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Background Amaranthus spp. are problematic weeds and competitors for nutrients in several crops, especially in soybean and corn. Resistance to glyphosate has been detected in several weed species, and a triple mutation in its EPSPS target gene was detected recently in Amaranthus hybridus. Objective The aim of this work was to develop a simple polymerase chain reaction (PCR) method to detect the EPSPS triple mutation in A. hybridus. Methods Two pairs of primers were designed for PCR-based detection of the EPSPS TAP-IVS triple mutation, which confers resistance to glyphosate, in A. hybridus. Results These sets of allele-specific primers were tested on five Amaranthus species and in 65 different field accessions. The PCR reaction using one set of the primers amplifies the wildtype (TAP) allele while the PCR reaction using the other pair of primers amplifies the triple mutation (IVS) allele. The presence of PCR products in both sets of primers identifies the heterozygous resistant individuals, and PCR product amplified only with the triple mutation set of primers identifies the homozygous resistant individuals. A DNA concentration test was performed and the recommend DNA amount to be used is 100 ng. Conclusions We developed and tested two sets of primers to detect the EPSPS TAP-IVS triple mutation and the results showed a 100% genotypic to phenotypic association. The triple mutation detection assay is easy to use and can be applied in a molecular laboratory with basic equipments. Early detection of resistance helps to better manage and control its spreading.

Significance Glyphosate is a nonselective herbicide used around the globe for weed control in glyphosate-resistant (GR) and noncrop situations. The extensive and exclusive use of glyphosate has led to the evolution of herbicide resistance in many crop weeds. The molecular target of glyphosate, the 5-enolpyruvlyshikimate-3-phosphate synthase (EPSPS) gene, confers resistance upon amplification and was first documented in GR Amaranthus palmeri. We now report that amplified EPSPS copies in GR A. palmeri are present in the form of extrachromosomal circular DNA molecules (eccDNAs) with various conformations. We discovered that eccDNAs are transmitted to the next generation by tethering to mitotic and meiotic chromosomes. These results represent a report of extrachromosomal structures that drive rapid adaptive evolution in higher organisms., Gene amplification has been observed in many bacteria and eukaryotes as a response to various selective pressures, such as antibiotics, cytotoxic drugs, pesticides, herbicides, and other stressful environmental conditions. An increase in gene copy number is often found as extrachromosomal elements that usually contain autonomously replicating extrachromosomal circular DNA molecules (eccDNAs). Amaranthus palmeri, a crop weed, can develop herbicide resistance to glyphosate [N-(phosphonomethyl) glycine] by amplification of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene, the molecular target of glyphosate. However, biological questions regarding the source of the amplified EPSPS, the nature of the amplified DNA structures, and mechanisms responsible for maintaining this gene amplification in cells and their inheritance remain unknown. Here, we report that amplified EPSPS copies in glyphosate-resistant (GR) A. palmeri are present in the form of eccDNAs with various conformations. The eccDNAs are transmitted during cell division in mitosis and meiosis to the soma and germ cells and the progeny by an as yet unknown mechanism of tethering to mitotic and meiotic chromosomes. We propose that eccDNAs are one of the components of McClintock’s postulated innate systems [McClintock B (1978) Stadler Genetics Symposium] that can rapidly produce soma variation, amplify EPSPS genes in the sporophyte that are transmitted to germ cells, and modulate rapid glyphosate resistance through genome plasticity and adaptive evolution.

The last patents protecting glyphosate herbicide tolerant soybean (Roundup Ready) expired in 2015 (US4940835, US5188642, US5804425, US5312910, US5352605, US5530196, US5627061, US5633435, US5717084, US5728925). This opens up a very big market for generic soybean. In this paper an overview of patent status will be developed which will offer records of claims, dates and owners in different world regions, including our own experience in transgenic soybean development and implementation of an open operating system will be an opportunity for applications developers from the perspective of the emerging generic transgenic crops agenda.

We report here on a high-quality draft genome sequence of Ochrobactrum haematophilum strain P6BS-III (DSM 106071), a Gram negative, non-sporulating bacterium isolated from a pastureland (Buenos Aires province, Argentina) which had been chronically exposed to the herbicide glyphosate. The genome of 5.25 Mb with a DNA G+C content of 56.63% size was estimated to contain 5,291 protein coding genes and 57 RNA genes. Genome analysis revealed the presence of the phn operon, which is involved in the phosphonate degradation pathway, and a class II 5-enolpyruvylshikimate-3-phosphate synthase (EPSP) that confers tolerance to glyphosate. Genes related to plant growth promotion traits are also present, and include genes for phosphorus metabolism, calcium phosphate and phytate solubilization, siderophore production, organic acid biosynthesis and indole acetic acid (IAA) production. Fil: Massot, Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; Argentina. Hasselt University; Bélgica Fil: Gkorezis, Panagiotis. Hasselt University; Bélgica Fil: McAmmond, Breanne. Thompson Rivers University; Canadá Fil: d´Haen, Jan. Hasselt University; Bélgica Fil: Van Hamme, Jonathan. Thompson Rivers University; Canadá Fil: Merini, Luciano Jose. Instituto Nacional de Tecnología Agropecuaria. Centro Regional La Pampa-San Luis. Estación Experimental Agropecuaria Anguil; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Vangronsveld, Jaco. Hasselt University; Bélgica Fil: Thijs, Sofie. Hasselt University; Bélgica

BACKGROUND:In California specialty cropping systems such as vineyards and orchards, Echinochloa colona is present as a summer annual weed. It is able to germinate throughout the growing season whenever favorable conditions are present, and management relies heavily on glyphosate applications. Glyphosate-resistant (GR) E. colona biotypes are present in the state, but the levels of resistance observed suggest that there may be differences in mechanisms of resistance among populations. RESULTS:Echinochloa colona lines collected from different regions of California's Central Valley presented resistance levels ranging from 1.4 to 4.3-fold compared to susceptible lines. No differences in the absorption and translocation of [14 C]-glyphosate were observed among lines. Resistant lines accumulated eight-fold less shikimic acid after treatment with 435 and 870 g a.e. ha-1 glyphosate compared to the most susceptible line. Sequencing of a region of the EPSPS gene revealed three single nucleotide changes leading to amino acid substitutions at Proline 106, including Pro106Leu, Pro106Thr and Pro106Ser. CONCLUSION:These results indicate that an altered target site in EPSPS is contributing to resistance in these lines and resistance has evolved independently, multiple times in the Central Valley of California. Additional research is needed to further understand the genomic contributions of resistance loci in this polyploid weed species. © 2018 Society of Chemical Industry.

The use of herbicides with different modes of action is the primary strategy used to control weeds possessing resistance to a single mechanism of action (MOA). However, this practice can lead to selection for generalist resistance mechanisms and may cause resistance to all MOAs. In this research, we characterized the resistance to diquat/paraquat (bipyridiliums) in an Epilobium ciliatum biotype (R1) collected in an olive orchard from Chile, where alternatives herbicides (2,4-D, glyphosate, glufosinate, flazasulfuron and pyraflufen-ethyl) with different MOAs were used, but they have also showed failure in controlling this species. Because the resistance/susceptibility patterns of the R1 biotype to glufosinate, 2,4-D and pyraflufen-ethyl were not clear, a recurrent resistance selection was carried out in field and greenhouse using these herbicides on R1 plants for three generations (R2 biotype). One biotype that was never treated with herbicides (S) was included as control. Results indicated that the S biotype was controlled at the field dose of all herbicides tested. The biotype R1 exhibited resistance to diquat, paraquat and flazasulfuron and natural tolerance to glyphosate. The R2 biotype displayed resistance to glufosinate, 2,4-D and pyraflufen-ethyl with LD50 (herbicide dose to kill 50% of plants) values higher than field doses in all assays. Physiological and biochemical studies determined the resistance to diquat of the R1 biotype, which was due to impaired translocation. The resistance to flazasulfuron in the R1 and R2 biotypes was confirmed by the low sensitivity of the acetolactate synthase (ALS) activity compared to the S biotype. The similar accumulation of shikimate in treated S, R1, and R2 plants with glyphosate supported the existence of innate tolerance to this herbicide in E. ciliatum. Resistance to glufosinate, 2,4-D and pyraflufen-ethyl in the R2 biotype, acquired after recurrent selection, was determined by low sensitivity of the glutamine synthetase, low accumulation of ethylene and protoporphyrinogen IX oxidase, respectively, in comparison to the S biotype. Epilobium ciliatum from Chilean olive orchards had resistance to only two MAOs (photosystem I and ALS inhibitors), but resistance to five MOAs could occur in the next cropping seasons, if alternatives to weed management, other than herbicides, are not included.

Fil: Marani, Mariela Mirta. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Centro Nacional Patagonico; Argentina

Incluye 3 ficheros de datos Imazamox and glyphosate represent two classes of herbicides that inhibit the activity of acetohydroxyacid synthase in the branched-chain amino acid biosynthesis pathway and the activity of 5-enolpyruvylshikimate-3-phosphate synthase in the aromatic amino acid biosynthesis pathway, respectively. However, it is still unclear how imazamox and glyphosate lead to plant death. Both herbicides inhibit amino-acid biosynthesis and were found to induce ethanol fermentation in plants, but an Arabidopsis mutant deficient in alcohol dehydrogenase 1 was neither more susceptible nor more resistant than the wild-type to the herbicides. In this study, we investigated the effects of the amino acid biosynthesis inhibitors, imazamox and glyphosate, on the pyruvate dehydrogenase bypass reaction and fatty acid metabolism in A. thaliana. We found that the pyruvate dehydrogenase bypass was upregulated following the treatment by the two herbicides. Our results suggest that the Arabidopsis aldehyde dehydrogenase 7B4 gene might be participating in the pyruvate dehydrogenase bypass reaction. We evaluated the potential role of the aldehyde dehydrogenase 7B4 upon herbicide treatment in the plant defence mechanism. Plants that overexpressed the ALDH7B4 gene accumulated less soluble sugars, starch, and fatty acids and grew better than the wild-type after herbicide treatment. We discuss how the upregulation of the ALDH7B4 alleviates the effects of the herbicides, potentially through the detoxification of the metabolites produced in the pyruvate dehydrogenase bypass. Miriam Gil-Monreal received funding from fellowships through Universidad Pública de Navarra. This work was financially supported by two grants from the Ministerio Español de Economía y Competitividad (AGL-2013-40567R and AGL-2016-77531R).

Glyphosate is the most widely used herbicide for its low cost and high efficiency. However, it is rarely applied directly in rice field due to its toxicity to rice. Therefore, glyphosate-tolerant rice can greatly decrease the cost of rice production and provide a more effective weed management strategy. Although, several approaches to develop transgenic rice with glyphosate tolerance have been reported, the agronomic performances of these plants have not been well evaluated, and the feasibility of commercial production has not been confirmed yet. Here, a novel glyphosate-tolerant gene cloned from the bacterium Isoptericola variabilis was identified, codon optimized (designated as I. variabilis-EPSPS (*)), and transferred into Zhonghua11, a widely used japonica rice cultivar. After systematic analysis of the transgene integration via PCR, Southern blot and flanking sequence isolation, three transgenic lines with only one intact I. variabilis-EPSPS (*) expression cassette integrated into intergenic regions were identified. Seed test results showed that the glyphosate tolerance of the transgenic rice was about 240 times that of wild type on plant medium. The glyphosate tolerance of transgenic rice lines was further evaluated based on comprehensive agronomic performances in the field with T3 and T5generations in a 2-year assay, which showed that they were rarely affected by glyphosate even when the dosage was 8400 g ha(-1). To our knowledge, this is the first demonstration of the development of glyphosate-tolerant rice lines based on a comprehensive analysis of agronomic performances in the field. Taken together, the results suggest that the selected glyphosate-tolerant rice lines are highly tolerant to glyphosate and have the possibility of commercial release. I. variabilis-EPSPS (*) also can be a promising candidate gene in other species for developing glyphosate-tolerant crops.

This is the peer reviewed version of the following article: Armendáriz, O., Gil-Monreal, M., Zulet, A., Zabalza, A. and Royuela, M. (2016), Both foliar and residual applications of herbicides that inhibit amino acid biosynthesis induce alternative respiration and aerobic fermentation in pea roots. Plant Biol J, 18: 382–390. doi:10.1111/plb.12412, which has been published in final form at http://dx.doi.org/10.1111/plb.12412. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. The objective of this work was to ascertain whether there is a general pattern of carbon allocation and utilisation in plants following herbicide supply, independent of the site of application: sprayed on leaves or supplied to nutrient solution. The herbicides studied were the amino acid biosynthesis-inhibiting herbicides (ABIH): glyphosate, an inhibitor of aromatic amino acid biosynthesis, and imazamox, an inhibitor of branched-chain amino acid biosynthesis. All treated plants showed impaired carbon metabolism; carbohydrate accumulation was detected in both leaves and roots of the treated plants. The accumulation in roots was due to lack of use of available sugars as growth was arrested, which elicited soluble carbohydrate accumulation in the leaves due to a decrease in sink strength. Under aerobic conditions, ethanol fermentative metabolism was enhanced in roots of the treated plants. This fermentative response was not related to a change in total respiration rates or cytochrome respiratory capacity, but an increase in alternative oxidase capacity was detected. Pyruvate accumulation was detected after most of the herbicide treatments. These results demonstrate that both ABIH induce the less-efficient, ATP-producing pathways, namely fermentation and alternative respiration, by increasing the key metabolite, pyruvate. The plant response was similar not only for the two ABIH but also after foliar or residual application. A. Zulet and M. Gil-Monreal received funding from fellowships through the Ministerio de Educación and Universidad Pública de Navarra, respectively. This work was financially supported by a grant from the Ministerio Español de Economía y Competitividad (AGL-2013-4067R).

Currently, there are 8 million new cases and 2 million deaths annually from tuberculosis, and it is expected that a total of 225 million new cases and 79 million deaths will occur between 1998 and 2030. The reemergence of tuberculosis as a public health threat, the high susceptibility of HIV-infected persons, and the proliferation of multi-drug-resistant strains have created a need to develop new antimycobacterial agents. The existence of homologues to the shikimate pathway enzymes has been predicted by the determination of the genome sequence of Mycobacterium tuberculosis. We have previously reported the cloning and overexpression of M. tuberculosis aroA-encoded EPSP synthase in both soluble and active forms, without IPTG induction. Here, we describe the purification of M. tuberculosis EPSP synthase (mtEPSPS) expressed in Escherichia coli BL21(DE3) host cells. Purification of mtEPSPS was achieved by a one-step purification protocol using an anion exchange column. The activity of the homogeneous enzyme was measured by a coupled assay using purified shikimate kinase and purine nucleoside phosphorylase proteins. A total of 53 mg of homogeneous enzyme could be obtained from 1L of LB cell culture, with a specific activity value of approximately 18 Umg(-1). The results presented here provide protein in quantities necessary for structural and kinetic studies, which are currently underway in our laboratory.

The synthesis of (±)-5-homoshikimic acid, its 5-fluoromethyl and difluoromethyl analogues are described from the cycloadducts of 5,6-dihydro-1- t -butyloxycarbonylpyridine and methyl acrylate. The adducts on ring opening afford methyl 5- t -butoxycarbonylmethyl-5,6-dihydrobenzoate, which may be 3,4- cis -dihydroxylated and converted in a series of steps into the title compounds

The synthesis of (±)-5-homoshikimic acid, its 5-fluoromethyl and difluoromethyl analogues are described from the cycloadducts of 5,6-dihydro-1- t -butyloxycarbonylpyridine and methyl acrylate. The adducts on ring opening afford methyl 5- t -butoxycarbonylmethyl-5,6-dihydrobenzoate, which may be 3,4- cis -dihydroxylated and converted in a series of steps into the title compounds

EPSP Synthase is an enzyme in the shikimate acid pathway1 found only in plants, fungi and bacteria. A novel 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase inhibitor has been prepared by one step reaction using 2- chloroethylphosphonic acids with amino acid in a dilute alkaline solution below 100C , It has been found that these compounds have herbicidal activities.

The mechanism of glyphosate tolerance was investigated in nine birdsfoot trefoil selections that exhibited a threefold difference in glyphosate tolerance. Single-stemmed ramets (vegetative clones) established from the nine selections were used to evaluate tolerance to glyphosate, spray retention, and14C-glyphosate absorption and translocation in growth chamber experiments. The tolerance of greenhouse-grown ramets correlated with the tolerance of field-grown plants, indicating that tolerance was not a function of plant size or affected by environment. The nine selections differed in spray retention and14C-glyphosate translocation but not in glyphosate absorption. The differences in retention and translocation were not correlated with the level of glyphosate tolerance but could contribute to the tolerance of an individual plant. The specific activity of 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) (EC 2.5.1.19) ranged from 1.3 to 3.5 nmol min−1mg−1protein among the nine selections assayed and was positively correlated with plant tolerance level. These results indicate that the primary mechanism of glyphosate tolerance in birdsfoot trefoil is based on the level of EPSPS activity.

5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the condensation of shikimate 3-phosphate (S3P) and phosphoenolpyruvate (PEP) to form EPSP, a precursor for the aromatic amino acids. This paper examines a recent claim [Studelska, D. R., McDowell, L. M., Espe, M. P., Klug, C. A., and Schaefer, J. (1997) Biochemistry 36, 15555-15560] that the mechanism of EPSP synthase involves two covalent enzyme-intermediates, in complete contrast to a large body of literature that has already proven the involvement of a single noncovalent intermediate. The evidence in the paper of Studelska et al. is examined closely, and unequivocal proof is provided that those authors' NMR assignments to covalent structures are in error, and that in fact the species they observed were simply the product EPSP and a side-product EPSP ketal. Since those authors used rotational-echo double-resonance (REDOR) solid-state NMR to measure intermolecular and intramolecular distances in the proposed covalent intermediates, we have used REDOR to measure the same distances in enzyme-free and enzyme-bound preparations of purified EPSP, and enzyme-free preparations of purified EPSP ketal. The distance between the shikimate ring phosphorus atom and C8 in enzyme-free EPSP is 6.6 +/- 0.1 A, which lengthens to 7.4 +/- 0.1 A in the presence of the enzyme, and in enzyme-free EPSP ketal is 5.6 +/- 0.1 A. These are entirely consistent with those measured by Studelska et al., which were 7.5 +/- 0.5 A for a putative enzyme-enolpyruvyl species and 6.1 +/- 0.3 A for a putative enzyme-ketal species.

A gene has been introduced into soybeans to confer tolerance to glyphosate, the active ingredient in the herbicide, Roundup (Monsanto Co., St. Louis, Mo.) 1,2 Soybean varieties expressing this trait will enable farmers to use Roundup herbicide within the crop during the growing season. Previous studies have confirmed the safety of the introduced protein (5-enolpyruvylshikimate-3-phosphate synthase [EPSPS]), which confers glyphosate tolerance? Over 1400 analyses have confirmed the compositional and nutritional equivalence of the soybeans produced from the glyphosate-tolerant soybean variety to the parental soybean variety. 2 The multiple, endogenous protein allergens previously identified in soybeans 34 represent an additional safety concern. The purpose of this study was to qualitatively and quantitatively compare the endogenous allergens in glyphosate-tolerant and commercial soybean varieties and tO determine whether the genetic engineering process itself affected these components.

ACP4 gene encoding CP4 EPSPS from genetically modifiedfeed through gastrointestinal tract to bacteria living in ani-mal gut we have observed beside full length gene alsofunctional truncated one present in bacteria [1]. Thecodon usage of both forms was originally optimized forplant [2]. Therefore we have used two different

Summary Shikimate kinase and 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase activities were detected in extracts of etiolated Phaseolus mungo seedlings. The existence of these enzymes was confirmed by identifying the reaction products as shikimate-3-phosphate and EPSP, respectively. Shikimate kinase requires ATP and Mg 2+ or Mn 2+ ions for activity.

Crystals of 5-enolpyruvylshikimate 3-phosphate synthase from Escherichia coli have been grown out of ammonium sulfate by the hanging drop method of vapor diffusion. The crystals belong to the hexagonal space group P6(1)22 or P6(5)22, with a = 124 A and c = 381 A, and diffract to 3.8 A resolution.