An artificially evolved gene for herbicide-resistant rice breeding.

Discovering and engineering herbicide-resistant genes is a crucial challenge in crop breeding. This study focuses on the 4-hydroxyphenylpyruvate dioxygenase Inhibitor Sensitive 1-Like (HSL) protein, prevalent in higher plants and exhibiting weak catalytic activity against many ß-triketone herbicides (ß-THs). The crystal structures of maize HSL1A complexed with ß-THs were elucidated, identifying four essential herbicide-binding residues and explaining the weak activity of HSL1A against the herbicides. Utilizing an artificial evolution approach, we developed a series of rice HSL1 mutants targeting the four residues. Then, these mutants were systematically evaluated, identifying the M10 variant as the most effective in modifying ß-THs. The initial active conformation of substrate binding in HSL1 was also revealed from these mutants. Furthermore, overexpression of M10 in rice significantly enhanced resistance to ß-THs, resulting in a notable 32-fold increase in resistance to methyl-benquitrione. In conclusion, the artificially evolved M10 gene shows great potential for the development of herbicide-resistant crops.

Standing genetic variation fuels rapid evolution of herbicide resistance in blackgrass.

Repeated herbicide applications in agricultural fields exert strong selection on weeds such as blackgrass (Alopecurus myosuroides), which is a major threat for temperate climate cereal crops. This inadvertent selection pressure provides an opportunity for investigating the underlying genetic mechanisms and evolutionary processes of rapid adaptation, which can occur both through mutations in the direct targets of herbicides and through changes in other, often metabolic, pathways, known as non-target-site resistance. How much target-site resistance (TSR) relies on de novo mutations vs. standing variation is important for developing strategies to manage herbicide resistance. We first generated a chromosome-level reference genome for A. myosuroides for population genomic studies of herbicide resistance and genome-wide diversity across Europe in this species. Next, through empirical data in the form of highly accurate long-read amplicons of alleles encoding acetyl-CoA carboxylase (ACCase) and acetolactate synthase (ALS) variants, we showed that most populations with resistance due to TSR mutations-23 out of 27 and six out of nine populations for ACCase and ALS, respectively-contained at least two TSR haplotypes, indicating that soft sweeps are the norm. Finally, through forward-in-time simulations, we inferred that TSR is likely to mainly result from standing genetic variation, with only a minor role for de novo mutations.

A Novel mechanism of herbicide action through disruption of pyrimidine biosynthesis.

A lead aryl pyrrolidinone anilide identified using high-throughput in vivo screening was optimized for efficacy, crop safety, and weed spectrum, resulting in tetflupyrolimet. Known modes of action were ruled out through in vitro enzyme and in vivo plant-based assays. Genomic sequencing of aryl pyrrolidinone anilide-resistant Arabidopsis thaliana progeny combined with nutrient reversal experiments and metabolomic analyses confirmed that the molecular target of the chemistry was dihydroorotate dehydrogenase (DHODH), the enzyme that catalyzes the fourth step in the de novo pyrimidine biosynthesis pathway. In vitro enzymatic and biophysical assays and a cocrystal structure with purified recombinant plant DHODH further confirmed this enzyme as the target site of this class of chemistry. Like known inhibitors of other DHODH orthologs, these molecules occupy the membrane-adjacent binding site of the electron acceptor ubiquinone. Identification of a new herbicidal chemical scaffold paired with a novel mode of action, the first such finding in over three decades, represents an important leap in combatting weed resistance and feeding a growing worldwide population.

An in-frame deletion mutation in the degron tail of auxin coreceptor IAA2 confers resistance to the herbicide 2,4-D in Sisymbrium orientale.

The natural auxin indole-3-acetic acid (IAA) is a key regulator of many aspects of plant growth and development. Synthetic auxin herbicides such as 2,4-D mimic the effects of IAA by inducing strong auxinic-signaling responses in plants. To determine the mechanism of 2,4-D resistance in a Sisymbrium orientale (Indian hedge mustard) weed population, we performed a transcriptome analysis of 2,4-D-resistant (R) and -susceptible (S) genotypes that revealed an in-frame 27-nucleotide deletion removing nine amino acids in the degron tail (DT) of the auxin coreceptor Aux/IAA2 (SoIAA2). The deletion allele cosegregated with 2,4-D resistance in recombinant inbred lines. Further, this deletion was also detected in several 2,4-D-resistant field populations of this species. Arabidopsis transgenic lines expressing the SoIAA2 mutant allele were resistant to 2,4-D and dicamba. The IAA2-DT deletion reduced binding to TIR1 in vitro with both natural and synthetic auxins, causing reduced association and increased dissociation rates. This mechanism of synthetic auxin herbicide resistance assigns an in planta function to the DT region of this Aux/IAA coreceptor for its role in synthetic auxin binding kinetics and reveals a potential biotechnological approach to produce synthetic auxin-resistant crops using gene-editing.

Metabolic resistance to acetolactate synthase inhibitors in Beckmannia syzigachne: identification of CYP81Q32 and its transcription regulation.

Frequent herbicide use selects for herbicide resistance in weeds. Cytochrome P450s are important detoxification enzymes responsible for herbicide resistance in plants. We identified and characterized a candidate P450 gene (BsCYP81Q32) from the problematic weed Beckmannia syzigachne to test whether it conferred metabolic resistance to the acetolactate synthase-inhibiting herbicides mesosulfuron-methyl, bispyribac-sodium, and pyriminobac-methyl. Transgenic rice overexpressing BsCYP81Q32 was resistant to the three herbicides. Equally, rice overexpressing the rice ortholog gene OsCYP81Q32 was more resistant to mesosulfuron-methyl. Conversely, an OsCYP81Q32 gene knockout generated using CRISPR/Cas9 enhanced mesosulfuron-methyl sensitivity in rice. Overexpression of the BsCYP81Q32 gene resulted in enhanced mesosulfuron-methyl metabolism in transgenic rice seedlings via O-demethylation. The major metabolite, demethylated mesosulfuron-methyl, was chemically synthesized and displayed reduced herbicidal effect in plants. Moreover, a transcription factor (BsTGAL6) was identified and shown to bind a key region in the BsCYP81Q32 promoter for gene activation. Inhibition of BsTGAL6 expression by salicylic acid treatment in B. syzigachne plants reduced BsCYP81Q32 expression and consequently changed the whole plant response to mesosulfuron-methyl. Sequence polymorphisms in an important region of the BsTGAL6 promoter may explain the higher expression of BsTGAL6 in resistant versus susceptible B. syzigachne plants. Collectively, the present study reveals the evolution of an herbicide-metabolizing and resistance-endowing P450 and its transcription regulation in an economically important weedy plant species.

Complete chloroplast genome and comparison of herbicides toxicity on Aeschynomene indica (Leguminosae) in upland direct-seeding paddy field.

BACKGROUND: Indian jointvetch (Aeschynomene indica) is a common and pernicious weed found in the upland direct-seeding rice fields in the lower reaches of the Yangtze River in China. However, there are few reports on the degree of harm, genetic characteristics, and management methods of this weed. The purpose of this study is to clarify the harm of Indian jointvetch to upland direct-seeding rice, analyze the genetic characteristics of this weed based on chloroplast genomics and identify its related species, and screen herbicides that are effective in managing this weed in upland direct-seeding rice fields. RESULTS: In a field investigation in upland direct-seeding rice paddies in Shanghai and Jiangsu, we determined that the plant height and maximum lateral distance of Indian jointvetch reached approximately 134.2 cm and 57.9 cm, respectively. With Indian jointvetch present at a density of 4/m2 and 8/m2, the yield of rice decreased by approximately 50% and 70%, respectively. We further obtained the first assembly of the complete chloroplast (cp.) genome sequence of Indian jointvetch (163,613 bp). There were 161 simple sequence repeats, 166 long repeats, and 83 protein-encoding genes. The phylogenetic tree and inverted repeat region expansion and contraction analysis based on cp. genomes demonstrated that species with closer affinity to A. indica included Glycine soja, Glycine max, and Sesbania cannabina. Moreover, a total of 3281, 3840, and 3838 single nucleotide polymorphisms were detected in the coding sequence regions of the cp. genomes of S. cannabina voucher IBSC, G. soja, and G. max compared with the A. indica sequence, respectively. A greenhouse pot experiment indicated that two pre-emergence herbicides, saflufenacil and oxyfluorfen, and two post-emergence herbicides, florpyrauxifen-benzyl and penoxsulam, can more effectively manage Indian jointvetch than other common herbicides in paddy fields. The combination of these two types of herbicides is recommended for managing Indian jointvetch throughout the entire growth period of upland direct-seeding rice. CONCLUSIONS: This study provides molecular resources for future research focusing on the identification of the infrageneric taxa, phylogenetic resolution, and biodiversity of Leguminosae plants, along with recommendations for reliable management methods to control Indian jointvetch.

Crystal structure of HPPD inhibitor sensitive protein from Oryza sativa.

4-hydroxyphenylpyruvate dioxygenase (HPPD) Inhibitor Sensitive 1 (HIS1) is an endogenous gene of rice, conferring broad-spectrum resistance to ß-triketone herbicides. Similar genes, known as HIS1-like genes (HSLs), exhibit analogous functions and can complement the herbicide-resistant characteristics endowed by HIS1. The identification of HIS1 and HSLs represents a valuable asset, as the intentional pairing of herbicides with resistance genes emerges as an effective strategy for crop breeding. Encoded by HIS1 is a Fe(II)/2-oxoglutarate-dependent oxygenase responsible for detoxifying ß-triketone herbicides through hydroxylation. However, the precise structure supporting this function remains unclear. This work, which determined the crystal structure of HIS1, reveals a conserved core motif of Fe(II)/2-oxoglutarate-dependent oxygenase and pinpoints the crucial residue dictating substrate preference between HIS1 and HSL.

Steerable Interfacial Assembly of 1D Amyloid-Like Protein Nanocomposites for Enhanced Nanoherbicide Utilization.

Conventional herbicide formulations suffer from serious problems such as easy drift, run-off and scouring into the environment, which pose enormous threats to human health and environmental safety. Herein, an innovative strategy is proposed to prepare oil-in-water nanoemulsions with long-term stability, enhanced droplet deposition, and improved nanoherbicide adhesion via steerable interfacial assembly of 1D amyloid-like protein nanocomposites. Bovine serum albumin (BSA) undergoes rapid amyloid-like aggregation upon reduction of its disulfide bond. The resulting phase-transitioned BSA (PTB) oligomers instantly self-assemble on the surface of cellulose nanofibers (CNF) to form the 1D PTB/CNF nanocomposites, which greatly expands the parameter space for interfacial assembly of amyloid-like proteins. The PTB/CNF nanocomposites exhibit excellent interfacial activity, enabling spontaneous adsorption at the oil-water interface to stabilize nanoemulsion. The excess PTB/CNF nanocomposites would also self-assemble at the air-aqueous interface upon spraying, resulting in efficient droplet deposition on (super)hydrophobic leaves. The deposited nanoherbicides show excellent resistance to wind/rain corrosion due to the robust amyloid-mediated adhesion, with a retention rate of more than 80% after severe scouring. Consequently, herbicide applications can be reduced by at least 30% compared to commercial emulsifiable concentrates, showing greater herbicidal efficiency. This study provides novel insights and approaches to promote sustainable agricultural development.

Intragenic Agrobacterium-mediated gene transfer mimics micro-translocations without foreign DNA.

MAIN CONCLUSION: Agrobacterium-mediated transformation of Nicotiana tabacum, using an intragenic T-DNA region derived entirely from the N. tabacum genome, results in the equivalence of micro-translocations within genomes. Intragenic Agrobacterium-mediated gene transfer was achieved in Nicotiana tabacum using a T-DNA composed entirely of N. tabacum DNA, including T-DNA borders and the acetohydroxyacid synthase gene conferring resistance to sulfonylurea herbicides. Genomic analysis of a resulting plant, with single locus inheritance of herbicide resistance, identified a single insertion of the intragenic T-DNA on chromosome 5. The insertion event was composed of three N. tabacum DNA fragments from other chromosomes, as assembled on the T-DNA vector. This validates that intragenic transformation of plants can mimic micro-translocations within genomes, with the absence of foreign DNA.

PfGSTF2 endows resistance to quizalofop-p-ethyl in Polypogon fugax by GSH conjugation.

Populations of Polypogon fugax have developed resistance to many acetyl-CoA carboxylase (ACCase)-inhibiting herbicides. This resistance threats the effectiveness and sustainability of herbicide use. In our previous research, a field P. fugax population exhibited GST-based metabolic resistance to the widely used ACCase-inhibiting herbicide quizalofop-p-ethyl. Here, in this current study, we identified and characterized two GST genes (named as PfGSTF2 and PfGSTF58) that showed higher expression levels in the resistant than the susceptible population. Transgenic rice calli overexpressing PfGSTF2, but not PfGSTF58, became resistant to quizalofop-p-ethyl and haloxyfop-R-methyl. This reflects similar cross-resistance pattern to what was observed in the resistant P. fugax population. Transgenic rice seedlings overexpressing PfGSTF2 also exhibited resistance to quizalofop-p-ethyl. In contrast, CRISPR/Cas9 knockout of the orthologue gene in rice seedlings increased their sensitivity to quizalofop-p-ethyl. LC-MS analysis of in vitro herbicide metabolism by Escherichia coli-expressed recombinant PfGSTF2 revealed that quizalofop (but not haloxyfop) was detoxified at the ether bond, generating the GSH-quizalofop conjugate and a propanoic acid derivative with greatly reduced herbicidal activity. Equally, these two metabolites accumulated at higher levels in the resistant population than the susceptible population. In addition, both recombinant PfGSTF2 and PfGSTF58 can attenuate cytotoxicity by reactive oxygen species (ROS), suggesting a role in plant defence against ROS generated by herbicides. Furthermore, the GST inhibitor (NBD-Cl) reversed resistance in the resistant population, and PfGSTF2 (but not PfGSTF58) responded to NBD-Cl inhibition. All these suggest that PfGSTF2 plays a significant role in the evolution of quizalofop resistance through enhanced herbicide metabolism in P. fugax.

Glyphosate effects on growth and biofilm formation in bee gut symbionts and diverse associated bacteria.

Biofilm formation is a common adaptation enabling bacteria to thrive in various environments and withstand external pressures. In the context of host-microbe interactions, biofilms play vital roles in establishing microbiomes associated with animals and plants and are used by opportunistic microbes to facilitate survival within hosts. Investigating biofilm dynamics, composition, and responses to environmental stressors is crucial for understanding microbial community assembly and biofilm regulation in health and disease. In this study, we explore in vivo colonization and in vitro biofilm formation abilities of core members of the honey bee (Apis mellifera) gut microbiota. Additionally, we assess the impact of glyphosate, a widely used herbicide with antimicrobial properties, and a glyphosate-based herbicide formulation on growth and biofilm formation in bee gut symbionts as well as in other biofilm-forming bacteria associated with diverse animals and plants. Our results demonstrate that several strains of core bee gut bacterial species can colonize the bee gut, which probably depends on their ability to form biofilms. Furthermore, glyphosate exposure elicits variable effects on bacterial growth and biofilm formation. In some instances, the effects correlate with the bacteria's ability to encode a susceptible or tolerant version of the enzyme inhibited by glyphosate in the shikimate pathway. However, in other instances, no such correlation is observed. Testing the herbicide formulation further complicates comparisons, as results often diverge from glyphosate exposure alone, suggesting that co-formulants influence bacterial growth and biofilm formation. These findings highlight the nuanced impacts of environmental stressors on microbial biofilms, with both ecological and host health-related implications. IMPORTANCE: Biofilms are essential for microbial communities to establish and thrive in diverse environments. In the honey bee gut, the core microbiota member Snodgrassella alvi forms biofilms, potentially aiding the establishment of other members and promoting interactions with the host. In this study, we show that specific strains of other core members, including Bifidobacterium, Bombilactobacillus, Gilliamella, and Lactobacillus, also form biofilms in vitro. We then examine the impact of glyphosate, a widely used herbicide that can disrupt the bee microbiota, on bacterial growth and biofilm formation. Our findings demonstrate the diverse effects of glyphosate on biofilm formation, ranging from inhibition to enhancement, reflecting observations in other beneficial or pathogenic bacteria associated with animals and plants. Thus, glyphosate exposure may influence bacterial growth and biofilm formation, potentially shaping microbial establishment on host surfaces and impacting health outcomes.

Introgression from local cultivars is a driver of agricultural adaptation in Argentinian weedy rice.

Weedy rice, a pervasive and troublesome weed found across the globe, has often evolved through fertilization of rice cultivars with little importance of crop-weed gene flow. In Argentina, weedy rice has been reported as an important constraint since the early 1970s, and, in the last few years, strains with herbicide-resistance are suspected to evolve. Despite their importance, the origin and genetic composition of Argentinian weedy rice as well its adaptation to agricultural environments has not been explored so far. To study this, we conducted genotyping-by-sequencing on samples of Argentinian weedy and cultivated rice and compared them with published data from weedy, cultivated and wild rice accessions distributed worldwide. In addition, we conducted a phenotypic characterization for weedy-related traits, a herbicide resistance screening and genotyped accessions for known mutations in the acetolactate synthase (ALS) gene, which confers herbicide resistance. Our results revealed large phenotypic variability in Argentinian weedy rice. Most strains were resistant to ALS-inhibiting herbicides with a high frequency of the ALS mutation (A122T) present in Argentinian rice cultivars. Argentinian cultivars belonged to the three major genetic groups of rice: japonica, indica and aus while weeds were mostly aus or aus-indica admixed, resembling weedy rice strains from the Southern Cone region. Phylogenetic analysis supports a single origin for aus-like South American weeds, likely as seed contaminants from the United States, and then admixture with local indica cultivars. Our findings demonstrate that crop to weed introgression can facilitate rapid adaptation to agriculture environments.

An efficient sulfadiazine selection scheme for stable transformation in the model liverwort Marchantia polymorpha.

Plant macroevolutionary studies leverage the phylogenetic position of non-flowering model systems like the liverwort Marchantia polymorpha to investigate the origin and evolution of key plant processes. To date, most molecular genetic studies in Marchantia rely on hygromycin and/or chlorsulfuron herbicide resistance markers for the selection of stable transformants. Here, we used a sulfonamide-resistant dihydropteroate synthase (DHPS) gene to enable sulfadiazine-based transformation selection in M. polymorpha. We demonstrate the reliability of sulfadiazine selection on its own and in combination with existing hygromycin and chlorsulfuron selection schemes through transgene stacking experiments. The utility of this system is further demonstrated through confocal microscopy of a triple transgenic line carrying fluorescent proteins labelling the plasma membrane, cortical microtubules, and the nucleus. Collectively, our findings and resources broaden the capacity to genetically manipulate the increasingly popular model liverwort M. polymorpha.

Herbicide-resistant HPPD variants identified via directed evolution.

Herbicides play a crucial role in boosting crop yields, yet the emergence of herbicide-resistant weeds and the susceptibility of crops to herbicides have posed significant challenges to their efficacy. ß-triketone herbicides specifically target the enzyme 4-Hydroxyphenylpyruvate dioxygenase (HPPD) essential for plant growth. Remarkably, few resistant weeds have been identified against these herbicides. In this study, we aimed to identify mutations within the cotton HPPD gene that confer resistance to mesotrione, a widely used triketone herbicide. Through the establishment of a high-throughput mutant screening system in E. coli, we identified four single nucleotide changes leading to amino acid substitutions in HPPD, resulting in mesotrione resistance while preserving native enzymatic activity. Various combinations of these mutations displayed synergistic effects on herbicide resistance. Additionally, the HPPD variants were able to complement the Arabidopsis athppd mutant, indicating their retention of sufficient native activity crucial for plant growth and development. Expression of these cotton HPPD variants in Arabidopsis resulted in heightened herbicide resistance. These findings offer critical insights into the target amino acids of HPPD for gene editing, paving the way for the development of herbicide-resistant cotton in the future.

Determination of acetochlor by UPLC-MS3 in cells and its application to a cellular pharmacokinetic study.

The aim of this work was to develop a fast, simple, and reliable UPLC-MS3 method for the sensitive detection of acetochlor in biological samples. In MS3 mode, the ion transition m/z 270.1 â†’ 224.1→148.1 was chosen for quantification with butachlor as the internal standard. In the UPLC system, separation was performed on a UPLC column (2.1 × 50 mm ID, 1.7 µm) with 0.1 % FA in water and acetonitrile as mobile phases. After simple protein precipitation via acetonitrile, the method was well validated with good linearity (0.5-20 ng/mL, r > 0.995), accuracy (-3.70 %-2.98 %), and precision (<15 %). The selectivity and sensitivity were improved obviously in MS3 mode than that in MRM mode. The developed UPLC-MS3 method was successfully applied to the cellular pharmacokinetics study of acetochlor in MCF-7 cells.

Laccase Immobilized on Arginine-Functionalized Boron Nitride Nanosheets for Enhanced Atrazine Degradation.

Enzyme-mediated systems have been widely employed for the biotransformation of environmental contaminants. However, the catalytic performance of free enzymes is restricted by the rapid loss of their catalytic activity, stability, and reusability. In this work, we developed an enzyme immobilization platform by elaborately anchoring fungal laccase onto arginine-functionalized boron nitride nanosheets (BNNS-Arg@Lac). BNNS-Arg@Lac showcased ∼75% immobilization yield and enhanced stability against fluctuating pH values and temperatures, along with remarkable reusability across six consecutive cycles, outperforming free natural laccase (nlaccase). A model pollutant, atrazine, was selected for a proof-of-concept demonstration, given the substantial environmental and public health concerns in agriculture runoff. BNNS-Arg@Lac-catalyzed atrazine degradation rate was nearly twice that of nlaccase. Moreover, BNNS-Arg@Lac consistently demonstrated superior atrazine degradation in synthetic agricultural wastewater and various mediator systems compared to nlaccase. Comprehensive product analysis unraveled distinct degradation pathways for BNNS-Arg@Lac and nlaccase. Overall, this research provides a foundation for the future development of enzyme-nanomaterial hybrids for degrading environmental chemicals and may unlock new potential for green and efficient resource recovery and waste management strategies.

Environmental Concentrations of Herbicide Prometryn Render Stress-Tolerant Corals Susceptible to Ocean Warming.

Global warming has caused the degradation of coral reefs around the world. While stress-tolerant corals have demonstrated the ability to acclimatize to ocean warming, it remains unclear whether they can sustain their thermal resilience when superimposed with other coastal environmental stressors. We report the combined impacts of a photosystem II (PSII) herbicide, prometryn, and ocean warming on the stress-tolerant coral Galaxea fascicularis through physiological and omics analyses. The results demonstrate that the heat-stress-induced inhibition of photosynthetic efficiency in G. fascicularis is exacerbated in the presence of prometryn. Transcriptomics and metabolomics analyses indicate that the prometryn exposure may overwhelm the photosystem repair mechanism in stress-tolerant corals, thereby compromising their capacity for thermal acclimation. Moreover, prometryn might amplify the adverse effects of heat stress on key energy and nutrient metabolism pathways and induce a stronger response to oxidative stress in stress-tolerant corals. The findings indicate that the presence of prometryn at environmentally relevant concentrations would render corals more susceptible to heat stress and exacerbate the breakdown of coral Symbiodiniaceae symbiosis. The present study provides valuable insights into the necessity of prioritizing PSII herbicide pollution reduction in coral reef protection efforts while mitigating the effects of climate change.

Position-specific carbon stable isotope analysis of glyphosate: isotope fingerprinting of molecules within a mixture.

Glyphosate [N-(phosphonomethyl) glycine] is a widely used herbicide and a molecule of interest in the environmental sciences, due to its global use in agriculture and its potential impact on ecosystems. This study presents the first position-specific carbon isotope (13C/12C) analyses of glyphosates from multiple sources. In contrast to traditional isotope ratio mass spectrometry (IRMS), position-specific analysis provides 13C/12C ratios at individual carbon atom positions within a molecule, rather than an average carbon isotope ratio across a mixture or a specific compound. In this work, glyphosate in commercial herbicides was analyzed with only minimal purification, using a nuclear magnetic resonance (NMR) spectroscopy method that detects 1H nuclei with bonds to either 13C or 12C, and isolates the signals of interest from other signals in the mixture. Results demonstrate that glyphosate from different sources can have significantly different intramolecular 13C/12C distributions, which were found to be spread over a wide range, with δ13C Vienna Peedee Belemnite (VPDB) values of -28.7 to -57.9‰. In each glyphosate, the carbon with a bond to the phosphorus atom was found to be depleted in 13C compared to the carbon at the C2 position, by 4 to 10‰. Aminomethylphosphonic acid (AMPA) was analyzed for method validation; AMPA contains only a single carbon position, so the 13C/12C results provided by the NMR method could be directly compared with traditional isotope ratio mass spectrometry. The glyphosate mixtures were also analyzed by IRMS to obtain their average 13C/12C ratios, for comparison with our position-specific results. This comparison revealed that the IRMS results significantly disguise the intramolecular isotope distribution. Finally, we introduce a 31P NMR method that can provide a position-specific 13C/12C ratio for carbon positions with a C-P chemical bond, and the results obtained by 1H and 31P for C3 carbon agree with one another within their analytical uncertainty. These analytical tools for position-specific carbon isotope analysis permit the isotopic fingerprinting of target molecules within a mixture, with potential applications in a range of fields, including the environmental sciences and chemical forensics.

Low-background interference detection of glyphosate, glufosinate, and AMPA in foods using UPLC-MS/MS without derivatization.

The abuse of herbicides has emerged as a great threat to food security. Herein, a low-background interference detection method based on UPLC-MS was developed for the simultaneous determination of glufosinate, glyphosate, and its metabolite aminomethylphosphonic acid (AMPA) in foods. Initially, this study proposed a simple and rapid pretreatment method, utilizing water extraction and PRiME HLB purification to isolate glyphosate, glufosinate, and AMPA from food samples. After the optimization of pretreatment conditions, the processed samples are then analyzed directly by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS) without pre-column derivatization. The method can effectively reduce interference from by-products of pre-column derivatization and background substrates of food sample, showing low matrix effects (ME) ranging from - 24.83 to 32.10%. Subsequently, the method has been validated by 13 kinds of food samples. The recoveries of the three herbicides in the food samples range from 84.2 to 115.6%. The limit of detection (LOD) is lower to 0.073 mg/kg, 0.017 mg/kg, and 0.037 mg/kg, respectively. Moreover, the method shows an excellent reproducibility with relative standard deviations (RSD) within 16.9%. Thus, the method can provide high trueness, reproducibility, sensitivity, and interference-free detection to ensure human health safety.

Impact of chronic exposure to field level glyphosate on the food consumption, survival, gene expression, gut microbiota, and metabolomic profiles of honeybees.

Glyphosate (GLY) is among the most widely used pesticides in the world. However, there are a lot of unknowns about chronic exposure to GLY's effects on Honeybee (HB) behavior and physiology. To address this, we carried out five experiments to study the impact of chronic exposure to 5 mg/kg GLY on sugar consumption, survival, gene expression, gut microbiota, and metabolites of HB workers. Our results find a significant decrease in sugar consumption and survival probability of HB after chronic exposure to GLY. Further, genes associated with immune response, energy metabolism, and longevity were conspicuously altered. In addition, a total of seven metabolites were found to be differentially expressed in the metabolomic profiles, mainly related the sucrose metabolism. There was no significant difference in the gut microbiota. Results suggest that chronic exposure to field-level GLY altered the health of HB and the intricate toxic mechanisms. Our data provided insights into the chronic effects of GLY on HB behavior in food intake and health, which represents the field conditions where HB are exposed to pesticides over extended periods.