoxification of small molecules by glutathione conjugation in crops, weeds and model plants. In this study, we introduce an easy and quick assay for photoaffinity labeling of GSTs to study GSTs globally in various plant species. The small-molecule probe contains glutathione, a photoreactive group and a minitag for coupling to reporter tags via click chemistry. Under UV irradiation, this probe quickly and robustly labels GSTs in crude protein extracts of different plant species. Purification and mass spectrometry (MS) analysis of labeled proteins from Arabidopsis identified 10 enriched GSTs from the Phi(F) and Tau(U) classes. Photoaffinity labeling of GSTs demonstrated GST induction in wheat seedlings upon treatment with safeners and in Arabidopsis leaves upon infection with avirulent bacteria. Treatment of Arabidopsis with salicylic acid (SA) analog benzothiadiazole (BTH) induces GST labeling independent of NPR1, the master regulator of SA. Six Phi- and Tau-class GSTs that are induced upon BTH treatment were identified, and their labeling was confirmed upon transient overexpression. These data demonstrate that GST photoaffinity labeling is a useful approach to studying GST induction in crude extracts of different plant species upon different types of stress.

Diazabicyclo derivatives as safeners protect cotton from injury caused by flumioxazin.

Flumioxazin, a protoporphyrinogen oxidase (PPO; EC 1.3.3.4) inhibitor, has been used in soybean, cotton, grapes, and many other crops to control broad leaf weeds. Unfortunately, it can cause damage to cotton. To ameliorate phytotoxicity of flumioxazin to cotton, this work assessed the protective effects of diazabicyclo derivatives as potential safeners in cotton. A bioactivity assay proved that the phytotoxicity of flumioxazin on cotton was alleviated by some of the compounds. In particular, the activity of glutathione S-transferases (GSTs) was significantly enhanced by Compound 32, which showed good safening activity against flumioxazin injury. The physicochemical properties and absorption, distribution, metabolism, excretion and toxicity (ADMET) predictions proved that the pharmacokinetic properties of Compound 32 are similar to those of the commercial safener BAS 145138. The present work demonstrated that diazabicyclo derivatives are potentially efficacious as herbicide safeners, meriting further investigation.

Dissipation, Fate, and Toxicity of Crop Protection Chemical Safeners in Aquatic Environments.

Safeners are a group of chemicals applied with herbicides to protect crop plants from potential adverse effects of agricultural products used to kill weeds in monocotyledonous crops. Various routes of dissipation of safeners from their point of applications were evaluated. Despite the large numbers of safeners (over 18) commercially available and the relatively large quantities (~2 × 106 kg/year) used, there is little information on their mobility and fate in the environment and occurrence in various environmental matrices. The only class of safeners for which a significant amount of information is available is dichloroacetamide safeners, which have been observed in some rivers in the USA at concentrations ranging from 42 to 190 ng/L. Given this gap in the literature, there is a clear need to determine the occurrence, fate, and bioavailability of other classes of safeners. Furthermore, since safeners are typically used in commercial formulations, it is useful to study them in relation to their corresponding herbicides. Common routes of dissipation for herbicides and applied safeners are surface run off (erosion), hydrolysis, photolysis, sorption, leaching, volatilization, and microbial degradation. Toxic potencies of safeners vary among organisms and safener compounds, ranging from as low as the LC50 for fish (Oncorhynchus mykiss) for isoxadifen-ethyl, which was 0.34 mg/L, to as high as the LC50 for Daphnia magna from dichlormid, which was 161 mg/L. Solubilities and octanol-water partition coefficients seem to be the principal driving force in understanding safener mobilities. This paper provides an up-to-date literature review regarding the occurrence, behaviour, and toxic potency of herbicide safeners and identifies important knowledge gaps in our understanding of these compounds and the potential risks posed to potentially impacted ecosystems.

Quinoxaline derivatives as herbicide safeners by improving Zea mays tolerance.

Isoxaflutole (IXF), a 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor, causes injury to crops leading to reductions in grain yield. In order to solve the phytotoxicity caused by IXF, the present work evaluated the protective response of the substituted quinoxaline derivatives as potential safeners on Zea mays. The bioassay results showed that all of the test compounds displayed protection against IXF. In particular, safener I-6 exhibited excellent safener activity against IXF injury via enhancing glutathione (GSH) content, glutathione S transferases (GSTs) and cytochrome P450 monooxygenases (CYP450) activity. The tested compounds induced the activity of CYP450 and GSTs in Z. mays. The physicochemical properties and ADMET properties of safener I-6, benoxacor and diketonitrile (DKN, IXF metabolite) were compared to predict pharmaceutical behavior. The present work demonstrates that the safener I-6 could be considered as a potential candidate for developing novel safeners in the future.

Stimulation action of mefenpyr-diethyl on soybean, wheat, and signal grass plants.

Mefenpyr-diethyl is a safener used for protection of cereal plants under applications of ACCase and ALS inhibitor herbicides. Current studies are describing safeners using a new approach, relating these products to stimulation action on plants. The objective of this work was to evaluate the stimulation action of mefenpyr-diethyl on soybean, wheat, and signal grass plants. The experiment was conducted in a greenhouse, under a completely randomized design, with four replications, in two seasons. Mefenpyr-diethyl (50 g a.i. ha-1) was applied on soybean plants (at V4 stage), and wheat and signal grass plants (both with 15 cm height). The variables evaluated were plant height, dry matter, and lipid content of the three species, and number of tillers of wheat and signal grass plants. The application of mefenpyr-diethyl in the first season increased the number of tillers of wheat and height of soybean plants. The soybean presented 24 and 14% more dry matter than the control in the first and second season, respectively, and 0.5% more lipid content in plants treated with mefenpyr-diethyl. These results show the stimulation action of mefenpyr-diethyl on wheat and soybean plants, denoting its potential for growth promotion and indicating the need for studies with this approach. No effect was found for the signal grass plants.

Effect of Fertiactyl® on the absorption and translocation of 14C-glyphosate in young eucalyptus plants.

Fertiactyl® is a foliar fertilizer with the potential to minimize the phytotoxicity effects caused by glyphosate drift in eucalyptus plants. As the interactions of the glyphosate and Fertiactyl® in tank mix on the plant behavior are not yet known, the objective was to evaluate the absorption and translocation of 14C-glyphosate, applied isolated and mixed in tank with Fertiactyl®, in young eucalyptus plants (clone I-144, Eucalyptus urophylla x E. grandis). The addition of Fertiactyl® to the mixture of 14C-glyphosate reduced the absorption by 94.3% in relation to the total absorbed at the end of the evaluation compared to plants treated only with 14C-glyphosate, i.e., Fertiactyl® protected the eucalyptus plants of the glyphosate intoxication by drift. The translocation rates from the treated leaves to the rest of the shoots and roots were low (<2% of the total recovered) in both treatments, suggest that restricted translocation is a mechanism of natural tolerance to glyphosate in plants of clone I-144. It is concluded that Fertiactyl®, mixed in the solution with glyphosate, protects young eucalyptus plants against glyphosate drift by reducing the amount of herbicide absorbed.

Effects of fenclorim on rice physiology, gene transcription and pretilachlor detoxification ability.

BACKGROUND: Fenclorim (Fen) can effectively protect rice from pretilachlor (Pre) injury, but its effects on rice have not been formally evaluated; thus, the Fen mode of action for alleviating the phytotoxicity caused by Pre in rice is not clear. This study aimed to examine the biochemical and physiological effects of Fen on rice and to determine the changes induced by Fen at the transcriptome level. RESULT: The chlorophyll content of rice plants was significantly affected by Pre but not by Fen. The activity of oxidative stress enzymes showed that Fen did not elicit any changes in oxidative stress; however, it reduced lipid peroxidation and oxidative damage induced by Pre. Fen did not affect the uptake of Pre but did affect its persistence in rice. In a transcriptome experiment, Fen upregulated genes in a detoxification pathway. Overall, 25 genes related to detoxification were identified, including P450, GST, and GT. Moreover, qRT-PCR analysis showed that four P450 genes, CYP71Y83, CYP71K14, CYP734A2 and CYP71D55, and two GST genes, GSTU16 and GSTF5, were upregulated by Fen and/or Pre. CONCLUSION: Our work indicates that Fen acts in antioxidative defense in addition to enhancing the metabolism of herbicides in rice.

New Lead Discovery of Herbicide Safener for Metolachlor Based on a Scaffold-Hopping Strategy.

The use of herbicide safeners can significantly alleviate herbicide injury to protect crop plants and expand the application scope of the existing herbicides in the field. Sanshools, which are well known as spices, are N-alkyl substituted compounds extracted from the Zanthoxylum species and have several essential physiological and pharmacological functions. Sanshools display excellent safener activity for the herbicide metolachlor in rice seedlings. However, the high cost of sanshools extraction and difficulties in the synthesis of their complicated chemical structures limit their utilization in agricultural fields. Thus, the present study designed and synthesized various N-alkyl amide derivatives via the scaffold-hopping strategy to solve the challenge of complicated structures and find novel potential safeners for the herbicide metolachlor. In total, 33 N-alkyl amide derivatives (2a-k, 3a-k, and 4a-k) were synthesized using amines and saturated and unsaturated fatty acids as starting materials through acylation and condensation. The identity of all the target compounds was well confirmed by 1H-NMR, 13C-NMR, and high-resolution mass spectrometry (HRMS). The primary evaluation of safener activities for the compounds by the agar method indicated that most of the target compounds could protect rice seedlings from injury caused by metolachlor. Notably, compounds 2k and 4k displayed excellent herbicide safener activities on plant height and demonstrated relatively similar activities to the commercialized compound dichlormid. Moreover, we showed that compounds 2k and 4k had higher glutathione S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and polyphenol oxidase (PPO) activities in rice seedlings, compared to the metolachlor treatment. In particular, 2k and 4k are safer for aquatic organisms than dichlormid. Results from the current work exhibit that compounds 2k and 4k have excellent crop safener activities toward rice and can, thus, be promising candidates for further structural optimization in rice protection.

Adsorption kinetics of the herbicide safeners, benoxacor and furilazole, to activated carbon and agricultural soils.

Chloroacetamide herbicides, namely acetochlor and metolachlor, are common herbicides used on corn and soybean fields. Dichloroacetamide safeners, namely benoxacor and furilazole, are commonly used in formulations containing chloroacetamide herbicides. Extensive reports on adsorption of chloroacetamide herbicides are available, yet little information exists regarding adsorption potential of co-applied safeners. Herein, the adsorption and desorption characteristics of selected herbicide safeners to granular activated carbon (GAC) and in agricultural soils are reported. Further, soil column studies were performed to understand the leaching behaviour of the herbicide Dual II Magnum. Equilibrium sorption experiments of safeners to three agricultural soils and one GAC showed that adsorption was best fitted by the Freundlich isotherm. The Freundlich adsorption constant, Kf, for benoxacor and furilazole sorption onto three agricultural soils ranged from 0.1 to 0.27 and 0.1 to 0.13 (mg/g) × (mg/L)ˆ(1/n), respectively. The Kf for benoxacor and furilazole to GAC was 6.4 and 3.4 (mg/g) × (mg/L)ˆ(1/n), respectively, suggesting more favorable sorption of benoxacor to GAC than furilazole to GAC. The sorption to soils was reversible as almost 40%-90% of both safeners was desorbed from three soils. These results were validated in four replicated soil column studies, where S-metolachlor was shown to leach similarly to the safener benoxacor, originating from the herbicide formulation. The leaching of S-metolachlor and benoxacor was influenced by soil texture. Cumulatively, these results show that safeners will move through the environment to surface waters similarly to the active ingredients in herbicides, but may be removed during drinking water treatment via GAC.

Fragment splicing-based design, synthesis and safener activity of novel substituted phenyl oxazole derivatives.

Fragment splicing is a primary strategy in the design and optimization of leading compound toward new skeleton with target bioactivity. Herein a series of novel substituted phenyl oxazole derivatives were designed via fragment analysis and coupling strategy that led to highly potent and bio-selective herbicide safener. The biological tests showed that most of the compounds could enhance the maize growth index, glutathione content and anti-reverse enzyme glutathione S-transferase activity in vivo. The molecular docking model exhibited that the novel compound could compete with chlorsulfuron binding to the herbicide target enzyme, which consequently attained the herbicide detoxification. Especially compound I-f displayed the best activities than commercial safener isoxadifen-ethyl and other compounds. The present work demonstrates that the synthesized compounds could be developed as potential candidates for the discovery of novel herbicide safeners in the future.

Rational design, synthesis and structure-activity relationship of novel substituted oxazole isoxazole carboxamides as herbicide safener.

A series of novel substituted oxazole isoxazole carboxamides derivatives were designed on the basis of active subunit combination. Forty-four novel compounds were synthesized by an efficient one-pot procedure under microwave irradiation. The bioactivity was evaluated as herbicide safener against the injury of chlorsulfuron. It was found that most of the synthesized compounds displayed remarkable protection against chlorsulfuron via enhanced glutathione content and glutathione S transferase activity. Especially compound I-11 exhibited better bioactivity than the safeners isoxadifen-ethyl and R-28725. Molecular docking simulations suggested that the target compounds could compete with chlorsulfuron in the active site of acetolactate synthase, which could explain the protective effects of safeners. The present work demonstrates that the target compounds containing oxazole isoxazole groups could be considered as potential candidates for developing novel safeners in the future.

Protective Responses Induced by Chiral 3-Dichloroacetyl Oxazolidine Safeners in Maize (Zea mays L.) and the Detoxification Mechanism.

Herbicide safeners selectively protect crops from herbicide injury while maintaining the herbicidal effect on the target weed. To some extent, the detoxification of herbicides is related to the effect of herbicide safeners on the level and activity of herbicide target enzymes. In this work, the expression of the detoxifying enzyme glutathione S-transferase (GST) and antioxidant enzyme activities in maize seedlings were studied in the presence of three potential herbicide safeners: 3-dichloroacetyl oxazolidine and its two optical isomers. Further, the protective effect of chiral herbicide safeners on detoxifying chlorsulfuron in maize was evaluated. All safeners increased the expression levels of herbicide detoxifying enzymes, including GST, catalase (CAT), and peroxidase (POD) to reduce sulfonylurea herbicide phytotoxicity in maize seedlings. Our results indicate that the R-isomer of 3-(dichloroacetyl)-2,2,5-trimethyl-1,3-oxazolidine can induce glutathione (GSH) production, GST activity, and the ability of GST to react with the substrate 1-chloro-2,4-dinitrobenzene (CDNB) in maize, meaning that the R-isomer can protect maize from damage by chlorsulfuron. Information about antioxidative enzyme activity was obtained to determine the role of chiral safeners in overcoming the oxidative stress in maize attributed to herbicides. The interaction of safeners and active target sites of acetolactate synthase (ALS) was demonstrated by molecular docking modeling, which indicated that both isomers could form a good interaction with ALS. Our findings suggest that the detoxification mechanism of chiral safeners might involve the induction of the activity of herbicide detoxifying enzymes as well as the completion of the target active site between the safener and chlorsulfuron.

Design, Synthesis, and Safener Activity of Novel Methyl (R)-N-Benzoyl/Dichloroacetyl-Thiazolidine-4-Carboxylates.

A series of novel methyl (R)-N-benzoyl/dichloroacetyl-thiazolidine-4-carboxylates were designed by active substructure combination. The title compounds were synthesized using a one-pot route from l-cysteine methyl ester hydrochloride, acyl chloride, and ketones. All compounds were characterized by IR, ¹H NMR, 13C NMR, and HRMS. The structure of 4q was determined by X-ray crystallography. The biological tests showed that the title compounds protected maize from chlorimuron-ethyl injury to some extent. The ALS activity assay showed that the title compounds increased the ALS activity of maize inhibited by chlorimuron-ethyl. Molecular docking modeling demonstrated that Compound 4e competed against chlorimuron-ethyl to combine with the herbicide target enzyme active site, causing the herbicide to be ineffective.

Isoxadifen safening mechanism in sweet corn genotypes with differential response to P450-metabolized herbicides.

Three sweet corn genotypes, two inbred lines (IBER001 and IBER002) and their hybrid (ER00X), differ in their phenotypic responses to several P450-metabolized herbicides, used in sweet corn, namely, foramsulfuron, iodosulfuron, rimsulfuron and tembotrione. Foramsulfuron is a sulfonylurea herbicide commonly formulated with the safener isoxadifen that is used for selective post-emergence weed control in corn. Our goal was to elucidate the mechanism of these genotypes' responses to foramsulfuron and safener isoxadifen and examine the heritability of those responses. IBER001 was sensitive to foramsulfuron+isoxadifen, with an ED50 of 3.6gaiha-1, while IBER002 and ER00X were tolerant with ED50 values of 808 and 700gaiha-1, respectively. ALS enzyme extracted from each of the different genotypes was equally sensitive to foramsulfuron. Pre-treatment with malathion, a known cytochrome P450 inhibitor, increased foramsulfuron injury in IBER002 and ER00X, but had no effect on those lines when isoxadifen was applied with the herbicide. Foramsulfuron-treated IBER001 was severely injured regardless of the presence of malathion and/or isoxadifen. Pre-treatment with malathion similarly increased the phytotoxicity of iodosulfuron+safener (mefenpyr) and rimsulfuron to the tolerant genotypes, but did not increase the level of injury caused by the tembotrione+isoxadifen treatment. Segregation of F2 and backcross progenies according to their responses to foramsulfuron+isoxadifen revealed a pattern of inheritance typical of a trait controlled by a single gene inheritance, with a recessive allele conferring sensitivity. Our results support the hypothesis that foramsulfuron selectivity is associated with P450 metabolism and that isoxadifen positively affects P450 activity. The sensitive genotype that does not respond to isoxadifen is presumably homozygous for a deficient or non-functioning P450 gene.

Solvent-Free Synthesis and Safener Activity of Sulfonylurea Benzothiazolines.

A series of novel sulfonylurea benzothiazolines was designed by splicing active groups and bioisosterism. A solvent-free synthetic route was developed for the sulfonylurea benzothiazoline derivatives via the cyclization and carbamylation. All compounds were characterized by IR, ¹H-NMR, 13C-NMR, HRMS. The biological activity tests indicated the compounds could protect maize against the injury caused by chlorsulfuron to some extent. The molecular docking result showed that the new compound competed with chlorsulfuron to bind with the herbicide target enzyme active site to attain detoxification.

A novel function of sanshools: the alleviation of injury from metolachlor in rice seedlings.

Szechuan peppers are extensively used as a spice and in traditional medicine in Asia, primarily because of its active compounds, sanshools (S). However, there is only limited mention in agriculture, and there are no papers reporting its use as an herbicide safener. In this study, we provide the first evidence that S can effectively alleviate rice-seedling injury from metolachlor (M). We observed that the M-treated (0.25 µM) rice seedlings, which were 56.0%, 66.0%, and 57.0% of the non-treated control in shoot height, root length, and fresh biomass, respectively, were recovered by S to 93.1%, 97.6%, and 94.8%, respectively. The emergence rate was enhanced to over 80% in the M+S treatment, whereas it was below 60% in the M treatment. This M+S mixture elevated the rice-seedling root activity to higher than 87.0% of the value for the non-treated control. The activity of glutathione transferases in the combined treatments approximately doubles that of the M treatment and quadruples that of the non-treated controls. This effect was positively correlated with the induced expression of OsGSTU3. Our results suggest that S may represent a new group of safeners and enable the possibility of using these compounds for improving plant production or protecting rice from the phytotoxicity of metolachlor.

Protective mechanisms of neral as a plant-derived safener against fenoxaprop-p-ethyl injury in rice.

BACKGROUND: The use of herbicide safeners effectively minimises crop damage while maintaining the full efficacy of herbicides. The present study aimed to assess the potential protective effects of neral (NR) as a safener, in order to mitigate injury caused by fenoxaprop-p-ethyl (FE) on rice. RESULTS: The alleviating effect of NR was similar to that of the safener isoxadifen-ethyl (IE). The root elongation of rice was significantly promoted under the FE + NR and FE + IE treatments, as compared to the FE treatment. The transcriptome analysis further suggested that the effects of NR treatment on plant metabolic pathways differed from those of IE treatment. In total, 895 and 47 up-differentially expressed genes induced by NR (NR-inducible genes) and IE (IE-inducible genes) were identified. NR-inducible genes were mainly enriched in phytohormone synthesis and signalling response, including 'response to brassinosteroid', 'response to jasmonic acid', 'response to ethylene', 'brassinosteroid metabolic process', 'brassinosteroid biosynthesis' and 'plant hormone signal transduction'. In contrast, IE-inducible genes were predominantly enriched in glutathione metabolism. The activity of glutathione S-transferase was found to be increased after IE treatment, whereas no significant increase was observed following NR treatment. Moreover, several transcription factor genes, such as those encoding AP2/ERF-ERF and (basic helix-loop-helix) bHLH were found to be significantly induced by NR treatment. CONCLUSION: This is the first report of the utilisation of NR as an herbicide safener. The results of this study suggest the toxicity of FE to rice is mitigated by NR through a distinct mechanism compared to IE. © 2023 Society of Chemical Industry.

Herbicide safener isoxadifen-ethyl associated with increased Goss's wilt severity in corn (Zea mays).

BACKGROUND: Goss's bacterial wilt and leaf blight (Goss's wilt), caused by the bacterium Clavibacter nebraskensis, is a corn disease that has been a top ten yield-reducing disease in North America in the past 15 years. Isoxadifen-ethyl is an herbicide safener that effectively increases cytochrome P450 activity in corn which enhances a plant's metabolism of herbicide molecules. Recent research found a potential link between isoxadifen-ethyl and increased Goss's wilt severity. RESULTS: The application of isoxadifen-ethyl increased (P = 0.014-0.046) area under disease progress curve (AUDPC) by 19%, 7%, and 9% at three environments, independent of accompanying herbicide or herbicide application timing. However, no significant differences in incidence of systemic wilt or corn grain yield occurred among treatments at any environment. CONCLUSION: These data provide evidence for an association between isoxadifen-ethyl safener and Goss's wilt in corn. The reason for this association is unknown, but the safener may affect plant or pathogen physiological mechanisms. While the increased disease severity did not result in decreased grain yield in these experiments, an increase in pathogen inoculum due to higher disease severity could influence Goss' wilt epidemics in future years. © 2024 Society of Chemical Industry.

Herbicide Safeners: From Molecular Structure Design to Safener Activity.

Herbicide safeners, highly effective antidotes, find widespread application in fields for alleviating the phytotoxicity of herbicides to crops. Designing new herbicide safeners remains a notable issue in pesticide research. This review focuses on discussing and summarizing the structure-activity relationships, molecular structures, physicochemical properties, and molecular docking of herbicide safeners in order to explore how different structures affect the safener activities of target compounds. It also provides insights into the application prospects of computer-aided drug design for designing and synthesizing new safeners in the future.

Design, Synthesis, and Biological Evaluation of Novel Purine Derivatives as Herbicide Safeners.

Mesosulfuron-methyl, an inhibitor of acetolactate synthase (ALS), has been extensively used in wheats. However, it can damage wheat (Triticum aestivum) and even lead to crop death. Herbicide safeners selectively shield crops from such damage without compromising weed control. To mitigate the phytotoxicity of mesosulfuron-methyl in crops, several purine derivatives were developed based on active substructure splicing. The synthesized title compounds underwent thorough characterization using infrared spectroscopy, 1H nuclear magnetic resonance (1H NMR), 13C nuclear magnetic resonance (13C NMR), and high-resolution mass spectrometry. We evaluated chlorophyll and glutathione contents as well as various enzyme activities to evaluate the safer activity of these compounds. Compounds III-3 and III-7 exhibited superior activity compared with the safener mefenpyr-diethyl. Molecular structure analysis, along with predictions of absorption, distribution, metabolism, excretion, and toxicity, indicated that compound III-7 shared pharmacokinetic traits with the commercial safener mefenpyr-diethyl. Molecular docking simulations revealed that compound III-7 competitively bound to the ALS active site with mesosulfuron-methyl, elucidating the protective mechanism of the safeners. Overall, this study highlights purine derivatives as potential candidates for novel safener development.