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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.

Growth of the toxic alga Prymnesium parvum is hormetically stimulated with environmentally relevant concentrations of glyphosate. The mechanisms of glyphosate hormesis in this species, however, are unknown. We evaluated the transcriptomic response of P. parvum to glyphosate at concentrations that stimulate maximum growth and where growth is not different from control values, the zero-equivalent point (ZEP). Maximum growth occurred at 0.1 mg l -1 and the ZEP was 2 mg l -1 . At 0.1 mg l -1 , upregulated transcripts outnumbered downregulated transcripts by one order of magnitude. Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses indicated that the upregulated transcriptome is primarily associated with metabolism and biosynthesis. Transcripts encoding heat shock proteins and co-chaperones were among the most strongly upregulated, and several others were associated with translation, Redox homeostasis, cell replication, and photosynthesis. Although most of the same transcripts were also upregulated at concentrations ≥ZEP, the proportion of downregulated transcripts greatly increased as glyphosate concentrations increased. At the ZEP, downregulated transcripts were associated with photosynthesis, cell replication, and anion transport, indicating that specific interference with these processes is responsible for the nullification of hormetic growth. Transcripts encoding the herbicidal target of glyphosate, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), were upregulated at concentrations ≥ZEP but not at 0.1 mg l -1 , indicating that disruption of EPSPS activity occurred at high concentrations and that nullification of hormetic growth involves the direct interaction of glyphosate with this enzyme. Results of this study may contribute to a better understanding of glyphosate hormesis and of anthropogenic factors that influence P. parvum biogeography and bloom formation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Abstract 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.

Key message: We have defined the conditions for citrus transformations using glyphosate as selection agent. This protocol results in high transformation rate and low incidence of chimeric shoots. Glyphosate, the most widely used herbicide in the world, specifically inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), an essential enzyme of the shikimate pathway. Various laboratory-generated or naturally evolved glyphosate-resistant EPSPS variants have been used to produce glyphosate-tolerant transgenic crops, enabling highly effective weed control in agriculture. In this study, we explored the potential of using a citrus EPSPS variant that mimics the previously reported Eleusine indica glyphosate-resistant TIPS (T102I + P106S) mutant for selection of transgenic citrus plants in the presence of glyphosate. We found that glyphosate did not suppress bud formation on 'Duncan' grapefruit seedling explants, but inhibited non-transgenic bud outgrowth to produce shoots in a concentration-dependent manner. At certain concentrations, glyphosate had dramatic effect on the transformation rate and the percentage of non-chimeric transgenic shoots in this newly developed selection system. Specifically, at 0, 10, 20, and 50 μM of glyphosate, the citrus TIPS EPSPS-based selection resulted in transformation rates of 4.02, 5.04, 14.46, and 40.78%, respectively, and 6.41, 23.96, 42.94, and 40.17% of non-chimeric transgenic shoots, respectively. These results indicate that the citrus TIPS EPSPS-glyphosate selection system is highly efficient and can be used as an alternative to antibiotic-based selection methods in citrus genetic transformation. Furthermore, the selection conditions defined in this study are expected to greatly facilitate the production of genetically modified, market-friendly citrus plants, such as cisgenic and intragenic plants. [ABSTRACT FROM AUTHOR]

BACKGROUND: Gene amplification has been shown to provide resistance to glyphosate in several weed species, including Hordeum glaucum populations in South Australia. The stability of 5‐enolpyruvylshikimate‐3‐phosphate synthase (EPSPS) gene copies in resistant populations in the presence or absence of glyphosate selection has not been determined. RESULTS: Applying glyphosate to a cloned plant resulted in an increase in resistance and EPSPS copy number in the progeny of that plant compared to the untreated clone. The LD50 (herbicide concentration required for 50% mortality) increased by 75% to 79% in the progeny of the treated clones compared to the untreated in both populations (YP‐17 and YP‐16). EPSPS copy number estimates were higher in treated individuals compared to untreated individuals with an average of seven copies compared to six in YP‐16 and 11 compared to six in YP‐17. There was a positive correlation (R2 = 0.78) between EPSPS copy number and LD50 of all populations. CONCLUSION: EPSPS gene copy number and resistance to glyphosate increased in H. glaucum populations under glyphosate selection, suggesting the number of EPSPS gene copies present is dependent on glyphosate selection. © 2021 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

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BACKGROUND: Bidens subalternans (greater beggarticks) is a tetraploid and troublesome weed infesting annual crops in most tropical regions of the world. A glyphosate‐resistant (GR) B. subalternans biotype was detected in a soybean field from Paraguay. A series of physiological and molecular analyses were conducted to elucidate its resistance mechanisms. RESULTS: The GR biotype had a high level of resistance (> 15‐fold LD50), relative to a glyphosate‐susceptible (GS) biotype. Shikimate accumulation was up to ten‐fold greater for GS compared with GR. We found no differences in sensitivity when plants were treated and kept under lower (10/4 °C) or higher temperatures (25/20 °C). GS and GR had the same relative EPSPS gene copy number, and similar glyphosate absorption and translocation rates. Neither biotype metabolized glyphosate. A double amino acid substitution (TIPT – Thr102Ile and Pro106Thr) was found in only one EPSPS allele from one of the two EPSPS homoeologs present in tetraploid GR B. subalternans. CONCLUSION: This is the first report of a TIPT double mutation conferring high levels of glyphosate resistance in a weed species. The presence of both wild‐type and TIPT mutant EPSPS on the polyploid genome of GR B. subalternans may offset a potential fitness cost, requiring additional research to confirm the absence of deleterious effects. © 2019 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

BACKGROUND: We previously identified a glyphosate‐resistant A. trifida phenotype from Wisconsin USA that showed a non‐rapid response to glyphosate. The mechanism of glyphosate resistance in this phenotype has yet to be elucidated. We conducted experiments to investigate non‐target‐site resistance and target‐site resistance mechanisms. The roles of glyphosate absorption, translocation, and metabolism in resistance of this phenotype have not been reported previously, nor have EPSPS protein abundance or mutations to the full‐length sequence of EPSPS. RESULTS: Whole‐plant dose–response results confirmed a 6.5‐level of glyphosate resistance for the resistant (R) phenotype compared to a susceptible (S) phenotype. Absorption and translocation of 14C‐glyphosate were similar between R and S phenotypes over 72 h. Glyphosate and AMPA concentrations in leaf tissue did not differ between R and S phenotypes over 96 h. In vivo shikimate leaf disc assays confirmed that glyphosate EC50 values were 4.6‐ to 5.4‐fold greater for the R than S phenotype. Shikimate accumulation was similar between phenotypes at high glyphosate concentrations (>1000 μM), suggesting that glyphosate entered chloroplasts and inhibited EPSPS. This finding was supported by results showing that EPSPS copy number and EPSPS protein abundance did not differ between R and S phenotypes, nor did EPSPS sequence at Gly101, Thr102, and Pro106 positions. Comparison of full‐length EPSPS sequences found five nonsynonymous polymorphisms that differed between R and S phenotypes. However, their locations were distant from the glyphosate target site and, therefore, not likely to affect enzyme‐glyphosate interaction. CONCLUSION: The results suggest that a novel mechanism confers glyphosate resistance in this A. trifida phenotype. © 2019 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS) overexpression, attempting to provide excess EPSPS to combine with glyphosate, is one way to improve glyphosate resistance of plants. The EPSPS in extremophiles which is selected by nature to withstand the evolutionary pressure may possess some potential-specific biological functions. In this study, we reported the cloning, expression and enzymatic characterization of a novel Class II EPSPS AroAT. maritima from Thermotoga maritima MSB8. The enzyme showed low sequence identities with other EPSPSs, and was one of the most thermostable EPSPSs so far, which showed the optimum enzyme activity at 80 °C. The enzyme maintains the activity below 50 °C and in a wide range of pH 4.0–10, which indicated its stability under rough environment, especially in tropical regions and alkaline soil. Excellent Ki/Km value of AroAT. maritima suggested that the enzyme showed powerful competitive binding capacity of PEP over glyphosate and high glyphosate tolerance. Furthermore, aroAT. maritima gene was transformed into Arabidopsis thaliana. The transgenic lines were resistant to 15 mM glyphosate, which proved the application value in the cultivation of glyphosate-tolerant plants. [ABSTRACT FROM AUTHOR]

Abstract Continuous use of glyphosate in citrus groves in the Gulf of Mexico region has selected for resistant Parthenium hysterophorus L. populations. In this study, the target-site and non-target-site resistance mechanisms were characterized in three putative glyphosate-resistant (GR) P. hysterophorus populations, collected in citrus groves from Acateno, Puebla (GR1 and GR2) and Martínez de la Torre, Veracruz (GR3), and compared with a susceptible population (GS). Based on plant mortality, the GR populations were 9.2–17.3 times more resistant to glyphosate than the GS population. The low shikimate accumulation in the GR population confirmed this resistance. Based on plant mortality and shikimate accumulation, the GR3 population showed intermediate resistance to glyphosate. The GR populations absorbed 15–28% less 14C-glyphosate than the GS population (78.7% absorbed from the applied) and retained 48.7–70.7% of 14C-glyphosate in the treated leaf, while the GS population translocated ~68% of absorbed herbicide to shoots and roots. The GR3 population showed the lowest translocation and absorption rates, but was found to be susceptible at the target site level. The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene sequence of the GR1 and GR2 populations showed the Pro106-Ser mutation, conferring 19- and 25-times more resistance in comparison to the GS population, respectively. Reduced absorption and impaired translocation conferred glyphosate resistance on the GR3 population, and contributed partially to the resistance of the GR1 and GR2 populations. Additionally, the Pro-106-Ser mutation increased the glyphosate resistance of the last two P. hysterophorus populations. Graphical abstract Unlabelled Image Highlights • Glyphosate resistance was confirmed in three P. hysterophorus populations from the Mexican citrus region. • Reduced absorption and translocation conferred resistance to the GR3 population. • These NTSR mechanisms also contributed partially in the resistance of the GR1 and GR2 populations. • Pro-106-Ser mutation governed glyphosate resistance in the GR1 and GR2 populations. • These results confirm the first case of glyphosate resistance of P. hysterophorus in Mexico. [ABSTRACT FROM AUTHOR]

Coincidence detector neurons of the medial superior olive (MSO) are sensitive to interaural time differences in the range of a few tens of microseconds. The biophysical basis for this remarkable acuity is a short integration time constant of the membrane, which is achieved by large low voltage-activated potassium and hyperpolarization-activated inward cation conductances. Additional temporal precision is thought to be achieved through a sub-cellular distribution of low voltage-activated potassium channel expression biased to the soma. To evaluate the contribution of potassium channels, we investigated the presence and sub-cellular distribution profile of seven potassium channel sub-units in adult MSO neurons of gerbils. We find that low- and high voltage-activated potassium channels are present with distinct sub-cellular distributions. Overall, low voltage-activated potassium channels appear to be biased to the soma while high voltage-activated potassium channels are more evenly distributed and show a clear expression at distal dendrites. Additionally, low voltage-activated potassium channel sub-units co-localize with glycinergic inputs while HCN1 channels co-localize more with high voltage-activated potassium channels. Functionally, high voltage-activated potassium currents are already active at low voltages near the resting potential. We describe a possible role of high voltage-activated potassium channels in modulating EPSPs in a computational model and contributing to setting the integration time window of coincidental inputs. Our data shows that MSO neurons express a large set of different potassium channels with distinct functional relevance. [ABSTRACT FROM AUTHOR]

Glyphosate, the most widely used herbicide in both agricultural and urban areas is toxic for plants and for many bacterial species. The mechanism of action of glyphosate is through the inhibition of the EPSP synthase, a key enzyme in the biosynthetic pathway of aromatic amino acids. Here we show that glyphosate induces the stringent response in Escherichia coli. Bacteria treated with glyphosate stop growing and accumulate ppGpp. Both growth arrest and ppGpp accumulation are restored to normal levels upon addition of aromatic amino acids. Glyphosate-induced ppGpp accumulation is dependent on the presence of the (p)ppGpp synthetase RelA. However, unlike other cases of amino acid starvation, pppGpp could not be discerned. In a gppA background both ppGpp and pppGpp accumulated when exposed to glyphosate. Conversely, the wild-type strain and gppA mutant treated with serine hydroxamate accumulated high levels of both ppGpp and pppGpp. Altogether, the data indicate that glyphosate induces amino acid starvation resulting in a moderate accumulation of ppGpp and a reversible stringent response. [ABSTRACT FROM AUTHOR]

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.

Small angle X-ray scattering (SAXS) is widely used as an efficient method to analysis the structural changes. Thus, the aim of this mini-review was to summarize important information about performing SAXS technique. For more clarification, a brief introduction of the fundamental history, scattering theory, and essential structure of SAXS were presented at first. But the main focus was placed on the application of SAXS for structural analysis studies, including carbohydrate polymers, protein, and synthetic compounds. Additionally, the simultaneous performance of SAXS with other instruments was addressed to show the potential application value of SAXS technique in structural analysis field. Furthermore, the differences between SAXS and other similar techniques, including synchrotron X-ray diffraction (SXD), wide-angle X-ray scattering (WAXS), scanning Electronic Microscopy (SEM), and wide-angle X-ray diffraction (WAXD), were discussed to save time and money for studies on structural analysis. Moreover, the practical points and future perspectives of utilizing SAXS technique were put forwards based on the diagnostic vision on the development direction of SAXS technique. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Determining the influence of soil environmental factors on the degradation of CP4-EPSPS protein from herbicide-tolerant (HT) corn residue is crucial for assessing the ecological risks of commercialized transgenic crops. We investigated the effects of soil temperature (15 °C, 25 °C, 35 °C), soil water content (20%, 33%, 50%), pH (5.0, 7.0, 9.0) and soil treatment (not sterilized, sterilized) on the degradation of CP4-EPSPS protein released from the leaves of the HT corn variety CC-2. An exponential model (first order) was fitted to the degradation dynamics of CP4-EPSPS protein and used to estimate the time required for 50% degradation (DT50) and for 90% degradation (DT90). The results showed that CP4-EPSPS protein released from CC-2 leaves presented similar degradation features in all treatments: a sharp decrease in the early stage followed by a slow decline in the middle and late stages was observed. In the late stage (more than 120 d after the experiment started), CP4-EPSPS protein released from CC-2 leaves was not detected in the soil using enzyme-linked immunosorbent assay (ELISA) tests. The DT50 values of CP4-EPSPS protein released from CC-2 leaves ranged from 0.92 d to 11.32 d, and the DT90 values ranged from 3.11 d to 36.59 d. The results suggest that soil temperature and soil sterilization significantly affected the degradation of CP4-EPSPS protein. Faster degradation rates were observed at higher temperature under non-sterilized conditions, but soil water content and pH did not clearly affect the degradation of CP4-EPSPS protein. These findings suggest that under appropriate soil temperature conditions, CP4-EPSPS protein from HT corn residue will not persist or accumulate in the soil. [ABSTRACT FROM AUTHOR]

Abstract: BACKGROUND: Susceptibility and the mechanism (s) governing tolerance/resistance to glyphosate were characterized in two putative‐glyphosate‐resistant Chloris barbata populations (R1 and R2), collected in Persian lime orchards from Colima State, Mexico, comparing them with one non‐treated population (referred to as S). RESULTS: Glyphosate doses required to reduce fresh weight or cause mortality by 50% were 4.2–6.4 times higher in resistant populations than in the S population. The S population accumulated 4.3 and 5.2 times more shikimate than the R2 and R1 populations, respectively. There were no differences in 14C‐glyphosate uptake between R and S populations, but the R plants translocated at least 12% less herbicide to the rest of plant and roots 96 h after treatment. Insignificant amounts of glyphosate were metabolized to aminomethyl phosphonate and glyoxylate in both R and S plants. The 5‐enolpyruvylshikimate‐3‐phosphate synthase gene of the R populations contained the Pro106‐Ser mutation, giving them a resistance 12 (R2) and 14.7 (R1) times greater at target‐site level compared with the S population. CONCLUSION: The Pro106‐Ser mutation governs the resistance to glyphosate of the R1 and R2 C barbata populations, but the impaired translocation could contribute to the resistance. These results confirm the first case of glyphosate resistance evolved in this species. © 2018 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Abstract: BACKGROUND: Glyphosate is a herbicide that acts by inhibition of the enzyme, 5‐enolpyruvylshikimate‐3‐phosphate synthase (EPSPS), involved in the catalysis of an essential step in the biosynthesis of aromatic amino acids. The objective of this study was the isolation of glyphosate‐resistant bacterial strains and subsequent characterization of the gene(s) encoding glyphosate resistance in these isolates. Using an enrichment culture technique, a glyphosate‐resistant bacterium, Ochrobactrum intermedium Sq20 was isolated from glyphosate‐contaminated indigenous soil and characterized. RESULTS: An open reading frame (ORF) comprising of 1353 bp potentially encoding aroAO. intermediumSq20 was amplified from O. intermedium Sq20. It showed 97% homology with aroA genes from other Ochrobactrum spp. Physicochemical characterization revealed that aroAO. intermediumSq20 encodes a polypeptide of 450 amino acids with a calculated molecular mass of 48.9782 kDa and an isoelectric point of 5.21. Secondary structure prediction of AroAO. intermediumSq20 demonstrated a high percentage of random coils and α helices. Methodical optimization and validation of the protein structure helped to build a reliable protein model indicating the presence of 91.8% amino acid residues in most favoured regions. In addition, strain Sq20 was found to be capable of complete degradation of glyphosate at 500 mg L–1 initial concentration as the sole carbon and energy source within 4 days. CONCLUSION: A glyphosate‐resistant bacterial strain O. intermedium Sq20 was discovered. Sequence analysis and structure modelling demonstrated that AroAO. intermediumSq20 closely resembles class II EPSPS and possesses high glyphosate resistance. This provides a good foundation for functional analysis of experimentally derived crystal structures. The cloning and characterization of AroAO. intermediumSq20 will further help in understanding its role at the molecular level and its potential use in the production of glyphosate‐resistant transgenic crops. © 2017 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Glyphosate is the most important herbicide globally, and horseweed (Conyza canadensis) has been one of the most commonly encountered weed species that has developed resistance to it in various parts of the world, including Greece. After glyphosate application, horseweed populations show a wide range of phenotypic plasticity in response to selection pressure. In previous work, we have proposed a herbicide resistance mechanism that is not due to a point mutation at the codon 106 of EPSP synthase but most likely due to a synchronized overexpression of EPSPS and the ABC transporter genes. In the current study, it is hypothesized that the observed phenotypic alterations and differential expression of the EPSPS gene could be attributed to epigenetic changes. DNA methylation plays a pivotal role in many biological procedures such as gene expression, differentiation, and cellular proliferation. Sodium bisulfite sequencing was used to detect epigenetic changes that occur at the C5 position of cytosine residues within CpGdi nucleotides in two horseweed populations (resistant vs. susceptible). Results show differential methylation pattern between the two populations. This work will elucidate the naturally increased resistance of C. Canadensis to glyphosate and set the bases for future development of techniques that restrict weed resistance to herbicides. [ABSTRACT FROM AUTHOR]

The evolution of weed-resistant species threatens the sustainable use of glyphosate, which is the most important herbicide widely used in agriculture worldwide. Moreover, the high glyphosate resistance (> 180-fold based on LD 50 ) of Eleusine indica found in Malaysia, which carries a double mutation in its 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), made the control of this species more difficult. By contrast, the same species carrying the same double mutation in EPSPS (T102I + P106S) but found in China only shows a resistance level of not more than 14-fold based on GR 50 . The resistance level of this population is four times higher than that of the population carrying a single mutation (P106L). Although the members of this population survive under a high glyphosate dosage of 10,080 g ae ha − 1 , their growth was significantly inhibited by glyphosate under the recommend dose (840 g ae ha − 1 ), where in the fresh weight was 85.4% of the control. EPSPS expression, relative copy number, and EPSPS activity in this population were similar to those of the susceptible population. In addition, the expression of two glutathione transferase (GST) genes ( GST-U8 and GST-23 ) and the enzyme activity of the GST in this population did not significantly differ from those of the susceptible population. This finding is important in elucidating the resistance of the naturally evolved glyphosate-resistant (GR) weed species carrying a double mutation in EPSPS to glyphosate. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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)—a superweed collected in a GR-soybean field from Cordoba, Argentina—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 Å (wild type) to 2.93 Å (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.

To survive at low temperatures, psychrophiles seem to produce cold-adapted enzymes with a high flexibility around active sites for high catalytic efficiency. To gain insights into the cold-adaptation of psychrophilic enzymes in atomic detail, we determined the crystal structure of 5-enolpyruvylshikimate-3-phosphate synthase ( Cps EPSPS) from Colwellia psychrerythraea , a psychrophilic bacterium. EPSPS is the primary target for the broad-spectrum herbicide, glyphosate, and a promising target for the development of antimicrobial and antiparasitic agents since it is absent in animals. The crystal structure of unliganded, open Cps EPSPS was determined at 2.2 Å resolution in space group P 2 1 with two protomers per asymmetric unit. Superposition of separate domain I and II of Cps EPSPS structure with those of Escherichia coli EPSPS ( Eco EPSPS) structure showed relatively small differences of RMSD values of 0.423 Å and 0.693 Å for domains I and II, respectively, implying the residues in ligand binding and catalysis of cold-adapted Cps EPSPS showed no significant flexibility. This result is conflicting to other cases of cold-adapted proteins. We also observed that hydrogen-bond forming residues in the surface of Eco EPSPS was mutated to non- or lesser hydrogen-bond forming one in Cps EPSPS, which makes the protein surface softer and eventually makes the protein more active at low temperature. In addition, domain rotation angle between open and closed states of Cps EPSPS was smaller than those of any EPSPSs whose structures are known. The restriction of the domain closure, which reduces the entropy cost of ligand binding and catalysis, may be a novel molecular adaptations of cold-adapted enzymes. [ABSTRACT FROM AUTHOR]

Enolpyruvyl transfer from phosphoenolpyruvate (PEP) to the hydroxyl group of shikimate-5-OH-3-phosphate (S3P) is catalyzed by 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase in a reaction that involves breaking the C-O bond of PEP. Catalysis involves an addition-elimination mechanism with the formation of a tetrahedral intermediate (THI). Experiments have elucidated the mechanism of THI formation and breakdown. However, the catalytic action of EPSP synthase and the individual roles of catalytic residues Asp313 and Glu341 remains unclear. We have used a hybrid quantum mechanical/molecular mechanical (QM/MM) approach to explore the free energy surface in a reaction catalyzed by EPSP synthase. The Glu341 was the most favorable acid/base catalyst. Our results indicate that the protonation of PEP C3 precedes the nucleophilic attack on PEP C2 in the addition mechanism. Also, the breaking of the C-O bond of THI to form an EPSP cation intermediate must occur before proton transfer from PEP C3 to Glu341 in the elimination mechanism. Analysis of the FES supports cationic intermediate formation during the reaction catalyzed by EPSP synthase. Finally, the computational model indicates a proton transfer shift (Hammond shift) from Glu341 to C3 for an enzyme-based reaction with the shifted transition state, earlier than in the reference reaction in water. [ABSTRACT FROM AUTHOR]

Glyphosate is considered the world’s most important herbicide, but widespread and continual use has resulted in the evolution of resistance. Kochia scoparia (kochia) has evolved resistance via tandem gene amplification of glyphosate’s target, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and resistant populations have been reported from the Canadian Prairies and the Northern Great Plains. Here, we evaluated the fitness costs of EPSPS amplification in kochia by comparing susceptible and resistant full siblings from segregating F2 populations generated from within six populations. Kochia was expected to be highly diverse because of strong gene flow; however, six of the seven field-collected parents with higher EPSPS copy number were homozygous. Under competitive greenhouse conditions, the EPSPS type of the line’s maternal parent showed persistent effects: delayed emergence, delayed flowering, and reductions in viable seed count and weight overall. High EPSPS copy number individuals had reduced seed count and weight, reduced competitive ability, and reduced final height in mixed stands, but better germination of the F3. However, all characteristics were highly variable and fitness costs were not constant across genetic backgrounds. In the absence of selection from glyphosate, kochia with increased EPSPS copy number will be at a competitive disadvantage in some genetic backgrounds. [ABSTRACT FROM AUTHOR]

Flexibility is involved in a wide range of biological processes, such as protein assembly and binding recognition. EPSP synthase is an enzyme that must undergo a large conformational change to accommodate its ligands into its binding cavity. However, although the structure of EPSP synthase has been determined, its plasticity has not been explored in depth. Therefore, in this work, we extensively examined the influence of the flexibility of Mycobacterium tuberculosis EPSP (MtEPSP) synthase on the function of this protein using classical and replica-exchange metadynamics simulations. We were able to identify five well-populated conformational clusters for MtEPSP synthase: two corresponding to open, one to ajar, and two to closed conformations. We also pinpointed three hydrophobic regions that are responsible for guiding transitions among these states. Taken together, the new findings presented here indicate how the hydrophobic regions modulate the flexibility of MtEPSP synthase, and they highlight the importance of considering these dynamic features in drug design projects employing this enzyme as a target. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

BACKGROUND Conyza spp. were the first broadleaf weeds reported to have evolved glyphosate resistance. Several mechanisms have been proposed for glyphosate resistance. In an effort to elucidate the mechanism of this resistance in Conyza bonariensis, possible target-site and non-target-site mechanisms were investigated in glyphosate-resistant ( GR) C. bonariensis biotypes. RESULTS Using differential glyphosate applications and analyses of shikimate accumulation, we followed the herbicide effect in different plant organs and monitored the herbicide's apparent mobility. We found high shikimate levels in the roots and young leaves of glyphosate-sensitive ( GS) plants, regardless of the site of application, whereas in GR plants, shikimate accumulated mainly in treated young leaves. 14C-glyphosate studies, however, revealed the expected source-to-sink translocation pattern in both GS and GR plants. Sequencing of the appropriate EPSPS DNA fragments of GR and GS plants revealed no alteration at the Pro106 position. CONCLUSION These data support the hypothesis that the glyphosate resistance of our C. bonariensis GR biotypes is associated with altered subcellular distribution of glyphosate, which keeps the herbicide sequestered away from the EPSPS target site in the chloroplast. © 2016 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

We report here our studies of the effects of the inhibitory transmitters glycine and GABA on monosynaptic EPSP recorded in response to microstimulation of presynaptic fibers (PF PSP) close to the bodies of motoneurons in the isolated frog spinal cord. Monosynaptic PF PSP had two components, mediated by AMPA/KA and NMDA receptors. Blockade of NMDA receptors was used to analyze the characteristics of the AMPA/KA and NMDA components of PF PSP. The amplitude of the NMDA component was 20% of PF PSP amplitude. The areas under the curves of the AMPA/KA and NMDA components were 78% and 22% respectively of the area of the PF PSP. Glycine and GABA had little effect on PF PSP, but significantly decreased the decay time constant (τ), by an average of 33.4 ± 4.0% ( n = 18) on application of glycine and by 40.2 ± 3.6% ( n = 18) on application of GABA. Blockade of NMDA receptors with DL-2-amino-5-phosophonovaleric acid (AP5) decreased the effects of glycine and GABA on the time constant. Effects were reversible and the amplitude and time characteristics of responses recovered in normal solution. Assessment of the inhibitory influences of glycine and GABA on each of the components of PF PSP showed that suppression of the NMDA component produced a greater proportionate effect than suppression of the AMPA/KA component. These results show that the inhibitory effects of glycine and GABA on the monosynaptic EPSP of motoneurons are mediated mainly by the NMDA component, with a small influence from the AMPA/KA component. [ABSTRACT FROM AUTHOR]

Carduus acanthoides L. is mainly a range-land weed, but in the 2010s has begun to invade GM crop production systems in Córdoba (Argentina), where glyphosate and 2,4-D have been commonly applied. In 2020, C. acanthoides was found with multiple resistance to these two herbicides. In this study, the mechanisms that confer multiple resistance to glyphosate and 2,4-D, were characterized in one resistant (R) population of C. acanthoides in comparison to a susceptible (S) population. No differences in 14C-herbicide absorption and translocation were observed between R and S populations. In addition, 14C-glyphosate was well translocated to the shoots (∼30%) and roots (∼16%) in both R and S plants, while most of 14C-2,4-D remained restricted in the treated leaf. Glyphosate metabolism did not contribute to resistance of the R population; however, as corroborated by malathion pretreatment, the mechanism of resistance to 2,4-D was enhanced metabolism (63% of the herbicide) mediated by cytochrome P450 (Cyt-P450). No differences were found in baseline EPSPS activity, copy number, and/or gene expression between the R and S populations, but a Pro-106-Ser mutation in EPSPS was present in the R population. Multiple resistances in the R population of C. acanthoides from Argentina were governed by target site resistance (a Pro-106 mutation for glyphosate) and non-target site resistance (Cyt-P450-based metabolic resistance for 2,4-D) mechanisms. This is the first case of resistance to glyphosate and 2,4-D confirmed for this weed in the world. [Display omitted] • Resistance mechanisms to glyphosate and 2,4-D were characterized in C. acanthoides. • Absorption, translocation, or metabolism did not participate in glyphosate resistance. • Population R presented metabolic resistance to 2,4-D mediated by cytochrome P450. • Pro-106-Ser mutation conferred for the resistance to glyphosate. • TSR and NTSR mechanisms were responsible for multiple resistances in C. acanthoides. [ABSTRACT FROM AUTHOR]

Glyphosate-resistant (GR) Kochia scoparia has evolved in dryland chemical fallow systems throughout North America and the mechanism of resistance involves 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene duplication. Agricultural fields in four states were surveyed for K. scoparia in 2013 and tested for glyphosate-resistance level and EPSPS gene copy number. Glyphosate resistance was confirmed in K. scoparia populations collected from sugarbeet fields in Colorado, Wyoming, and Nebraska, and Montana. Glyphosate resistance was also confirmed in K. scoparia accessions collected from wheat-fallow fields in Montana. All GR samples had increased EPSPS gene copy number, with median population values up to 11 from sugarbeet fields and up to 13 in Montana wheat-fallow fields. The results indicate that glyphosate susceptibility can be accurately diagnosed using EPSPS gene copy number. [ABSTRACT FROM AUTHOR]

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 T5 generations in a two-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.

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. [ABSTRACT FROM AUTHOR]

Enolpyruvylshikimate-3-phosphate synthase (EPSPS) catalyzes a critical step in the biosynthesis of a number of aromatic metabolites. An essential prokaryotic enzyme and the molecular target of the herbicide glyphosate, EPSPSs are the subject of both pharmaceutical and commercial interest. Two EPSPS classes that exhibit low sequence homology, differing substrate/glyphosate affinities, and distinct cation activation properties have previously been described. Here, we report structural studies of the monovalent cation-binding class II Coxiella burnetii EPSPS (cbEPSPS). Three cbEPSPS crystal structures reveal that the enzyme undergoes substantial conformational changes that alter the electrostatic potential of the active site. A complex with shikimate-3-phosphate, inorganic phosphate (Pi), and K+ reveals that ligand induced domain closure produces an unusual cation-binding site bordered on three sides by the N-terminal domain, C-terminal domain, and the product Pi. A crystal structure of the class I Vibrio cholerae EPSPS (vcEPSPS) clarifies the basis of differential class I and class II cation responsiveness, showing that in class I EPSPSs a lysine side chain occupies the would-be cation-binding site. Finally, we identify distinct patterns of sequence conservation at the domain-domain interface and propose that the two EPSPS classes have evolved to differently optimize domain opening-closing dynamics. [ABSTRACT FROM AUTHOR]

The enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the sixth step of the seven-step shikimate pathway. Chorismate, the product of the pathway, is a precursor for the biosynthesis of aromatic amino acids, siderophores and metabolites such as folate, ubiquinone and vitamin K. The shikimate pathway is present in bacteria, fungi, algae, plants and apicomplexan parasites, but is absent in humans. The EPSP synthase enzyme produces 5-enolpyruvylshikimate 3-phosphate and phosphate from phosphoenolpyruvate and shikimate 3-phosphate via a transferase reaction, and is the target of the herbicide glyphosate. The Acinetobacter baumannii gene encoding EPSP synthase, aroA, has previously been demonstrated to be essential during host infection for the growth and survival of this clinically important drug-resistant ESKAPE pathogen. Prephenate dehydrogenase is also encoded by the bifunctional A. baumannii aroA gene, but its activity is dependent upon EPSP synthase since it operates downstream of the shikimate pathway. As part of an effort to evaluate new antimicrobial targets, recombinant A. baumannii EPSP (AbEPSP) synthase, comprising residues Ala301-Gln756 of the aroA gene product, was overexpressed in Escherichia coli, purified and crystallized. The crystal structure, determined to 2.37 Å resolution, is described in the context of a potential antimicrobial target and in comparison to EPSP synthases that are resistant or sensitive to the herbicide glyphosate. [ABSTRACT FROM AUTHOR]

This study investigated the effects of different GM maize crops on the abundances of rove beetles. It also investigated competition between rove beetles and other epigeic arthropod predatory groups (ground beetles and spiders). Altogether a total of 5926 rove beetle individuals belonging to 25 species were collected. Whilst the average number of rove beetle individuals per replicate per sampling date varied and, moreover, decreased each year, there were no observed differences in rove beetle abundance when compared between isogenic and GM maize plots within the study period. Furthermore, no clear trends on positive correlations between weed coverage and rove beetle abundances were detected. Altogether a total number of 76,070 ground beetle individuals and 11,126 spiders (including all Araneae) were collected with pitfall traps. Analyses revealed a strong negative relationship between rove beetles and the other two groups in each treatment and year. According to the results the effect of interspecific competition on rove beetle abundance in GM maize is much higher than the effects of treatments including both Bt toxins and extra glyphosate applications. [ABSTRACT FROM AUTHOR]

Background: 5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS) is the sixth and penultimate enzyme in the shikimate biosynthesis pathway, and is the target of the herbicide glyphosate. The EPSPS genes of allohexaploid wheat (Triticum aestivum, AABBDD) have not been well characterized. Herein, the three homoeologous copies of the allohexaploid wheat EPSPS gene were cloned and characterized. Methods: Genomic and coding DNA sequences of EPSPS from the three related genomes of allohexaploid wheat were isolated using PCR and inverse PCR approaches from soft white spring "Louise'. Development of genomespecific primers allowed the mapping and expression analysis of TaEPSPS-7A1, TaEPSPS-7D1, and TaEPSPS-4A1 on chromosomes 7A, 7D, and 4A, respectively. Sequence alignments of cDNA sequences from wheat and wheat relatives served as a basis for phylogenetic analysis. Results: The three genomic copies of wheat EPSPS differed by insertion/deletion and single nucleotide polymorphisms (SNPs), largely in intron sequences. RT-PCR analysis and cDNA cloning revealed that EPSPS is expressed from all three genomic copies. However, TaEPSPS-4A1 is expressed at much lower levels than TaEPSPS-7A1 and TaEPSPS-7D1 in wheat seedlings. Phylogenetic analysis of 1190-bp cDNA clones from wheat and wheat relatives revealed that: 1) TaEPSPS-7A1 is most similar to EPSPS from the tetraploid AB genome donor, T. turgidum (99.7 % identity); 2) TaEPSPS-7D1 most resembles EPSPS from the diploid D genome donor, Aegilops tauschii (100 % identity); and 3) TaEPSPS-4A1 resembles EPSPS from the diploid B genome relative, Ae. speltoides (97.7 % identity). Thus, EPSPS sequences in allohexaploid wheat are preserved from the most two recent ancestors. The wheat EPSPS genes are more closely related to Lolium multiflorum and Brachypodium distachyon than to Oryza sativa (rice). Conclusions: The three related EPSPS homoeologues of wheat exhibited conservation of the exon/intron structure and of coding region sequence, but contained significant sequence variation within intron regions. The genome-specific primers developed will enable future characterization of natural and induced variation in EPSPS sequence and expression. This can be useful in investigating new causes of glyphosate herbicide resistance. [ABSTRACT FROM AUTHOR]

To date, only aroA variants derived from Agrobacterium tumefaciens CP4 have been used to generate the commercial glyphosate-resistant crops currently available in the market. Therefore, it is of interest to increase aroA gene diversity and seek new glyphosate-tolerant genes for developing glyphosate-tolerant crops. In this study, a novel aroA gene encoding 5-enolpyruvylshikimate-3-phosphate synthase from Paracoccus denitrificans was isolated using the genomic library construction and complementary screening. Furthermore, the transgenic Arabidopsis plants with the aroA gene from P. denitrificans were obtained to confirm the potential of the novel aroA gene in developing glyphosate-resistant crops. [ABSTRACT FROM AUTHOR]

BACKGROUND Echinochloa colona is an annual weed affecting field crops and orchards in California. An E. colona population carrying a mutation in the EPSPS gene endowing resistance to glyphosate, the most widely used non-selective herbicide, was recently identified in the Northern Sacramento Valley of California. Plants from this population, from a suspected glyphosate-resistant ( GR) population, and from one susceptible (S) population collected in the Northern Sacramento Valley of California, were used to generate three GR and one S selfed lines to study possible mechanisms involved in glyphosate resistance. RESULTS Based on the amount of glyphosate required to kill 50% of the plants ( LD50), GR lines were 4-9-fold more resistant than S plants and accumulated less shikimate after glyphosate treatment. GR and S lines did not differ in glyphosate absorption, translocation or metabolism. A different target-site mutation was found in each of two of the GR lines corresponding to Pro106Thr and Pro106Ser substitutions; the mutations were found in different homoeologous EPSPS genes. No mutation was found in the third GR line, which exhibited 1.4-fold higher basal EPSPS activity and a fivefold greater LD50 than S plants. Quantitative RT-PCR revealed that GR lines had similar or lower EPSPS expression than S plants. CONCLUSION It is demonstrated that individuals with different glyphosate resistance mechanisms can coexist in the same population, individuals from different populations may carry different resistance mechanisms and different mechanisms can act in concert within single E. colona plants. However, other plant factors or resistance mechanisms appear to modulate plant expression of EPSPS sensitivity to glyphosate. © 2014 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

The objective of this work was to characterize the resistance mechanisms and the primary metabolism of a multiple resistant (MR) population of Amaranthus palmeri to glyphosate and to the acetolactate synthase (ALS) inhibitor pyrithiobac. All MR plants analysed were glyphosate-resistant due to 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene amplification. Resistance to pyrithiobac was more variable among individuals and was related to point mutations at five positions in the ALS gene sequence: A122, A205, W574, S653 and G654. All MR plants were heterozygous for W574, the most abundant mutation. In nontreated plants, the presence of mutations did not affect ALS functionality, and plants with the W574L mutation showed the highest ALS resistance level to pyrithiobac. The accumulation of the transcripts corresponding to several genes of the aromatic amino acid (AAA) and branched-chain amino acid (BCAA) pathways detected in nontreated MR plants indicated additional effects of EPSPS gene amplification and ALS mutations. The physiological performance of the MR population after treatment with glyphosate and/or pyrithiobac was compared with that of a sensitive (S) population. The increase induced in total soluble sugars, AAA or BCAA content by both herbicides was higher in the S population than in the MR population. Physiological effects were not exacerbated after the mixture of both herbicides in S or in MR populations. This study provides new insights into the physiology of a multiple resistant A. palmeri , which could be very useful for achieving effective management of this weed. • EPSPS amplification was detected in all plants of the multiple resistant population. • Simple or double ALS gene mutations were detected at positions A122, A205, W574, S653 and G654 in resistant plants • W574 was the most abundant position and the one providing the highest resistance trait. • Resistant plants showed altered expression of the aromatic and branched-chain amino acid pathways under basal conditions. • Multiresistant plants showed lower physiological effects after glyphosate and/or pyrithiobac than treated sensitive plants. [ABSTRACT FROM AUTHOR]

Glyphosate is a systemic, non-selective herbicide widely used in agriculture worldwide. It acts as an inhibitor of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase by interrupting the synthesis of essential aromatic amino acids. This pathway is not present in animals, although some studies have shown that the herbicide glyphosate can affect fish reproduction. In this study, the effect of glyphosate on sperm quality of the fish Danio rerio was investigated after 24 and 96 h of exposure at concentrations of 5 mg/L and 10 mg/L. The spermatic cell concentration, sperm motility and motility period were measured employing conventional microscopy. The mitochondrial functionality, membrane integrity and DNA integrity were measured by fluorescence microscopy using specific probes. No significant differences in sperm concentration were observed; however, sperm motility and the motility period were reduced after exposure to both glyphosate concentrations during both exposure periods. The mitochondrial functionality and membrane and DNA integrity were also reduced at the highest concentration during both exposure periods. The results showed that glyphosate can induce harmful effects on reproductive parameters in D. rerio and that this change would reduce the fertility rate of these animals. [ABSTRACT FROM AUTHOR]

The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is a key enzyme in the aromatic amino acid biosynthetic pathway in microorganisms and plants, which catalyzes the formation of 5-enolpyruvylshikimate-3-phosphate (EPSP) from shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP). In this study, a novel AroA-encoding gene was identified from the deep sea bacterium Alcanivorax sp. L27 through screening the genomic library and termed as AroAA.sp. A phylogenetic analysis revealed that AroAA.sp (1317 bp and 438 amino acids) is a class II AroA. This enzyme exhibited considerable activity between pH 5.5 and pH 8.0 and notable activity at low temperatures. The KM for PEP and IC50 [glyphosate] values (the concentration of glyphosate that inhibited enzyme activity by 50%) of AroAA.sp were 78 μM and 1.5 mM, respectively. Furthermore, site-directed mutagenesis revealed that the G100A mutant had a 30-fold increase in the IC50 [glyphosate] value; while the L105P mutant showed only 20% catalytic activity compared to wild-type AroAA.sp. The specific activity of the wild-type AroAA.sp, the G100A mutant and the L105P mutant were 7.78 U/mg, 7.26 U/mg and 1.76 U/mg, respectively. This is the first report showing that the G100A mutant of AroA displays considerably improved glyphosate resistance and demonstrates that Leu105 is essential for the enzyme's activity. [ABSTRACT FROM AUTHOR]

A general model of the catalytic mechanism for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPs) has already been proposed. But whether shikimate-3-phosphate (S3P) alone can cause EPSPs' conformation changes, and whether the binding site of phosphoenolpyruvate (PEP) and glyphosate is the same are still in debate. In this paper, DsaroA gene amplified and cloned from Dunaliella salina (our laboratory's early study) was used for DsEPSPs expression and purification. Then the DsEPSP conformation changes as it bind with different substrates were detected by fluorimetry. The results show that we obtained the DsEPSPs by prokaryotic expression and purification, and the S3P binding with DsEPSPs alone cannot cause DsEPSPs to form 'close' conformation directly. However, when S3P exits, DsEPSPs did have a trend to change to the 'close' conformation. Then the 'close' conformation can be formed completely with the addition of phosphoenolpyruvate (PEP) or glyphosate. The inorganic phosphorus can help S3P to induce two domains of DsEPSPs to form 'close' conformation. Besides, when DsEPSPs binds with S3P, in 295 nm, only the intensity of emission peak decreases, however, in 280 nm, not only the peak intensity reduces but also the blue-shift phenomenon takes place. The reason for blue-shift phenomenon was the distribution of aromatic amino acids in EPSPs. EPSPs is a good target for novel antibiotics and herbicides, because of shikimic acid pathway is only present in plants and microorganisms, completely absent in mammals, fish, birds, reptiles, and insects. The results demonstrate that the binding of substrates to EPSPs causes a conformational change from an open form to a closed form, that might be important for designing of novel antimicrobial and herbicidal agents that block closure of the enzyme. [ABSTRACT FROM AUTHOR]

The main strategy for resistance to the herbicide glyphosate in plants is the overexpression of an herbicide insensitive, bacterial 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). A glyphosate resistance strategy based on the ability to degrade the herbicide can be useful to reduce glyphosate phytotoxicity to the crops. Here we present the characterization of glyphosate resistance in transgenic alfalfa (Medicago sativa L.) expressing a plant-optimized variant of glycine oxidase (GO) from Bacillus subtilis, evolved in vitro by a protein engineering approach to efficiently degrade glyphosate. Two constructs were used, one with (GOTP+) and one without (GOTP-) the pea rbcS plastid transit peptide. Molecular and biochemical analyses confirmed the stable integration of the transgene and the correct localization of the plastid-imported GO protein. Transgenic alfalfa plants were tested for glyphosate resistance both in vitro and in vivo. Two GOTP+ lines showed moderate resistance to the herbicide in both conditions. Optimization of expression of this GO variant may allow to attain sufficient field resistance to glyphosate herbicides, thus providing a resistance strategy based on herbicide degradation [ABSTRACT FROM AUTHOR]