Enzyme 5-enolpyruvylshikimate-3-phosphate Synthase Research Articles

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

Cysteine proteases are activated in sensitive Amaranthus palmeri populations upon treatment with herbicides inhibiting amino acid biosynthesis.

The herbicides glyphosate and pyrithiobac inhibit the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in the aromatic amino acid biosynthetic pathway and acetolactate synthase (ALS) in the branched-chain amino acid biosynthetic pathway, respectively. Here we characterise the protease activity profiles of a sensitive (S), a glyphosate-resistant (GR) and a multiple-resistant (MR) population of Amaranthus palmeri in response to glyphosate and pyrithiobac. Amino acid accumulation and cysteine protease activities were induced with both herbicides in the S population and with pyrithiobac in the GR population, suggesting that the increase in cysteine proteases is responsible for the increased degradation of the available proteins and the observed increase in free amino acids. Herbicides did not induce any changes in the proteolytic activities in the populations with target-site resistance, indicating that this effect was only induced in sensitive plants.

Effects of the herbicide glyphosate [n-(phosphonomethyl) glycine] on biodiversity and organisms in the soil

Glyphosate is an organophosphate herbicide manufactured by Monsanto, which eliminates annual and perennial weeds by inhibiting the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) involved in the production of aromatic amino acids in plants and microorganisms. As this herbicide is used extensively, there is a lot of research on its effect on plants, animals and microbes, and human health. Glyphosate contaminates different ecosystems by spray drift, volatilization, and erosion by wind of it adsorbed on soil particles. Soil and aquatic microbiota play a significant role in this process. This molecule is resistant to abiotic degradation. Degradation by microbes is important. The aim of this review is to provide a concise and comprehensive survey of certain relevant aspects related to its effect on the biodiversity in soil. The effect on human health is also discussed.

First report of glyphosate resistance in an Amaranthus palmeri population from Europe

AbstractThe invasive weed Amaranthus palmeri is spreading throughout Spain, with Catalonia being one of the most affected regions. For this species, acetolactate synthase‐inhibiting herbicide‐resistant populations have been reported, and now glyphosate resistance is also suspected. Glyphosate targets and inhibits the enzyme 5‐enolpyruvylshikimate‐3‐phosphate synthase (EPSPS), but A. palmeri has evolved different resistant mechanisms leading to plant survival. One of the most effective is the EPSPS overexpression due to copy number variation (CNV). Gene copies accumulate within the EPSPS cassette that is an extrachromosomal circular DNA displaying unique structural polymorphisms. This study aims to determine the response to glyphosate of a suspected resistant population collected from a roadside and investigate the resistance mechanism involved. The herbicide bioassay confirmed that 40% of the plants survived glyphosate applied at 540 g a.i. ha−1. No known mutations endowing glyphosate resistance were found at EPSPS amongst confirmed resistant plants, while in most of them (70%) specific molecular markers revealed the presence of the EPSPS cassette. All these results indicate that this population is glyphosate resistant and it is very likely that the EPSPS gene CNV is the main resistance mechanism. This is the first case of glyphosate resistance in A. palmeri in Europe whose introduction is likely due to importation of contaminated seed with glyphosate‐resistant Palmer amaranth from the Americas. This introduction poses a significant danger to summer crops in our continent.

Does Glyphosate Affect the Human Microbiota?

Glyphosate is the world’s most widely used agrochemical. Its use in agriculture and gardening has been proclaimed safe because humans and other animals do not have the target enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). However, increasing numbers of studies have demonstrated risks to humans and animals because the shikimate metabolic pathway is present in many microbes. Here, we assess the potential effect of glyphosate on healthy human microbiota. Our results demonstrate that more than one-half of human microbiome are intrinsically sensitive to glyphosate. However, further empirical studies are needed to determine the effect of glyphosate on healthy human microbiota.

Quantification of the Potential Impact of Glyphosate-Based Products on Microbiomes

Glyphosate-based products (GBP) are the most common broad-spectrum herbicides worldwide. The target of glyphosate is the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in the shikimate pathway, which is virtually universal in plants. The inhibition of the enzyme stops the production of three essential amino acids: phenylalanine, tyrosine, and tryptophan. EPSPS is also present in fungi and prokaryotes, such as archaea and bacteria; thus, the use of GBP may have an impact on the microbiome composition of soils, plants, herbivores, and secondary consumers. This article aims to present general guidelines to assess the effect of GBP on microbiomes from field experiments to bioinformatics analyses and provide a few testable hypotheses. Two field experiments are presented to test the GBP on non-target organisms. First, plant-associated microbes from 10 replicated control and GBP treatment plots simulating no-till cropping are sampled and analyzed. In the second experiment, samples from experimental plots fertilized by either poultry manure containing glyphosate residues or non-treated control manure were obtained. Bioinformatics analysis of EPSPS protein sequences is utilized to determine the potential sensitivity of microbes to glyphosate. The first step in estimating the effect of GBP on microbiomes is to determine their potential sensitivity to the target enzyme (EPSPS). Microbial sequences can be obtained either from public repositories or by means of PCR amplification. However, in the majority of field studies, microbiome composition has been determined based on universal DNA markers such as the 16S rRNA and the internal transcribed spacer (ITS). In these cases, sensitivity to glyphosate can only be estimated through a probabilistic analysis of EPSPS sequences using closely related species. The quantification of the potential sensitivity of organisms to glyphosate, based on the EPSPS enzyme, provides a robust approach for further experiments to study target and non-target resistant mechanisms.

Quantification of the Potential Impact of Glyphosate-Based Products on Microbiomes.

Glyphosate-based products (GBP) are the most common broad-spectrum herbicides worldwide. The target of glyphosate is the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in the shikimate pathway, which is virtually universal in plants. The inhibition of the enzyme stops the production of three essential amino acids: phenylalanine, tyrosine, and tryptophan. EPSPS is also present in fungi and prokaryotes, such as archaea and bacteria; thus, the use of GBP may have an impact on the microbiome composition of soils, plants, herbivores, and secondary consumers. This article aims to present general guidelines to assess the effect of GBP on microbiomes from field experiments to bioinformatics analyses and provide a few testable hypotheses. Two field experiments are presented to test the GBP on non-target organisms. First, plant-associated microbes from 10 replicated control and GBP treatment plots simulating no-till cropping are sampled and analyzed. In the second experiment, samples from experimental plots fertilized by either poultry manure containing glyphosate residues or non-treated control manure were obtained. Bioinformatics analysis of EPSPS protein sequences is utilized to determine the potential sensitivity of microbes to glyphosate. The first step in estimating the effect of GBP on microbiomes is to determine their potential sensitivity to the target enzyme (EPSPS). Microbial sequences can be obtained either from public repositories or by means of PCR amplification. However, in the majority of field studies, microbiome composition has been determined based on universal DNA markers such as the 16S rRNA and the internal transcribed spacer (ITS). In these cases, sensitivity to glyphosate can only be estimated through a probabilistic analysis of EPSPS sequences using closely related species. The quantification of the potential sensitivity of organisms to glyphosate, based on the EPSPS enzyme, provides a robust approach for further experiments to study target and non-target resistant mechanisms.

A Review on Impact of Glyphosate on Development of Cancer

Pesticides are a vast mixture of compounds used to control pests like plants, moulds, and insects. In agriculture, non-agricultural vegetation management, and crop desiccant harvesting aid, chemicals from every major functional family of pesticides, such as insecticides, herbicides, fungicides, and fumigants, were frequently used. Herbicides are one of the most effective tools for farmers to obtain optimal crop yields when used correctly. Glyphosate (N-(phosphonomethyl) glycine) is a broad-spectrum weed killer that is used all over the world in agriculture and forestry. Glyphosate's herbicidal activity in plants is to disrupt the shikimic acid pathway's generation of branched-chain amino acids by preventing the binding of phosphoenolpyruvate (PEP) to the enzyme 5-enolpyruvylshikimate 3-phosphate synthase. This causes a deficiency in aromatic amino acid synthesis and, as a result, weeds mortality. Glyphosate exposure through food, drinking water, wind, water erosion, and other environmental pathways has been linked to human health issues as a carcinogen, mutagen, and reproductive toxicity. Glyphosate has a wide range of tumorigenic effects in biological systems, and epidemiological evidence suggests that glyphosate use on crops is linked to a wide range of cancers, including liver cancer, breast cancer, thyroid cancer, pancreatic cancer, kidney cancer, bladder cancer, and myeloid cancer. The shikimate pathway enzymes, intermediates, and derivative amino acids, which have been associated to genotoxicity and carcinogenicity, are thought to have a role in most cancer pathologies. This review summarises glyphosate's function in cancer pathology, including the ability of the glyphosate circuit to induce cancer and implications for future therapeutic methods.

Transcriptomic analysis reveals the transcription factors involved in regulating the expression of EPSPS gene, which confers glyphosate resistance of goosegrass (Eleusine indica)

Glyphosate inhibits the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and overexpression of the EPSPS gene is one of the molecular mechanisms conferring glyphosate resistance in weeds. A regulatory sequence of EPSPS gene was isolated previously, and an alteration in its 5´-untranslated region (UTR) pyrimidine (Py)-rich stretch element is involved in the regulation of EPSPS expression in glyphosate-resistant (GR) Eleusine indica. However, the transcription factors involved in this regulatory sequence remain to be elucidated. In this study, we investigated the regulatory network of EPSPS overexpression associated genes in a GR E. indica population by RNA-seq. The differentially expressed transcript analyses revealed that glyphosate treatment caused an increase in the expression of 2752 unigenes and a decrease in the expression of 4025 unigenes in the GR E. indica, compared to the glyphosate-susceptible (GS) E. indica. Among them, 1373 unigenes were identified to be co-expressed with the EPSPS gene in GR E. indica. GO and KEGG pathway analyses showed that the up-regulated unigenes were mainly enriched in chloroplasts and associated with the shikimate biosynthesis pathway, chlorophy II and peroxisome metabolism processes. Notably, the expression of a Shikimate kinase which catalyzed the conversion of Shikimate to Shikimate 3-phosphate (S3P, a substrate of EPSPS), was also up-regulated. Eight transcription factors were identified as likely to be involved in the regulation of the EPSPS expression, and three of them (ARF2, ARF8 and BPC6) showed more binding sites because of a (CT)n insertion of the 5´-UTR Py-rich stretch element in GR. However, the yeast one-hybrid assay illustrated that ARF8 and BPC6 could bind to the 5´-UTR Py-rich stretch element of wild type EPSPS, but could not bind to the mutated form. Our data suggests that the transcriptional regulation of EPSPS expression is complex and was significantly altered in GR E. indica. These discoveries provide new references for further study of the EPSPS overexpression mechanism that endows glyphosate resistance.

Can Control of Glyphosate Susceptible and Resistant Conyza sumatrensis Populations Be Dependent on the Herbicide Formulation or Adjuvants?

In this work, we studied the effect of three glyphosate formulations (isopropylamine, ammonium and potassium salts) and two non-ionic adjuvants on the resistance response of two resistant (R1, R2) and one susceptible population of the highly invasive Asteraceae, Conyza sumatrensis, from Southern France vineyards. Only in R1, an amino acid substitution (Pro106Thr) was found in the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). The two adjuvants, in a similar fashion, significantly reduced GR50 values for every population and glyphosate formulation. Without adjuvants, glyphosate as potassium salt was the only formulation able to significantly reduce the GR50 values of every population. For every population, the two adjuvants improved, indistinguishably, leaf retention of the herbicidal solution and the potassium salt formulation led to the highest retention, both with and without the adjuvant added. Uptake responses paralleled those of retention and adjuvant addition was more effective in increasing foliar uptake of the lower performing formulations (isopropylamine and ammonium salts). The allocation pattern of glyphosate among plant compartments was only dependent on population, with R2 retaining most glyphosate in the treated leaf, clearly suggesting the occurrence of a Non-Target Site Resistance (NTSR) mechanism. Results indicate that control of weed populations possessing NTSR mechanisms of resistance to glyphosate may be improved through adequate selection of formulation and adjuvant use.

Increased Glyphosate-Induced Gene Expression in the Shikimate Pathway Is Abolished in the Presence of Aromatic Amino Acids and Mimicked by Shikimate.

The herbicide glyphosate inhibits the plant enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in the aromatic amino acid (AAA) biosynthetic pathway, also known as the shikimate pathway. Amaranthus palmeri is a fast-growing weed, and several populations have evolved resistance to glyphosate through increased EPSPS gene copy number. The main objective of this study was to elucidate the regulation of the shikimate pathway and determine whether the regulatory mechanisms of glyphosate-sensitive and glyphosate-resistant plants were different. Leaf disks of sensitive and resistant (due to EPSPS gene amplification) A. palmeri plants were incubated for 24 h with glyphosate, AAA, glyphosate + AAA, or several intermediates of the pathway: shikimate, quinate, chorismate and anthranilate. In the sensitive population, glyphosate induced shikimate accumulation and induced the gene expression of the shikimate pathway. While AAA alone did not elicit any change, AAA applied with glyphosate abolished the effects of the herbicide on gene expression. It was not possible to fully mimic the effect of glyphosate by incubation with any of the intermediates, but shikimate was the intermediate that induced the highest increase (three-fold) in the expression level of the genes of the shikimate pathway of the sensitive population. These results suggest that, in this population, the lack of end products (AAA) of the shikimate pathway and shikimate accumulation would be the signals inducing gene expression in the AAA pathway after glyphosate application. In general, the effects on gene expression detected after the application of the intermediates were more severe in the sensitive population than in the resistant population. These results suggest that when EPSPS is overexpressed, as in the resistant population, the regulatory mechanisms of the AAA pathway are disrupted or buffered. The mechanisms underlying this behavior remain to be elucidated.

New Case of False-Star-Grass (Chloris distichophylla) Population Evolving Glyphosate Resistance

Chloris distichophylla, suspected of glyphosate resistance (GR), was collected from areas of soybean cultivation in Rio Grande do Sul, Brazil. A comparison was made with a susceptible population (GS) to evaluate the resistance level, mechanisms involved, and control alternatives. Glyphosate doses required to reduce the dry weight (GR50) or cause a mortality rate of 50% (LD50) were around 5.1–3 times greater in the GR population than in the GS population. The shikimic acid accumulation was around 6.2-fold greater in GS plants than in GR plants. No metabolized glyphosate was found in either GR or GS plants. Both populations did not differ in the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) basal activity or in vitro inhibition of EPSPS activity by glyphosate (I50). The maximum glyphosate absorption was observed at 96 hours after treatment (HAT), which was twofold higher in the GS plants than in the GR plants. This confirms the first case of glyphosate resistance in C. distichophylla. In addition, at 96 HAT, the GS plants translocated more 14C-glyphosate than the GR ones. The best options for the chemical control of both C. distichophylla populations were clethodim, quizalofop, paraquat, glufosinate, tembotrione, diuron, and atrazine. The first case of glyphosate resistance in C. distichophylla was due to impaired uptake and translocation. Chemical control using multiple herbicides with different modes of action (MOA) could be a tool used for integrated weed management (IWM) programs.

Preliminary Environmental Risk Assessment of Genetically Modified Oilseed Rape MON 88302 for Food and Feed Uses, Import and Processing under Regulation (EC) No 1829/2003 (Application EFSA/GMO/BE/2011/101)

Preliminary Environmental Risk Assessment of Genetically Modified Oilseed Rape MON 88302 for Food and Feed Uses, Import and Processing under Regulation (EC) No 1829/2003 (Application EFSA/GMO/BE/2011/101)

Molecular cloning and metabolomic characterization of the 5-enolpyruvylshikimate-3-phosphate synthase gene from Baphicacanthus cusia

BackgroundIndigo alkaloids, such as indigo, indirubin and its derivatives, have been identified as effective antiviral compounds in Baphicacanthus cusia. Evidence suggests that the biosynthesis of indigo alkaloids in plants occurs via the shikimate pathway. The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is involved in plant metabolism; however, its underlying putative mechanism of regulating the production of indigo alkaloids is currently unknown.ResultsOne gene encoding EPSPS was isolated from B. cusia. Quantitative real-time PCR analysis revealed that BcEPSPS was expressed at the highest level in the stem and upregulated by methyl jasmonate (MeJA), salicylic acid (SA) and abscisic acid (ABA) treatment. The results of subcellular localization indicated that BcEPSPS is mainly expressed in both the plastids and cytosol, which has not been previously reported. An enzyme assay revealed that the heterogeneously expressed BcEPSPS protein catalysed the generation of 5-enolpyruvyl shikimate-3-phosphate. The overexpression of BcEPSPS in Isatis indigotica hairy roots resulted in the high accumulation of indigo alkaloids, such as indigo, secologanin, indole and isorhamnetin.ConclusionsThe function of BcEPSPS in catalysing the production of EPSP and regulating indigo alkaloid biosynthesis was revealed, which provided a distinct view of plant metabolic engineering. Our findings have practical implications for understanding the effect of BcEPSPS on active compound biosynthesis in B. cusia.

Surface Functionalization by Hydrophobin-EPSPS Fusion Protein Allows for the Fast and Simple Detection of Glyphosate.

Glyphosate, the most widely used pesticide worldwide, is under debate due to its potentially cancerogenic effects and harmful influence on biodiversity and environment. Therefore, the detection of glyphosate in water, food or environmental probes is of high interest. Currently detection of glyphosate usually requires specialized, costly instruments, is labor intensive and time consuming. Here we present a fast and simple method to detect glyphosate in the nanomolar range based on the surface immobilization of glyphosate’s target enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) via fusion to the hydrophobin Ccg2 and determination of enzyme activity with a malachite green assay, which is a common photometric technique to measure inorganic phosphate (Pi). The assay demonstrates a new approach for a fast and simple detection of pesticides.

Do plants pay a fitness cost to be resistant to glyphosate?

We reviewed the literature to understand the effects of glyphosate resistance on plant fitness at the molecular, biochemical and physiological levels. A number of correlations between enzyme characteristics and glyphosate resistance imply the existence of a plant fitness cost associated with resistance-conferring mutations in the glyphosate target enzyme, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). These biochemical changes result in a tradeoff between the glyphosate resistance of the EPSPS enzyme and its catalytic activity. Mutations that endow the highest resistance are more likely to decrease catalytic activity by reducing the affinity of EPSPS for its natural substrate, and/or slowing the velocity of the enzyme reaction, and are thus very likely to endow a substantial plant fitness cost. Prediction of fitness costs associated with EPSPS gene amplification and overexpression can be more problematic. The validity of cost prediction based on the theory of evolution of gene expression and resource allocation has been cast into doubt by contradictory experimental evidence. Further research providing insights into the role of the EPSPS cassette in weed adaptation, and estimations of the energy budget involved in EPSPS amplification and overexpression are required to understand and predict the biochemical and physiological bases of the fitness cost of glyphosate resistance.

Improvement of Glyphosate Resistance through Concurrent Mutations in Three Amino Acids of the Pantoea sp. 5-Enolpyruvylshikimate-3-Phosphate Synthase.

Glyphosate inhibits the target enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in the shikimate pathway. A mutant of EPSPS from Pantoea sp. was identified using site-directed mutagenesis (SDM). The mutant significantly improved glyphosate resistance. The mutant had mutations in three amino acids: Gly97 to Ala, Thr 98 to Ile and Pro 102 to Ser. These mutation sites in E.coli have been studied as significant active sites of glyphosate resistance. However, in our research they were found to jointly contribute to the improvement of glyphosate tolerance. In addition, the level of glyphosate tolerance in transgenic Arabidopsis confirmed the potentiality of the mutant in breeding glyphosate-resistant plants.

Alterations in the 5' untranslated region of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene influence EPSPS overexpression in glyphosate-resistant Eleusine indica.

The herbicide glyphosate inhibits the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Overexpression of the EPSPS gene is one of the molecular mechanisms conferring glyphosate resistance in weeds, but the transcriptional regulation of this gene is poorly understood. The EPSPS gene was found to be significantly up-regulated following glyphosate treatment in a glyphosate-resistant Eleusine indica population from southern China. To further investigate the regulation of EPSPS overexpression, the promoter of the EPSPS gene from this E. indica population was cloned and analyzed. Two upstream regulatory sequences, Epro-S (862 bp) and Epro-R (877 bp), of EPSPS were obtained from glyphosate-susceptible (S) and -resistant (R) E. indica plants, respectively, by high-efficiency thermal asymmetric interlaced polymerase chain reaction (HiTAIL-PCR). The Epro-S and Epro-R sequences were 99% homologous, except for two insertions (3 and12 bp) in the R sequence. The 12-base insertion in the Epro-R sequence was located in the 5' untranslated region (UTR) pyrimidine nucleotide-rich (Py-rich) stretch element. Promoter activity tests showed that the 12-base insertion resulted in significant enhancement of Epro-R promoter activity, whereas the 3-base insertion had little effect on Epro-R promoter activity. Alterations in the 5' UTR Py-rich stretch element of EPSPS are responsible for glyphosate-induced EPSPS overexpression. Thus, EPSPS transcriptional regulation confers glyphosate resistance in this E. indica population. © 2018 Society of Chemical Industry.

An intragenic approach to confer glyphosate resistance in chile (Capsicum annuum) by introducing an in vitro mutagenized chile EPSPS gene encoding for a glyphosate resistant EPSPS protein.

Chile pepper (Capsicum annuum) is an important high valued crop worldwide, and when grown on a large scale has problems with weeds. One important herbicide used is glyphosate. Glyphosate inactivates the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), a key enzyme in the synthesis of aromatic amino acids. A transgenic approach towards making glyphosate resistant plants, entails introducing copies of a gene encoding for glyphosate-resistant EPSPS enzyme into the plant. The main objective of our work was to use an intragenic approach to confer resistance to glyphosate in chile which would require using only chile genes for transformation including the selectable marker. Tobacco was used as the transgenic system to identify different gene constructs that would allow for the development of the intragenic system for chile, since chile transformation is inefficient. An EPSPS gene was isolated from chile and mutagenized to introduce substitutions that are known to make the encoded enzyme resistant to glyphosate. The promoter for EPSPS gene was isolated from chile and the mutagenized chile EPSPS cDNA was engineered behind both the CaMV35S promoter and the EPSPS promoter. The leaves from the transformants were checked for resistance to glyphosate using a cut leaf assay. In tobacco, though both gene constructs exhibited some degree of resistance to glyphosate, the construct with the CaMV35S promoter was more effective and as such chile was transformed with this gene construct. The chile transformants showed resistance to low concentrations of glyphosate. Furthermore, preliminary studies showed that the mutated EPSPS gene driven by the CaMV35S promoter could be used as a selectable marker for transformation. We have shown that an intragenic approach can be used to confer glyphosate-resistance in chile. However, we need a stronger chile promoter and a mutated chile gene that encodes for a more glyphosate resistant EPSPS protein.

Protein identification before and after glyphosate exposure in Lolium multiflorum genotypes.

Weeds reduce crop yields, and among the methods used to control these plants, the use of chemicals is preferred. However, the repeated application of herbicides with the same mechanism of action selects for resistant populations. The aim of this study was to evaluate glyphosate resistance in Lolium multiflorum (Lam.) and relate the resistance to protein expression in the absence and presence of the herbicide using a metabolic-proteomic approach. Glyphosate resistance was confirmed, with a sevenfold difference in resistance between susceptible and resistant genotypes. Among the possible mechanisms affecting resistance, mutations in the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), herbicide differential translocation and overexpression of EPSPS are suggested. Susceptible plants had higher growth than did resistant plants in the absence of the herbicide, in addition to greater expression of protein groups related to photosynthesis and to tolerance to biotic and abiotic stresses. With application of glyphosate, resistant plants maintained their metabolism and began to express EPSPS and other candidate proteins related to herbicide resistance. In the absence of glyphosate, the susceptible plants would replace the resistant plants over time, and abiotic or biotic stresses would accelerate this process. Resistance in plants resulted from a combination of target-site and non-target-site resistance mechanisms. We identified several candidate proteins that could be investigated in future studies on glyphosate resistance. © 2017 Society of Chemical Industry.