Overproduction of 5-enolphyruvylshikimate-3-phosphate synthase in glyphosate tolerant Petunia hybrida cell line. Arch Biochem Biophys
Analysis of a Petunia hybrida cell culture (MP4-G) resistant to 1 mM glyphosate revealed a 15- to 20-fold increased level of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase in the herbicide-tolerant strain. Immunoblotting and enzyme kinetic measurements established that the increased EPSP synthase activity resulted from overproduction of a herbicide-sensitive form of the enzyme. Homogeneous enzyme preparations were obtained from the herbicide-tolerant cell line by sequential ion-exchange, hydroxyapatite, hydrophobic-interaction, and molecular sieve chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and molecular sieve chromatography established the Petunia enzyme to be a monomeric protein with Mr 49,000-55,800. Km values for phosphoenolpyruvate and shikimate 3-phosphate were about 14 and 18 microM, respectively. Glyphosate inhibited the enzyme competitively with phosphoenolpyruvate (Ki = 0.17 microM). These experiments provide further evidence that EPSP synthase is a major site of glyphosate action in plant cells.
Available from: Xiao Yang
- "In previous studies, this mechanism was evaluated as a possible strategy for glyphosate resistance in commercial agriculture (Steinrucken et al., 1986; Klee et al., 1987). Scientists at Monsanto selected glyphosateresistant cell lines in Petunia hybrida that overproduced EPSPS (Steinrucken et al., 1986). They then cloned the epsps gene from these lines and inserted it back into P. hybrida plants with the cauliflower mosaic virus 35S promoter (Shah et al., 1986). "
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ABSTRACT: Understanding evolutionary interactions among crops and weeds can facilitate effective weed management. For example, gene flow from crops to their wild or weedy relatives can lead to rapid evolution in recipient populations. In rice (Oryza sativa), transgenic herbicide resistance is expected to spread to conspecific weedy rice (Oryza sativa f. spontanea) via hybridization.
Here, we studied fitness effects of transgenic over-expression of a native 5-enolpyruvoylshikimate-3-phosphate synthase (epsps) gene developed to confer glyphosate resistance in rice. Controlling for genetic background, we examined physiological traits and field performance of crop–weed hybrid lineages that segregated for the presence or absence of this novel epsps transgene.
Surprisingly, we found that transgenic F2 crop–weed hybrids produced 48–125% more seeds per plant than nontransgenic controls in monoculture- and mixed-planting designs without glyphosate application. Transgenic plants also had greater EPSPS protein levels, tryptophan concentrations, photosynthetic rates, and per cent seed germination compared with nontransgenic controls.
Our findings suggest that over-expression of a native rice epsps gene can lead to fitness advantages, even without exposure to glyphosate. We hypothesize that over-expressed epsps may be useful to breeders and, if deployed, could result in fitness benefits in weedy relatives following transgene introgression.
Available from: ncbi.nlm.nih.gov
- "The same substitution of the corresponding site, using site-directed mutagenesis, conferred high glyphosate tolerance to plant EPSPS enzymes (Padgette et al., 1991). Overexpression of a herbicide-sensitive form of EPSPS was demonstrated to be responsible for glyphosate resistance in a petunia (Petunia hybrida) cell line (MP4-G; Steinrucken et al., 1986). "
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ABSTRACT: 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) is a key enzyme in the shikimate pathway and is targeted by the wide-spectrum herbicide glyphosate. Here, we describe the use of a selection system based on directed evolution to select glyphosate-resistant mutants of EPSPS. Using this system, the rice (Oryza sativa) EPSPS gene, mutagenized by Error-Prone polymerase chain reaction, was introduced into an EPSPS-deficient Escherichia coli strain, AB2829, and transformants were selected on minimal medium by functional complementation. Three mutants with high glyphosate resistance were identified in three independent glyphosate selection experiments. Each mutant contained a C(317)-->T transition within the EPSPS coding sequence, causing a change of proline-106 to leucine (P106L) in the protein sequence. Glyphosate resistance assays indicated a 3-fold increase in glyphosate resistance of E. coli expressing the P106L mutant. Affinity of the P106L mutant for glyphosate and phosphoenolpyruvate was decreased about 70-fold and 4.6-fold, respectively, compared to wild-type EPSPS. Analysis based on a kinetic model demonstrates that the P106L mutant has a high glyphosate resistance while retaining relatively high catalytic efficiency at low phosphoenolpyruvate concentrations. A mathematical model derived from the Michaelis-Menten equation was used to characterize the effect of expression level and selection conditions on kinetic (Ki and Km) variation of the mutants. This prediction suggests that the expression level is an important aspect of the selection system. Furthermore, glyphosate resistance of the P106L mutant was confirmed in transgenic tobacco (Nicotiana tabacum), demonstrating the potential for using the P106L mutant in transgenic crops.
Available from: Milvia Luisa Racchi
- "Concentrations corresponding to the estimated ID50 values for whole plant extracts ranged from 5 to 10 pM, a range similar to those previously reported for. a maize cell culture (Forlani et al ., 1992) and for EPSP synthase from dicotyledonous species (see for instance Rubin et al ., 1984; Steinriicken et al ., 1986 ; Sellin et al ., 1992) . The confidence limits largely overlapped, thus ruling out the possibility that the "
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ABSTRACT: Variation in susceptibility to the safe broad-spectrum herbicide glyphosate was investigated in maize. Eleven inbred lines, grown in a growth chamber, were evaluated for their tolerance to the herbicide at 2.4 mM (0.2 kg a.i. in 400 I ha-1 of water). Following treatment with glyphosate at the three-leaf stage, significant variation in damage, expressed as visual injury ratings scored 7, 14 and 21 days after the application of the herbicide, was found. Effects on dry weight and shoot height were consistent with visual scores and the carbon-exchange rate was found to be a sensitive index of differential injury.
Biochemical characterization of 5-enol-pyruvyl-shikimate-3-phosphate (EPSP) synthase, the main target of the herbicide, ruled out the possibility that this differential susceptibility was due to variations in the sensitivity of the enzyme. On the contrary, a positive correlation was found between in vivo tolerance and EPSP synthase levels, measured at different stages during seedling growth. This result suggests that a naturally occurring difference in EPSP synthase levels in the tissues may contribute to the differential response observed in vivo in maize inbreds.
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