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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., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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 LD 50 (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 (R 2  = 0.78) between EPSPS copy number and LD 50 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., (© 2021 Society of Chemical Industry.)

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.

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 AroA T. 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 K i /K m value of AroA T. maritima suggested that the enzyme showed powerful competitive binding capacity of PEP over glyphosate and high glyphosate tolerance. Furthermore, aroA T. 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.

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 14 C-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 ATA 102 , GTC 103 and TCA 106 instead of ACA 102 , GCG 103 , and CCA 106 , 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.

5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS) catalyzes the transfer of a carboxyvinyl group from phosphoenolpyruvate (PEP) to shikimate-3-phosphate and in plants is the target of the herbicide glyphosate. EPSPSs with high catalytic efficiency and insensitivity to glyphosate are of microbial origin, including the enzyme from Agrobacterium strain CP4, in which insensitivity is conferred by an active site alanine. In the sequence context of plant EPSPSs, alanine in place of glycine at the equivalent position interferes with the binding of both glyphosate and PEP. We show here that iterative optimization of maize EPSPS containing the G101A substitution yielded variants on par with CP4 in terms of catalytic activity in the presence of glyphosate. The improvement relative to G101A alone was entirely due to reduction in K m for PEP from 333 to 18 μm, versus 9.5 μm for native maize EPSPS. A large portion of the reduction in K m was conferred by two down-sizing substitutions (L97C and V332A) within 8 Å of glyphosate, which together reduced K m for PEP to 43 μm Although the original optimization was conducted with maize EPSPS, contextually homologous substitutions conferred similar properties to the EPSPSs of other crops. We also discovered a variant having the known glyphosate-desensitizing substitution P106L plus three additional ones that reduced the K m for PEP from 47 μm, observed with P106L alone, to 10.3 μm The improvements obtained with both Ala 101 and Leu 106 have implications regarding glyphosate-tolerant crops and weeds., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article., (© 2019 Dong et al.)

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 14 C-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., (© 2018 Society of Chemical Industry.)

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., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

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 10,080gaeha -1 , their growth was significantly inhibited by glyphosate under the recommend dose (840gaeha -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., (Copyright © 2017. Published by Elsevier Inc.)

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 (CpsEPSPS) 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 CpsEPSPS was determined at 2.2 Å resolution in space group P2 1 with two protomers per asymmetric unit. Superposition of separate domain I and II of CpsEPSPS structure with those of Escherichia coli EPSPS (EcoEPSPS) 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 CpsEPSPS 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 EcoEPSPS was mutated to non- or lesser hydrogen-bond forming one in CpsEPSPS, 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 CpsEPSPS 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., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

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. 14 C-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., (© 2016 Society of Chemical Industry.)

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., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A new 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene from Malus domestica (MdEPSPS) was cloned and characterized by rapid amplification of cDNA ends to identify an EPSPS gene appropriate for the development of transgenic glyphosate-tolerant plants. However, wild-type MdEPSPS is not suitable for the development of transgenic glyphosate-tolerant plants because of its poor glyphosate resistance. Thus, we performed DNA shuffling on MdEPSPS, and one highly glyphosate-resistant mutant with mutations in eight amino acids (N63D, N86S, T101A, A187T, D230G, H317R, Y399R and C413A.) was identified after five rounds of DNA shuffling and screening. Among the eight amino acid substitutions on this mutant, only two residue changes (T101A and A187T) were identified by site-directed mutagenesis as essential and additive in altering glyphosate resistance, which was further confirmed by kinetic analyses. The single-site A187T mutation has also never been previously reported as an important residue for glyphosate resistance. Furthermore, transgenic rice was used to confirm the potential of MdEPSPS mutant in developing glyphosate-resistant crops., (© 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.)

Glyphosate-tolerant soybeans (GTSs), the first biotechnologically improved soybeans to be marketed, became available commercially in 1996. The safety of GTSs was assessed in 2 ways: study of the introduced protein and of the soybean seed and selected processing fractions. Because soybeans are a major source of protein in most farm animals’ diets, animal feeding studies in wholesomeness were done to complement the analyses. Analysis of the expressed protein in GTSs [3-phosphoshikimate 1-carboxyvinyltransferase (EC 2.5.1.19)] showed it to be readily digestible and to possess no allergenic concerns. In addition, comparison of the composition of seeds and selected processing fractions from 2 GTS lines with the parental line showed that the lines are equivalent. Feeding studies in various animal species confirmed that the feeding value of GTS lines is comparable with that of the parental line. These studies support the conclusion that GTSs are as safe and nutritious as traditional soybeans marketed currently and can be incorporated safely into feed and food products in the near future. [ABSTRACT FROM AUTHOR]