Development of a double-antibody sandwich ELISA for quantification of mutated EPSPS gene expression in rice.

Glyphosate resistance is a critically important trait for genetically modified (GM) crops. Mutation of the rice EPSPS gene results in a high level of glyphosate resistance, presenting significant potential for the development of glyphosate-tolerant crops. The resistance of rice to glyphosate is correlated with the expression levels of resistance genes. Therefore, developing a convenient, stable, and sensitive method for quantifying the OsmEPSPS protein is crucial for the development of glyphosate-resistant crops. We developed a double-antibody sandwich quantitative ELISA (DAS-ELISA) using a specific monoclonal antibody (mAb) for OsmEPSPS capture and an HRP-conjugated anti-OsmEPSPS rabbit polyclonal antibody (pAb). The method could be used to detect OsmEPSPS within a linear range of 16-256 ng/mL with robust intra- and inter-batch duplicability (%CV values: 0.17 %-7.24 %). OsmEPSPS expression in the transgenic rice lines (54.44-445.80 µg/g) was quantified using the DAS-ELISA. Furthermore, the expression of the OsmEPSPS gene was validated through Western blotting. This study demonstrated the reliability and stability of the DAS-ELISA for OsmEPSPS detection in GM rice.

Evolving dual-trait EPSP synthase variants using a synthetic yeast selection system.

The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) functions in the shikimate pathway which is responsible for the production of aromatic amino acids and precursors of other essential secondary metabolites in all plant species. EPSPS is also the molecular target of the herbicide glyphosate. While some plant EPSPS variants have been characterized with reduced glyphosate sensitivity and have been used in biotechnology, the glyphosate insensitivity typically comes with a cost to catalytic efficiency. Thus, there exists a need to generate additional EPSPS variants that maintain both high catalytic efficiency and high glyphosate tolerance. Here, we create a synthetic yeast system to rapidly study and evolve heterologous EPSP synthases for these dual traits. Using known EPSPS variants, we first validate that our synthetic yeast system is capable of recapitulating growth characteristics observed in plants grown in varying levels of glyphosate. Next, we demonstrate that variants from mutagenesis libraries with distinct phenotypic traits can be isolated depending on the selection criteria applied. By applying strong dual-trait selection pressure, we identify a notable EPSPS mutant after just a single round of evolution that displays robust glyphosate tolerance (Ki of nearly 1 mM) and improved enzymatic efficiency over the starting point (~2.5 fold). Finally, we show the crystal structure of corn EPSPS and the top resulting mutants and demonstrate that certain mutants have the potential to outperform previously reported glyphosate-resistant EPSPS mutants, such as T102I and P106S (denoted as TIPS), in whole-plant testing. Altogether, this platform helps explore the trade-off between glyphosate resistance and enzymatic efficiency.

Quantitative Detection of the CP4-EPSPS Protein in Genetically Modified Soybeans Using a Novel Fluorescence Immunoassay Assay.

Protein-based detection methods, enzyme-linked immunosorbent assays (ELISAs) and lateral flow strips, have been widely used for rapid, specific, and sensitive detection of genetically modified organisms (GMOs). However, the traditional ELISA method for the quantitative detection of GMOs has certain limitations. Herein, a quantum dot (QD)-based fluorescence-linked immunosorbent assay was developed using QDs as fluorescent markers for the detection of glyphosate-resistant protein (CP4-EPSPS) in the MON89788 soybean. The end-point fluorescent detection system was carried out using QDs conjugated with a goat anti-rabbit secondary antibody. Compared with the conventional sandwich ELISA method, the newly developed fluorescence-linked immunosorbent assay was highly sensitive and accurate for detecting the CP4-EPSPS protein. The quantified linearity was achieved in the range of 0.05-5% (w/w) for the MON89788 soybean sample. The recovery of protein extracted from mixed MON89788 soybean samples ranged from 87.67% to 116.83%. The limits of detection and limits of quantification were 0.7101 and 2.152 pg/mL, respectively. All of the results indicated that the QD-based fluorescence-linked immunosorbent assay was a highly specific and sensitive method for monitoring the CP4-EPSPS protein in GMOs.

Assessment of the potential risks in SD rats gavaged with genetically modified yeast containing the cp4-epsps gene.

Introduction: Despite the absence of definitive evidence indicating that the cp4-epsps gene and its resultant recombinant proteins have significant harmful effects on either human or animal health, the safety assessment of genetically modified (GM) crops expressing the CP4-EPSPS proteins has been controversial. This study endeavor was aimed at evaluating the potential risks posed by the CP4-EPSPS protein in transgenic crops, thereby contributing to the advancement of risk assessment methodologies in the context of genetically engineered crops. Methods: To ascertain the appropriate daily dosages for oral gavage administration, the expression levels of the CP4-EPSPS protein in a recombinant yeast were quantified. Subsequently, physiological and biochemical analysis, metabolomics, and metagenomic analysis were conducted based on a 90-day Sprague-Dawley (SD) rats feeding experiment, respectively, thereby enhancing the depth and precision of our risk assessment framework. Results: The results from the physiological and biochemical analysis, organ pathological, blood metabolism, gut microbiota, and correlation analysis of metabolites and gut microbiota revealed several biomarkers for further risk assessment. These biomarkers include clinical biochemical indexes such as total bilirubin (TBIL), direct bilirubin (DBIL), creatine kinase (CK), and lactate dehydrogenase (LDH); metabolites like Methionine, 2-Oxovaleric acid, and LysoPC (16:0); and gut microbiota including Blautia wexlerae, Holdemanella biformis, Dorea sp. CAG 317, Coriobacteriaceae and Erysipelotrichaceae. Conclusion: In conclusion, the risk can be significantly reduced by directly consuming inactivated recombinant CP4-EPSPS. Therefore, in everyday life, the risk associated with consuming GM foods containing recombinant CP4-EPSPS is substantially reduced after heat treatment.

The Effect of Nucleo-Olivary Stimulation on Climbing Fiber EPSPs in Purkinje Cells.

Climbing fibers, connecting the inferior olive and Purkinje cells, form the nervous system's strongest neural connection. These fibers activate after critical events like motor errors or anticipation of rewards, leading to bursts of excitatory postsynaptic potentials (EPSPs) in Purkinje cells. The number of EPSPs is a crucial variable when the brain is learning a new motor skill. Yet, we do not know what determines the number of EPSPs. Here, we measured the effect of nucleo-olivary stimulation on periorbital elicited climbing fiber responses through in-vivo intracellular Purkinje cell recordings in decerebrated ferrets. The results show that while nucleo-olivary stimulation decreased the probability of a response occurring at all, it did not reduce the number of EPSPs. The results suggest that nucleo-olivary stimulation does not influence the number of EPSPs in climbing fiber bursts.

Unraveling the atomic mechanisms underlying glyphosate insensitivity in EPSPS: implications of distal mutations.

5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS), as an indispensable enzyme in the shikimate pathway, is the specific target of grasser killer glyphosate (GPJ). GPJ is a competitive inhibitor of phosphoenolpyruvate (PEP), which is the natural substrate of EPSPS. A novel Ls-EPSPS gene variant discovered from Liliaceae, named ELs-EPSPS, includes five distal mutations, E112V, D142N, T351S, D425G, and R496G, endowing high GPJ insensitivity. However, the implicit molecular mechanism of the enhanced tolerance/insensitivity of GPJ in ELs-EPSPS is not fully understood. Herein, we try to interpret the hidden molecular mechanism using computational methods. Computational results reveal the enhanced flexibility of apo EPSPS upon mutations. The enhanced affinity of the initial binding substrate shikimate-3-phosphate (S3P), and the higher probability of second ligands PEP/GPJ entering the pocket are observed in the ELs-EPSPS-S3P system. Docking and MD results further confirmed the decreased GPJ-induced EPSPS inhibition upon mutations. And, the alterations of K98 and R179 side-chain orientations upon mutations are detrimental to GPJ binding at the active site. Additionally, the oscillation of side chain K98, in charge of PEP location, improves the proximity effect for substrates in the dual-substrate systems upon mutations. Our results clarify that the enhanced GPJ tolerance of EPSPS is achieved from decreased competitive inhibition of GPJ at the atomic perspective, and this finding further contributes to the cultivation of EPSPS genes with higher GPJ tolerance/insensitivity and a mighty renovation for developing glyphosate-resistant crops.Communicated by Ramaswamy H. Sarma.

CRISPR/Cas9-mediated homology donor repair base editing system to confer herbicide resistance in maize (Zea mays L.).

Weed infestation is a significant concern to crop yield loss, globally. The potent broad-spectrum glyphosate (N-phosphomethyl-glycine) has a widely utilized herbicide, acting on the shikimic acid pathway within chloroplast by inhibiting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). This crucial enzyme plays a vital role in aromatic amino acid synthesis. Repurposing of CRISPR/Cas9-mediated gene-editing was the inflection point for generating novel crop germplasm with diverse genetic variations in essential agronomic traits, achieved through the introduction of nucleotide substitutions at target sites within the native genes, and subsequent induction of indels through error-prone non-homologous end-joining DNA repair mechanisms. Here, we describe the development of efficient herbicide-resistant maize lines by using CRISPR/Cas9 mediated site-specific native ZmEPSPS gene fragment replacement via knock-out of conserved region followed by knock-in of desired homologous donor repair (HDR-GATIPS-mZmEPSPS) with triple amino acid substitution. The novel triple substitution conferred high herbicide tolerance in edited maize plants. Transgene-free progeny harbouring the triple amino acid substitutions revealed agronomic performances similar to that of wild-type plants, suggesting that the GATIPS-mZmEPSPS allele substitutions are crucial for developing elite maize varieties with significantly enhanced glyphosate resistance. Furthermore, the aromatic amino acid contents in edited maize lines were significantly higher than in wild-type plants. The present study describing the introduction of site-specific CRISPR/Cas9- GATIPS mutations in the ZmEPSPS gene via genome editing has immense potential for higher tolerance to glyphosate with no yield penalty in maize.

Response characterization and target site mechanism study in glyphosate-resistant populations of Lolium multiflorum L. from Brazil.

Italian ryegrass (Lolium multiflorum L.) is an invasive species widely spread in croplands worldwide. The intensive use of glyphosate has resulted in the selection of resistance to this herbicide in Italian ryegrass. This work characterized the response to glyphosate of Italian ryegrass populations from the South and Southwest regions of Paraná, Brazil. A total of 44 Italian ryegrass populations were collected in farming areas, and were classified for glyphosate resistance with 75% of populations resistant to gloyphosate. Of these, 3 resistant (VT05AR, MR20AR and RN01AR) and three susceptible (VT07AS, MR05AS and RN01AS) of these populations were selected to determine the resistance level and the involvement of the target site mechanisms for glyphosate resistance. Susceptible populations GR50 ranged from 165.66 to 218.17 g.e.a. ha-1 and resistant populations from 569.37 to 925.94, providing RI ranging from 2.88 and 4.70. No mutation in EPSPS was observed in the populations, however, in two (MR20AR and RN02AR) of the three resistant populations, an increase in the number of copies of the EPSPs gene (11 to 57×) was detected. The number of copies showed a positive correlation with the gene expression (R2 = 0.86) and with the GR50 of the populations (R2 = 0.81). The increase in EPSPS gene copies contributes to glyphosate resistance in Italian ryegrass populations from Brazil.

In maize, co-expression of GAT and GR79-EPSPS provides high glyphosate resistance, along with low glyphosate residues.

Herbicide tolerance has been the dominant trait introduced during the global commercialization of genetically modified (GM) crops. Herbicide-tolerant crops, especially glyphosate-resistant crops, offer great advantages for weed management; however, despite these benefits, glyphosate-resistant maize (Zea mays L.) has not yet been commercially deployed in China. To develop a new bio-breeding resource for glyphosate-resistant maize, we introduced a codon-optimized glyphosate N-acetyltransferase gene, gat, and the enolpyruvyl-shikimate-3-phosphate synthase gene, gr79-epsps, into the maize variety B104. We selected a genetically stable high glyphosate resistance (GR) transgenic event, designated GG2, from the transgenic maize population through screening with high doses of glyphosate. A molecular analysis demonstrated that single copy of gat and gr79-epsps were integrated into the maize genome, and these two genes were stably transcribed and translated. Field trials showed that the transgenic event GG2 could tolerate 9000 g acid equivalent (a.e.) glyphosate per ha with no effect on phenotype or yield. A gas chromatography-mass spectrometry (GC-MS) analysis revealed that, shortly after glyphosate application, the glyphosate (PMG) and aminomethylphosphonic acid (AMPA) residues in GG2 leaves decreased by more than 90% compared to their levels in HGK60 transgenic plants, which only harbored the epsps gene. Additionally, PMG and its metabolic residues (AMPA and N-acetyl-PMG) were not detected in the silage or seeds of GG2, even when far more than the recommended agricultural dose of glyphosate was applied. The co-expression of gat and gr79-epsps, therefore, confers GG2 with high GR and a low risk of herbicide residue accumulation, making this germplasm a valuable GR event in herbicide-tolerant maize breeding. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-023-00114-8.

Co-expression of GR79 EPSPS and GAT generates high glyphosate-resistant alfalfa with low glyphosate residues.

Weed competition seriously threatens the yield of alfalfa, the most important forage legume worldwide, thus generating herbicide-resistant alfalfa varieties is becoming a necessary cost-effective strategy to assist farmers for weed control. Here, we report the co-expression of plant codon-optimized forms of GR79 EPSPS (pGR79 EPSPS) and N-acetyltransferase (pGAT) genes, in alfalfa, via Agrobacterium-mediated transformation. We established that the pGR79 EPSPS-pGAT co-expression alfalfa lines were able to tolerate up to tenfold higher commercial usage of glyphosate and produced approximately ten times lower glyphosate residues than the conventional cultivar. Our findings generate an elite herbicide-resistant germplasm for alfalfa breeding and provide a promising strategy for developing high-glyphosate-resistant and low-glyphosate-residue forages. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-023-00119-3.

Identification and Expression Analysis of EPSPS and BAR Families in Cotton.

Weeds seriously affect the yield and quality of crops. Because manual weeding is time-consuming and laborious, the use of herbicides becomes an effective way to solve the harm caused by weeds in fields. Both 5-enolpyruvyl shikimate-3-phosphate synthetase (EPSPS) and acetyltransferase genes (bialaphos resistance, BAR) are widely used to improve crop resistance to herbicides. However, cotton, as the most important natural fiber crop, is not tolerant to herbicides in China, and the EPSPS and BAR family genes have not yet been characterized in cotton. Therefore, we explore the genes of these two families to provide candidate genes for the study of herbicide resistance mechanisms. In this study, 8, 8, 4, and 5 EPSPS genes and 6, 6, 5, and 5 BAR genes were identified in allotetraploid Gossypium hirsutum and Gossypium barbadense, diploid Gossypium arboreum and Gossypium raimondii, respectively. Members of the EPSPS and BAR families were classified into three subgroups based on the distribution of phylogenetic trees, conserved motifs, and gene structures. In addition, the promoter sequences of EPSPS and BAR family members included growth and development, stress, and hormone-related cis-elements. Based on the expression analysis, the family members showed tissue-specific expression and differed significantly in response to abiotic stresses. Finally, qRT-PCR analysis revealed that the expression levels of GhEPSPS3, GhEPSPS4, and GhBAR1 were significantly upregulated after exogenous spraying of herbicides. Overall, we characterized the EPSPS and BAR gene families of cotton at the genome-wide level, which will provide a basis for further studying the functions of EPSPS and BAR genes during growth and development and herbicide stress.

Subchronic feeding study of glyphosate-tolerant maize GG2 with the gr79-epsps and gat genes in Wistar Han RCC rats.

The genetically modified (GM) maize GG2 contains gr79-epsps and gat genes, conferring glyphosate tolerance. The present study aimed to investigate potential effects of maize GG2 in a 90-day subchronic feeding study on Wistar Han RCC rats. Maize grains from GG2 or non-GM maize were incorporated into diets at concentrations of 25% and 50% and administered to Wistar Han RCC rats (n = 10/sex/group) for 90 days. The basal-diet group of rats (n = 10/sex/group) were fed with common commercialized rodent diet. Compared with rats fed with the corresponding non-GM maize and the basal-diet, no biologically relevant differences were observed in rats fed with the maize GG2, according to the results of body weight/gain, feed consumption/utilization, clinical signs, mortality, ophthalmology, clinical pathology (hematology, prothrombin time, urinalysis, serum chemistry), organ weights, and gross and microscopic pathology. Under the conditions of this study, these results indicated that maize GG2 is as safe as the non-GM maize in this 90-day feeding study.

Divergence in Glyphosate Susceptibility between Steinchisma laxum Populations Involves a Pro106Ser Mutation.

The characterization of the mechanisms conferring resistance to herbicides in weeds is essential for developing effective management programs. This study was focused on characterizing the resistance level and the main mechanisms that confer resistance to glyphosate in a resistant (R) Steinchisma laxum population collected in a Colombian rice field in 2020. The R population exhibited 11.2 times higher resistance compared to a susceptible (S) population. Non-target site resistance (NTSR) mechanisms that reduced absorption and impaired translocation and glyphosate metabolism were not involved in the resistance to glyphosate in the R population. Evaluating the target site resistance mechanisms by means of enzymatic activity assays and EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) gene sequencing, the mutation Pro106Ser was found in R plants of S. laxum. These findings are crucial for managing the spread of S. laxum resistance in Colombia. To effectively control S. laxum in the future, it is imperative that farmers use herbicides with different mechanisms of action in addition to glyphosate and adopt Integrate Management Programs to control weeds in rice fields of the central valleys of Colombia.

Functional characterization of novel mutations in the conserved region of EPSPS for herbicide resistance in pigeonpea: structure-based coherent design.

Prospectively, agroecosystems for the growth of crops provide the potential fertile, productive, and tropical environment which attracts infestation by weedy plant species that compete with the primary crop plants. Infestation by weed is a major biotic stress factor faced by pigeonpea that hampers the productivity of the crop. In the modern era with the development of chemicals the problem of weed infestation is dealt with armours called herbicides. The most widely utilized, post-emergent, broad-spectrum herbicide has an essential active ingredient called glyphosate. Glyphosate mechanistically inhibits a chloroplastic enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) by competitively interacting with the PEP binding site which hinders the shikimate pathway and the production of essential aromatic amino acids (Phe, Tyr, Trp) and other secondary metabolites in plants. Moreover, herbicide spray for weed management is lethal to both the primary crop and the weeds. Therefore, it is critical to develop herbicide-resistant crops for field purposes to reduce the associated yield and economic losses. In this study, the in-silico analysis drove the selection and validation of the point mutations in the conserved region of the EPSPS gene, which confers efficient herbicide resistance to mutated-CcEPSPS enzyme along with the retention of the normal enzyme function. An optimized in-silico validation of the target mutation before the development of the genome-edited resistant plant lines is a prerequisite for testing their efficacy as a proof of concept. We validated the combination of GATIPS mutation for its no-cost effect at the enzyme level via molecular dynamic (MD) simulation.Communicated by Ramaswamy H. Sarma.

HIGHLIGHTSWeed infestation is a major biotic stress factor and a consistent problem in agriculture.Development of glyphosate-resistant mutation is crucial to minimize the yield loss in agriculturally or nutritionally important crops for field application.Present in-silico approach is a proof-of-concept for validation of the selected glyphosate-resistant mutations.The current study has validated the combination of GATIPS mutation for its glyphosate-resistant phenotype and no negative cost effect at the enzyme simulation level.

Two electrochemiluminescence immunosensors for the sensitive and quantitative detection of the CP4-EPSPS protein in genetically modified crops.

Two different models of electrochemiluminescence (ECL) immunosensors for the sensitive and quantitative detection of the CP4-EPSPS protein in genetically modified (GM) crops were proposed in this study. One was a signal-reduced ECL immunosensor based on nitrogen-doped graphene, graphitic carbon nitride and polyamide-amine (GN-PAMAM-g-C3N4) composites as the electrochemically active substance. The other model was a signal-enhanced ECL immunosensor based on a GN-PAMAM modified electrode for the detection of CdSe/ZnS quantum dots (QDs)-labeled antigens. The ECL signal responses of the reduced and enhanced immunosensors linearly decreased as the increase of the soybean RRS and RRS-QDs content in the range of 0.05% to 1.5% and 0.025% to 1.0%, with the limits of detection of 0.03% and 0.01% (S/N = 3), respectively. Both of the ECL immunosensors showed good specificity, stability, accuracy, and reproducibility in the analysis of real samples. The results indicate that the two immunosensors provide an ultra-sensitive and quantitative approach for the determination of the CP4-EPSPS protein. Due to their outstanding performances, the two ECL immunosensors could be useful tools for achieving the effective regulation of GM crops.

Safety Assessment of the CP4 EPSPS and NPTII Proteins in Eucalyptus.

Glyphosate herbicide treatment is essential to sustainable Eucalyptus plantation management in Brazil. Eucalyptus is highly sensitive to glyphosate, and Suzano/FuturaGene has genetically modified eucalyptus to tolerate glyphosate, with the aim of both protecting eucalyptus trees from glyphosate application damage and improving weed management. This study presents the biosafety results of the glyphosate-tolerant eucalyptus event 751K032, which expresses the selection marker neomycin phosphotransferase II (NPTII) enzyme and CP4-EPSPS, a glyphosate-tolerant variant of plant 5-enolpyruvyl-shikimate-3-phosphate synthase enzyme. The transgenic genetically modified (GM) event 751K032 behaved in the plantations like conventional non-transgenic eucalyptus clone, FGN-K, and had no effects on arthropods and soil microorganisms. The engineered NPTII and CP4 EPSPS proteins were heat-labile, readily digestible, and according to the bioinformatics analyses, unlikely to cause an allergenic or toxic reaction in humans or animals. This assessment of the biosafety of the glyphosate-tolerant eucalyptus event 751K032 concludes that it is safe to be used for wood production.

Potent Antifungal Functions of a Living Modified Organism Protein, CP4-EPSPS, against Pathogenic Fungal Cells.

Various proteins introduced into living modified organism (LMO) crops function in plant defense mechanisms against target insect pests or herbicides. This study analyzed the antifungal effects of an introduced LMO protein, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from Agrobacterium sp. strain CP4 (CP4-EPSPS). Pure recombinant CP4-EPSPS protein, expressed in Escherichia coli, inhibited the growth of human and plant fungal pathogens (Candida albicans, C. tropicalis, C. krusei, Colletotrichum gloeosporioides, Fusarium solani, F. graminearum, and Trichoderma virens), at minimum inhibitory concentrations (MICs) that ranged from 62.5 to 250 µg/mL. It inhibited fungal spore germination as well as cell proliferation on C. gloeosporioides. Rhodamine-labeled CP4-EPSPS accumulated on the fungal cell wall and within intracellular cytosol. In addition, the protein induced uptake of SYTOX Green into cells, but not into intracellular mitochondrial reactive oxygen species (ROS), indicating that its antifungal action was due to inducing the permeability of the fungal cell wall. Its antifungal action showed cell surface damage, as observed from fungal cell morphology. This study provided information on the effects of the LMO protein, EPSPS, on fungal growth.

Extrachromosomal DNA-mediated glyphosate resistance in Italian ryegrass.

BACKGROUND: An Italian ryegrass population from Arkansas, USA developed glyphosate resistance due to 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene amplification. The plants in this population with approximately 70 EPSPS copies were used in the present study for the physical mapping of amplified copies of EPSPS gene to determine the possible mechanism of EPSPS gene amplification conferring glyphosate resistance in Italian ryegrass. RESULT: Fluorescence in situ hybridization (FISH) analysis of glyphosate resistant (GR) Italian ryegrass plants with approximately 70 EPSPS copies displayed EPSPS hybridization signals randomly on most of the metaphase chromosomes. Glyphosate susceptible (GS) Italian ryegrass plants with one EPSPS copy displayed single prominent EPSPS hybridization signal, which was co-localized with 5S rDNA locus along with few additional signals on the outside of chromosomes. Pulsed-field gel electrophoresis (PFGE) followed by DNA blot using EPSPS gene as a probe identified a prominent EPSPS hybridization around the 400 kb region in GR DNA samples, but not in GS DNA samples. CONCLUSION: We report the extrachromosomal DNA-mediated glyphosate resistance in Italian ryegrass. Physical mapping of amplified copies of EPSPS gene in Italian ryegrass by FISH gives us a clue that the amplified copies of EPSPS gene may be present in the extrachromosomal DNA elements. Further analysis by PFGE followed by DNA blotting revealed that the extrachromosomal DNA containing EPSPS is approximately 400 kb similar in size with that of eccDNA replicon in Amaranthus palmeri. © 2023 Society of Chemical Industry.

Yeast of Eden: microbial resistance to glyphosate from a yeast perspective.

First marketed as RoundUp, glyphosate is history's most popular herbicide because of its low acute toxicity to metazoans and broad-spectrum effectiveness across plant species. The development of glyphosate-resistant crops has led to increased glyphosate use and consequences from the use of glyphosate-based herbicides (GBH). Glyphosate has entered the food supply, spurred glyphosate-resistant weeds, and exposed non-target organisms to glyphosate. Glyphosate targets EPSPS/AroA/Aro1 (orthologs across plants, bacteria, and fungi), the rate-limiting step in the production of aromatic amino acids from the shikimate pathway. Metazoans lacking this pathway are spared from acute toxicity and acquire their aromatic amino acids from their diet. However, glyphosate resistance is increasing in non-target organisms. Mutations and natural genetic variation discovered in Saccharomyces cerevisiae illustrate similar types of glyphosate resistance mechanisms in fungi, plants, and bacteria, in addition to known resistance mechanisms such as mutations in Aro1 that block glyphosate binding (target-site resistance (TSR)) and mutations in efflux drug transporters non-target-site resistance (NTSR). Recently, genetic variation and mutations in an amino transporter affecting glyphosate resistance have uncovered potential off-target effects of glyphosate in fungi and bacteria. While glyphosate is a glycine analog, it is transported into cells using an aspartic/glutamic acid (D/E) transporter. The size, shape, and charge distribution of glyphosate closely resembles D/E, and, therefore, glyphosate is a D/E amino acid mimic. The mitochondria use D/E in several pathways and mRNA-encoding mitochondrial proteins are differentially expressed during glyphosate exposure. Mutants downstream of Aro1 are not only sensitive to glyphosate but also a broad range of other chemicals that cannot be rescued by exogenous supplementation of aromatic amino acids. Glyphosate also decreases the pH when unbuffered and many studies do not consider the differences in pH that affect toxicity and resistance mechanisms.

Unravelling the Phytotoxic Effects of Glyphosate on Sensitive and Resistant Amaranthus palmeri Populations by GC-MS and LC-MS Metabolic Profiling.

Glyphosate, the most successful herbicide in history, specifically inhibits the activity of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19), one of the key enzymes in the shikimate pathway. Amaranthus palmeri is a driver weed in agriculture today that has evolved glyphosate-resistance through increased EPSPS gene copy number and other mechanisms. Non-targeted GC-MS and LC-MS metabolomic profiling was conducted to examine the innate physiology and the glyphosate-induced perturbations in one sensitive and one resistant (by EPSPS amplification) population of A. palmeri. In the absence of glyphosate treatment, the metabolic profile of both populations was very similar. The comparison between the effects of sublethal and lethal doses on sensitive and resistant populations suggests that lethality of the herbicide is associated with an amino acid pool imbalance and accumulation of the metabolites of the shikimate pathway upstream from EPSPS. Ferulic acid and its derivatives were accumulated in treated plants of both populations, while quercetin and its derivative contents were only lower in the resistant plants treated with glyphosate.