Absence Of Glyphosate Research Articles

Genetically engineered rice endogenous 5-enolpyruvoylshikimate-3-phosphate synthase (epsps) transgene alters phenology and fitness of crop-wild hybrid offspring

Genetically engineered (GE) rice endogenous epsps (5-enolpyruvoylshikimate-3-phosphate synthase) gene overexpressing EPSPS can increase glyphosate herbicide-resistance of cultivated rice. This type of epsps transgene can enhance the fecundity of rice crop-weed hybrid offspring in the absence of glyphosate, stimulating great concerns over undesired environmental impacts of transgene flow to populations of wild relatives. Here, we report the substantial alteration of phenology and fitness traits in F1-F3 crop-wild hybrid descendants derived from crosses between an epsps GE rice line and two endangered wild rice (Oryza rufipogon) populations, based on the common-garden field experiments. Under the glyphosate-free condition, transgenic hybrid lineages showed significantly earlier tillering and flowering, as well as increased fecundity and overwintering survival/regeneration abilities. In addition, a negative correlation was observed between the contents of endogenous EPSPS of wild, weedy, and cultivated rice parents and fitness differences caused by the incorporation of the epsps transgene. Namely, a lower level of endogenous EPSPS in the transgene-recipient populations displayed a more pronounced enhancement in fitness. The altered phenology and enhanced fitness of crop-wild hybrid offspring by the epsps transgene may cause unwanted environmental consequences when this type of glyphosate-resistance transgene introgressed into wild rice populations through gene flow.

Effects of over-expressing a native gene encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) on glyphosate resistance in Arabidopsis thaliana.

Widespread overuse of the herbicide glyphosate, the active ingredient in RoundUp®, has led to the evolution of glyphosate-resistant weed biotypes, some of which persist by overproducing the herbicide’s target enzyme, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). EPSPS is a key enzyme in the shikimic acid pathway for biosynthesis of aromatic amino acids, lignin, and defensive compounds, but little is known about how overproducing EPSPS affects downstream metabolites, growth, or lifetime fitness in the absence of glyphosate. We are using Arabidopsis as a model system for investigating phenotypic effects of overproducing EPSPS, thereby avoiding confounding effects of genetic background or other mechanisms of herbicide resistance in agricultural weeds. Here, we report results from the first stage of this project. We designed a binary vector expressing a native EPSPS gene from Arabidopsis under control of the CaMV35S promoter (labelled OX, for over-expression). For both OX and the empty vector (labelled EV), we obtained nine independent T3 lines. Subsets of these lines were used to characterize glyphosate resistance in greenhouse experiments. Seven of the nine OX lines exhibited enhanced glyphosate resistance when compared to EV and wild-type control lines, and one of these was discarded due to severe deformities. The remaining six OX lines exhibited enhanced EPSPS gene expression and glyphosate resistance compared to controls. Glyphosate resistance was correlated with the degree of EPSPS over-expression for both vegetative and flowering plants, indicating that glyphosate resistance can be used as a surrogate for EPSPS expression levels in this system. These findings set the stage for examination of the effects of EPSPS over-expression on fitness-related traits in the absence of glyphosate. We invite other investigators to contact us if they wish to study gene expression, downstream metabolic effects, and other questions with these particular lines.

Interactive effects of temperature and glyphosate on the behavior of blue ridge two-lined salamanders (Eurycea wilderae).

The objective of the present study was to evaluate the potential interactive effects of stream temperatures and environmentally relevant glyphosate-based herbicide concentrations on movement and antipredator behaviors of larval Eurycea wilderae (Blue Ridge two-lined salamander). Larval salamanders were exposed to 1 of 4 environmentally relevant glyphosate concentrations (0.00 µg acid equivalent [a.e.]/L, 0.73 µg a.e./L, 1.46 µg a.e./L, and 2.92 µg a.e./L) at either ambient (12 °C) or elevated (23 °C) water temperature. Behaviors observed included the exploration of a novel habitat, use of refuge, habitat selection relative to a potential predator, and burst movement distance. In the absence of glyphosate, temperature consistently affected movement and refuge-use behavior, with individuals moving longer distances more frequently and using refuge less at warm temperatures; however, when glyphosate was added, the authors observed inconsistent effects of temperature that may have resulted from differential toxicity at various temperatures. Larval salamanders made shorter, more frequent movements and demonstrated reduced burst distance at higher glyphosate concentrations. The authors also found that lower glyphosate concentrations sometimes had stronger effects than higher concentrations (i.e., nonmonotonic dose responses), suggesting that standard safety tests conducted only at higher glyphosate concentrations might overlook important sublethal effects on salamander behavior. These data demonstrate that sublethal effects of glyphosate-based herbicides on natural behaviors of amphibians can occur with short-term exposure to environmentally relevant concentrations. Environ Toxicol Chem 2016;35:2297-2303. © 2016 SETAC.

Control of Johnsongrass in the Absence of Glyphosate in Midsouth Cotton Production Systems

Johnsongrass populations that are resistant to 5-enolpyruvyl-3-shikimate synthase (EPSPS)–, acetyl coenzyme A carboxylase (ACCase)–, or acetolactate synthase (ALS)–inhibiting herbicides are increasingly common throughout the midsouth. Three trials were conducted in 2012, 2013, and 2014 in Fayetteville, AR and Alexandria, LA to evaluate strategies with and without ALS- and ACCase inhibitors for management of rhizomatous johnsongrass in the absence of glyphosate. Fluometuron or fluometuron plus pyrithiobac applied PRE followed by (fb) EPOST, MPOST, and LAYBY tank mixtures containing multiple effective mechanisms of action (MOA) controlled johnsongrass at least 90%. Simplifying the program by removing a herbicide or eliminating an application timing reduced control, and increased vegetative and sexual reproduction of johnsongrass. To manage severe infestations or escapes glufosinate plus clethodim fb glufosinate plus clethodim or clethodim plus pyrithiobac fb clethodim) effectively controlled 15-cm johnsongrass. However, johnsongrass control was reduced when ALS and ACCase inhibitors were tank mixed, especially for the second POST application, compared to ACCase inhibitors alone. Effective herbicide programs are available to growers to control johnsongrass in the absence of glyphosate, but the use of PRE herbicides followed by multiple applications of POST herbicides is critical for successful management.

Growth and Reproduction of Glyphosate-Resistant and Susceptible Populations of Kochia scoparia.

Evolution of glyphosate-resistant kochia is a threat to no-till wheat-fallow and glyphosate-resistant (GR) cropping systems of the US Great Plains. The EPSPS (5-enol-pyruvylshikimate-3-phosphate synthase) gene amplification confers glyphosate resistance in the tested Kochia scoparia (L.) Schrad populations from Montana. Experiments were conducted in spring to fall 2014 (run 1) and summer 2014 to spring 2015 (run 2) to investigate the growth and reproductive traits of the GR vs. glyphosate-susceptible (SUS) populations of K. scoparia and to determine the relationship of EPSPS gene amplification with the level of glyphosate resistance. GR K. scoparia inbred lines (CHES01 and JOP01) exhibited 2 to 14 relative copies of the EPSPS gene compared with the SUS inbred line with only one copy. In the absence of glyphosate, no differences in growth and reproductive parameters were evident between the tested GR and SUS inbred lines, across an intraspecific competition gradient (1 to 170 plants m-2). GR K. scoparia plants with 2 to 4 copies of the EPSPS gene survived the field-use rate (870 g ha-1) of glyphosate, but failed to survive the 4,350 g ha-1 rate of glyphosate (five-times the field-use rate). In contrast, GR plants with 5 to 14 EPSPS gene copies survived the 4,350 g ha-1 of glyphosate. The results from this research indicate that GR K. scoparia with 5 or more EPSPS gene copies will most likely persist in field populations, irrespective of glyphosate selection pressure.

Glyphosate effects on soil rhizosphere-associated bacterial communities

Glyphosate is one of the most widely used herbicides in agriculture with predictions that 1.35millionmetric tons will be used annually by 2017. With the advent of glyphosate tolerant (GT) cropping more than 10years ago, there is now concern for non-target effects on soil microbial communities that has potential to negatively affect soil functions, plant health, and crop productivity. Although extensive research has been done on short-term response to glyphosate, relatively little information is available on long-term effects. Therefore, the overall objective was to investigate shifts in the rhizosphere bacterial community following long-term glyphosate application on GT corn and soybean in the greenhouse. In this study, rhizosphere soil was sampled from rhizoboxes following 4 growth periods, and bacterial community composition was compared between glyphosate treated and untreated rhizospheres using next-generation barcoded sequencing. In the presence or absence of glyphosate, corn and soybean rhizospheres were dominated by members of the phyla Proteobacteria, Acidobacteria, and Actinobacteria. Proteobacteria (particularly gammaproteobacteria) increased in relative abundance for both crops following glyphosate exposure, and the relative abundance of Acidobacteria decreased in response to glyphosate exposure. Given that some members of the Acidobacteria are involved in biogeochemical processes, a decrease in their abundance could lead to significant changes in nutrient status of the rhizosphere. Our results also highlight the need for applying culture-independent approaches in studying the effects of pesticides on the soil and rhizosphere microbial community.

Noncompetitive Growth and Fecundity of Wisconsin Giant Ragweed Resistant to Glyphosate

Glyphosate-resistant giant ragweed has been confirmed in several Midwestern states. In some cases, weed resistance to glyphosate has been shown to carry a fitness penalty. Previous research has found that a glyphosate-resistant giant ragweed biotype from Indiana with a rapid necrosis response to glyphosate displayed early, rapid growth in the absence of glyphosate, flowered earlier, but produced 25% less seed than a sensitive biotype, suggesting that there may be a fitness penalty associated with the rapid necrosis resistance trait. In Wisconsin, we have recently identified a giant ragweed accession with a 6.5-fold level of resistance to glyphosate that does not demonstrate the rapid necrosis response. Our objective was to determine the noncompetitive growth and fecundity of the resistant accession in the absence of glyphosate, relative to a sensitive accession from a nearby field border population. In greenhouse experiments, plant height, leaf area, and dry shoot biomass were similar between the resistant and sensitive accessions during vegetative growth to the onset of flowering. The instantaneous relative growth rate, instantaneous net assimilation rate, and instantaneous leaf area ratio also did not differ between accessions. However, fecundity of resistant plants (812 seeds plant−1) was greater (P = 0.008) than sensitive plants (425 seeds plant−1). The percentage of intact viable seeds, intact nonviable seeds, and empty involucres did not differ between resistant and sensitive accessions. These results indicate that resistance of this accession of giant ragweed to glyphosate has not affected its growth and development relative to a sensitive accession. The greater fecundity and similar viability of resistant plants relative to sensitive plants suggests that in the absence of selection by glyphosate, the frequency of the resistance trait for glyphosate may increase in the giant ragweed field population over time.

Morpho-Physiological Characterization of Glyphosate-Resistant and -Susceptible Horseweed (Conyza canadensis) Biotypes of US Midsouth

Horseweed is traditionally considered a non-cropland weed. However, populations resistant to glyphosate have eventually become established in no-till agronomic cropping systems. Growth chamber and greenhouse experiments were conducted to compare selected biological and physi- ological parameters of glyphosate-resistant (GR) and -susceptible (GS) horseweed biotypes from Mississippi with a broader goal of fitness characterization in these biotypes. Vegetative growth parameters (number of leaves, rosette diameter and area, shoot and root fresh weights) were recorded weekly from 5 to 11 wk after emergence and reproductive attributes (days to bolting (production of a flowering stalk) and flowering) and senescence were measured for both GR and GS biotypes under high (24˚C/20˚C) and low (18˚C/12˚C) temperature regimes, both with a 13-h light period. Physiological traits such as net photosynthesis, phenolic content, and cell membrane thermostability, all in the presence and absence of glyphosate, and leaf content of divalent cations such as Ca 2+ and Mg 2+ were assayed in the two biotypes under the high temperature regime. All horseweed vegetative growth parameters except root fresh weight were higher in the high tem- perature regime compared to that in low temperature regime in both biotypes. Number of leaves, rosette diameter and area, shoot and root fresh weight were 40 vs. 35, 9.3 vs. 8.7 cm, 51 vs. 43 cm 2 , 3.7 vs. 3.2 g, and 3.5 vs. 4.2 g under high and low temperature conditions, respectively, when av- * Corresponding author.

Inhibition effect of glyphosate on the acute and subacute toxicity of cadmium to earthworm Eisenia fetida.

The acute and subacute toxicities of cadmium (Cd) to earthworm Eisenia fetida in the presence and absence of glyphosate were studied. Although Cd is highly toxic to E. fetida, the presence of glyphosate markedly reduced the acute toxicity of Cd to earthworm; both the mortality rate of the earthworms and the accumulation of Cd decreased with the increase of the glyphosate/Cd molar ratio. The subcellular distribution of Cd in E. fetida tissues showed that internal Cd was dominant in the intact cells fraction and the heat-stable proteins fraction. The presence of glyphosate reduced the concentration of Cd in all fractions, especially the intact cells. During a longer period of exposure, the weight loss of earthworm and the total Cd absorption was alleviated by glyphosate. Thus, the herbicide glyphosate can reduce the toxicity and bioavailability of Cd in the soil ecosystems at both short- and long-term exposures.

Occurrence of glyphosate in water bodies derived from intensive agriculture in a tropical region of southern Mexico.

Glyphosate is an agrochemical widely used to control weeds. However, glyphosate spreads to water bodies by spray-drift, run-off and leaching, potentially causing detrimental effects on non-target biota. There is no information on the occurrence of this herbicide in water bodies near crop fields in Mexico, although it is the most commonly used pesticide in this country. To fill this gap, we quantified glyphosate in water bodies from twenty-three locations, including natural protected areas and agricultural areas in southern Mexico, during the dry and the rainy seasons. We expected (1) higher concentrations during the dry season due to reduced dilution by precipitation and, (2) absence of glyphosate in the protected areas. In agreement with our expectation, concentration of glyphosate was higher during the dry season (up to 36.7 μg/L). Nonetheless, glyphosate was detected in all samples-including natural protected areas. These results emphasize the need for an evaluation of the impact of glyphosate on native species as well as regulate its use.

Comment on ‘A novel 5‐enolpyruvoylshikimate‐3‐phosphate (EPSP) synthase transgene for glyphosate resistance stimulates growth and fecundity in weedy rice (Oryza sativa) without herbicide’ by Wang et al. (2014)

Comment on ‘A novel 5‐enolpyruvoylshikimate‐3‐phosphate (EPSP) synthase transgene for glyphosate resistance stimulates growth and fecundity in weedy rice (<i><scp>O</scp>ryza sativa</i>) without herbicide’ by Wang <i>et al</i>. (2014)

Impact of Genetic Background in Fitness Cost Studies: An Example from Glyphosate-Resistant Palmer Amaranth

Since its discovery in 2005, glyphosate-resistant Palmer amaranth has become a major problem for many farmers in the southern United States. One mechanism of resistance found in a Georgia population of glyphosate-resistant Palmer amaranth is amplification of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene throughout the genome, with some resistant plants containing and expressing more than 100EPSPSgenes. Such high numbers ofEPSPSgenes and protein production could result in a fitness cost to resistant plants due to (1) metabolic cost of overproduction of this enzyme and (2) disruption of other genes after insertion of theEPSPSgene. A greenhouse experiment was set up to investigate differences in growth and reproduction between glyphosate-susceptible and -resistant Palmer amaranth plants. Measurements included growth rate, plant height/volume ratio, final biomass, photosynthetic rate, inflorescence length, pollen viability, and seed set. This study found no significant fitness costs for plants with the resistance trait. This study also provided a clear example of how controlling for genetic background is important in fitness cost studies and how potentially misleading results can be obtained if only a few fitness traits are measured. These results indicate that glyphosate-resistant Palmer amaranth plants with highEPSPSgene copy numbers are likely to persist in field populations, even in the absence of glyphosate, potentially leading to long-term loss of glyphosate as a control option for Palmer amaranth.

No fitness cost of glyphosate resistance endowed by massive EPSPS gene amplification in Amaranthus palmeri

Amplification of the EPSPS gene has been previously identified as the glyphosate resistance mechanism in many populations of Amaranthus palmeri, a major weed pest in US agriculture. Here, we evaluate the effects of EPSPS gene amplification on both the level of glyphosate resistance and fitness cost of resistance. A. palmeri individuals resistant to glyphosate by expressing a wide range of EPSPS gene copy numbers were evaluated under competitive conditions in the presence or absence of glyphosate. Survival rates to glyphosate and fitness traits of plants under intra-specific competition were assessed. Plants with higher amplification of the EPSPS gene (53-fold) showed high levels of glyphosate resistance, whereas less amplification of the EPSPS gene (21-fold) endowed a lower level of glyphosate resistance. Without glyphosate but under competitive conditions, plants exhibiting up to 76-fold EPSPS gene amplification exhibited similar height, and biomass allocation to vegetative and reproductive organs, compared to glyphosate susceptible A. palmeri plants with no amplification of the EPSPS gene. Both the additive effects of EPSPS gene amplification on the level of glyphosate resistance and the lack of associated fitness costs are key factors contributing to EPSPS gene amplification as a widespread and important glyphosate resistance mechanism likely to become much more evident in weed plant species.

Inhibition Mechanisms of Zn Precipitation on Aluminum Oxide by Glyphosate: A 31P NMR and Zn EXAFS Study

In this research, the effects of glyphosate (GPS) on Zn sorption/precipitation on γ-alumina were investigated using a batch technique, Zn K-edge EXAFS, and (31)P NMR spectroscopy. The EXAFS analysis revealed that, in the absence of glyphosate, Zn adsorbed on the aluminum oxide surface mainly as bidentate mononuclear surface complexes at pH 5.5, whereas Zn-Al layered double hydroxide (LDH) precipitates formed at pH 8.0. In the presence of glyphosate, the EXAFS spectra of Zn sorption samples at pH 5.5 and 8.0 were very similar, both of which demonstrated that Zn did not directly bind to the mineral surface but bonded with the carboxyl group of glyphosate. Formation of γ-alumina-GPS-Zn ternary surface complexes was further suggested by (31)P solid state NMR data which indicated the glyphosate binds to γ-alumina via a phosphonate group, bridging the mineral surface and Zn. Additionally, we showed the sequence of additional glyphosate and Zn can influence the sorption mechanism. At pH 8, Zn-Al LDH precipitates formed if Zn was added first, and no precipitates formed if glyphosate was added first or simultaneously with Zn. In contrast, at pH 5.5, only γ-alumina-GPS-Zn ternary surface complexes formed regardless of whether glyphosate or Zn was added first or both were added simultaneously.

In Vitro Cytotoxic Effect of Glyphosate Mixture Containing Surfactants

We investigated whether glyphosate influences the cellular toxicity of the surfactants TN-20 and LN-10 on the mouse fibroblast-like cells, alveolar epithelial cells, and a heart cell line. The cytotoxicity of TN-20 and LN-10 (0.4-100 µM), in the presence or absence of glyphosate was determined by assessing membrane integrity. TN-20 toxicity was significantly lower in the presence of 50 µM glyphosate for the fibroblast-like cell (6.25 µM; 3.9% ± 3.4% vs -4.8% ± 0.7%), for the alveolar cells (0.78 µM; 5.7% ± 0.9% vs 0.1% ± 0.6%), and for the heart cell line (25.0 µM; 7.9% ± 3.0% vs 19.4% ± 0.7%) compared to that of TN-20 alone. The cellular toxicity of LN-10 towards the fibroblast-like cells was found to be increased in the presence of 50 µM glyphosate when LN-10 concentrations of 50 µM (31.3% ± 3.9% vs 19.2% ± 0.9%) and 100 µM (62.1% ± 3.4% vs 39.0% ± 0.7%) were compared to that of LN-10 alone. These results suggest that the mixture toxicity may be a factor in glyphosate-surfactant toxicity in patients with acute glyphosate herbicide intoxication.

Fate of Glyphosate-Resistant Giant Ragweed (Ambrosia trifida) in the Presence and Absence of Glyphosate

Glyphosate-resistant giant ragweed has become an increasing problem, and the potential spread of these biotypes is a threat to production agriculture and to the long-term utility of glyphosate and glyphosate-resistant crops. The fate of glyphosate resistance in a giant ragweed population is dependent on the fitness of the resistant biotype. Our objective was to determine the fitness of glyphosate-resistant giant ragweed in the absence and presence of glyphosate. We compared the growth and seed production of glyphosate-resistant (GR) and glyphosate-susceptible (GS) giant ragweed under field conditions in the absence of glyphosate. A greenhouse study was also conducted to determine the survival of GR and GS giant ragweed and their open-pollinated progeny from the field study under glyphosate-induced selection pressure. In the absence of glyphosate, GR giant ragweed displayed rapid, early season growth, but 50 d after planting, the biotypes were similar in height, shoot weight, and leaf area. During reproduction, the GR biotype flowered earlier and produced 25% less seed than the GS biotype. In the presence of glyphosate, an outcrossing rate of 31% was detected between GR and GS biotypes because 61% of progeny were resistant to glyphosate at 840 g ae ha−1. A second application 14 d later at 2,520 g ae ha−1completely removed the GS alleles from the population, whereas several homozygous and heterozygous GR plants survived and produced seed. These results indicate GR will persist in the population when subjected to glyphosate and that glyphosate resistance in giant ragweed has the potential to spread rapidly in our current agricultural ecosystem.

Electrochemical detection of glyphosate herbicide using horseradish peroxidase immobilized on sulfonated polymer matrix

This paper describes the use of horseradish peroxidase (HRP) based biosensor for novel detection of glyphosate herbicide. The biosensor was prepared by electrochemically depositing poly(2,5-dimethoxyaniline) (PDMA) doped with poly(4-styrenesulfonic acid) (PSS) onto the surface of a gold electrode followed by electrostatic attachment of the enzyme HRP onto the PDMA-PSS composite film. Fourier transform infrared (FTIR) and UV–Vis spectrometry inferred that HRP was not denatured during its immobilization on PDMA-PSS composite film. The biosensing principle was based on the determination of the cathodic responses of the immobilized HRP to H 2O 2, before and after incubation in glyphosate standard solutions. Glyphosate inhibited the activity of HRP causing a decrease in its response to H 2O 2. The determination of glyphosate was achieved in the range of 0.25–14.0 µg L − 1 with a detection limit of 1.70 μg L − 1 . The apparent Michaelis–Menten constant (calculated for the HRP/PDMA-PSS biosensor in the presence and absence of glyphosate was found to be 7.73 µM and 7.95 µM respectively.

Distribuição de glyphosate e acúmulo de nutrientes em biótipos de azevém

Objetivou-se neste trabalho avaliar o acúmulo de nutrientes e a translocação de glyphosate em biótipos de azevém. Para isso, foram montados dois ensaios: no primeiro aplicou-se 14C-glyphosate, adicionando 10 µL da calda sobre a face adaxial da primeira folha com lígula totalmente visível, quando as plantas de azevém apresentavam três perfilhos. A quantidade de glyphosate absorvido, translocado e exsudado foi avaliada 64 horas após aplicação, por meio da medição da radiação emitida pelo 14C-glyphosate, em espectrômetro de cintilação líquida. O glyphosate foi quantificado em folha de aplicação, perfilhos, raízes e na solução nutritiva onde foram cultivados os biótipos de azevém. No segundo experimento, aplicou-se o glyphosate (480 g ha-1) tanto no biótipo sensível quanto no resistente. Após dez dias da aplicação, a parte área e as raízes das plantas foram coletadas e secas em estufa, sendo determinados os teores de macronutrientes. No primeiro ensaio, verificou-se exsudação radicular em ambos os biótipos, nos quais a quantidade de glyphosate exsudada foi semelhante, não ultrapassando 5% do total que penetrou na planta. No perfilho principal do biótipo sensível, comparado ao resistente, foi observada maior concentração do produto marcado. O biótipo resistente apresentou maior acúmulo de produto marcado na folha de aplicação; no sensível, a maior parte do glyphosate foi encontrada nas raízes. Com relação ao segundo ensaio, na presença de herbicida o biótipo sensível apresentou menor teor de fósforo tanto na parte aérea quanto na planta. Os biótipos resistente e sensível, sem aplicação de herbicida, tenderam a apresentar maiores teores de N total e N inorgânico na parte aérea e na planta como um todo, quando comparados aos tratamentos em que foi realizada a aplicação do produto. Ambos os biótipos mostraram a mesma capacidade de absorção e acúmulo de macronutrientes na ausência do produto.

Structural Basis of Glyphosate Tolerance Resulting from Mutations of Pro101 in Escherichia coli 5-Enolpyruvylshikimate-3-phosphate Synthase

Glyphosate, the world's most used herbicide, is a massive success because it enables efficient weed control with minimal animal and environmental toxicity. The molecular target of glyphosate is 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), which catalyzes the sixth step of the shikimate pathway in plants and microorganisms. Glyphosate-tolerant variants of EPSPS constitute the basis of genetically engineered herbicide-tolerant crops. A single-site mutation of Pro(101) in EPSPS (numbering according to the enzyme from Escherichia coli) has been implicated in glyphosate-resistant weeds, but this residue is not directly involved in glyphosate binding, and the basis for this phenomenon has remained unclear in the absence of further kinetic and structural characterization. To probe the effects of mutations at this site, E. coli EPSPS enzymes were produced with glycine, alanine, serine, or leucine substituted for Pro(101). These mutant enzymes were analyzed by steady-state kinetics, and the crystal structures of the substrate binary and substrate.glyphosate ternary complexes of P101S and P101L EPSPS were determined to between 1.5- and 1.6-A resolution. It appears that residues smaller than leucine may be substituted for Pro(101) without decreasing catalytic efficiency. Any mutation at this site results in a structural change in the glyphosate-binding site, shifting Thr(97) and Gly(96) toward the inhibitor molecule. We conclude that the decreased inhibitory potency observed for glyphosate is a result of these mutation-induced long-range structural changes. The implications of our findings concerning the development and spread of glyphosate-resistant weeds are discussed.

Investigation of copper dissolution in the presence of glyphosate using hydrodynamic voltammetry and chronoamperometry

The copper dissolution was investigated in phosphate buffer in the presence of glyphosate using hydrodynamic voltammetry and chronoamperometry. The electrochemical studies showed that the addition of glyphosate in the phosphate buffer increases the electrochemical dissolution of copper electrode and this phenomenon was associated to the Cu(II) complexation by glyphosate. The apparent rate constant of the copper dissolution process (1.2 × 10 − 2 cm mol − 1 s − 1 ) and the diffusion coefficient of the complex (0.74 × 10 − 5 cm 2 s − 1 ), were obtained. It was observed that during the chronoamperometric experiments, in the absence of glyphosate, the electrode surface was covered by blue passive film, probably copper phosphate; nevertheless, in the presence of glyphosate, there was a gradual development of a blue color in the solution, indicating the copper dissolution and formation of the Cu(II)–glyphosate complex.