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Background Indigo 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. Results One 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. Conclusions The 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.

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.

The introduction of glyphosate-resistant (GR) crops revolutionized weed management, however, the improper use of this technology has selected for a wide range of weeds resistant to glyphosate, referred to as superweeds. We characterized the high glyphosate resistance level of an Amaranthus hybridus population (GRH)&mdash, a superweed collected in a GR-soybean field from Cordoba, Argentina&mdash, as well as the resistance mechanisms that govern it in comparison to a susceptible population (GSH). The GRH population was 100.6 times more resistant than the GSH population. Reduced absorption and metabolism of glyphosate, as well as gene duplication of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) or its overexpression did not contribute to this resistance. However, GSH plants translocated at least 10% more 14C-glyphosate to the rest of the plant and roots than GRH plants at 9 h after treatment. In addition, a novel triple amino acid substitution from TAP (wild type, GSH) to IVS (triple mutant, GRH) was identified in the EPSPS gene of the GRH. The nucleotide substitutions consisted of ATA102, GTC103 and TCA106 instead of ACA102, GCG103, and CCA106, respectively. The hydrogen bond distances between Gly-101 and Arg-105 positions increased from 2.89 &Aring, (wild type) to 2.93 &Aring, (triple-mutant) according to the EPSPS structural modeling. These results support that the high level of glyphosate resistance of the GRH A. hybridus population was mainly governed by the triple mutation TAP-IVS found of the EPSPS target site, but the impaired translocation of herbicide also contributed in this resistance.

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

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. This work was funded by the CONACYT-242088 (Mexico) and AGL2016-78944-R (Spain) projects. The authors are grateful for the technical collaboration of Rafael A. Roldán-Gómez.

During research we have utilized recombinant enzyme 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS), conferring resistance to glyphosate for GM soybean, for the hen immunization and obtaining specific yolk antibodies IgY. Stages of ELISA development that can detect at least 0,1 % of GM-soybean resistant to glyphosate were present

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.

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.