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In the presence of the nonselective herbicide glyphosate (N-Iphosphonomethyliglycine), buckwheat

The broad-spectrum, non-selective herbicide glyphosate [N-(phosphonomethyl)glycine] is a potent inhibitor of highly purified 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (3-phosphoshikimate 1 -carboxyvinyltransferase, EC 2.5.1.19) of Klebsiella pneumoniae. The inhibition is competitive with phosphoenolpyruvate (PEP) with Ki = 1 FM at pH 6.8 and non-competetive with shikimate 3-phosphate, EPSP, and inorganic phosphate. Nonherbicidal analogues of glyphosate, such as aminomethylphosphonic acid, bis-N-(phosphonomethy1)glycine and iminodiacetic acid, do not inhibit the enzyme. Inhibition of EPSP synthase by glyphosate strongly increases with increasing pH. Glyphosate protects the enzyme against inactivation by phenylglyoxal, 3-bromopyruvate, and N-ethylmaleimide. It is proposed that glyphosate binds to the PEP-binding site of EPSP synthase as a transitionstate analogue of PEP. Other PEP-utilizing enzymes were not found to be subject to inhibition by glyphosate.In the preceding paper [I] the purification and some physical and kinetic properties of the shikimate pathway enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase of Klebsiellu pneumoniue have been described. That effort was undertaken as a sequel to our initial discovery that EPSP synthase is subject to inhibition by the herbicide glyphosate [N-(phosphonomethy1)-glycine, Fig. 31 [2]. This compound is the herbicidal component of Roundup@, which is widely used as a broad-spectrum, non-selective post-emergence weedkiller. In the first published report on the mode of action of glyphosate [3] an interference of glyphosate with the biosynthesis of the aromatic amino acids in the bacterium Rhizobium japonicum and the higher plant Lemna gibba was deduced from the observation that aromatic amino acids, in particular phenylalanine, alleviate the inhibition of growth by glyphosate. In agreement with this hypothesis we observed an inhibition of the synthesis of chorismate-derived primary and secondary metabolites, as well as a massive accumulation of shikimic acid, in plants exposed to glyphosate [4 -61. As the biosynthesis of chorismate proceeds via the sequence shikimate+shikimate 3-phosphate (S3P)+5-enolpyruvylshikimate 3-phosphate (EPSP)-+chorismate [7], the inhibition of EPSP synthase by glyphosate [2] offered a satisfactory explanation for these observations. The potent inhibition of EPSP synthase by glyphosate has been confirmed for the enzymes of Escherichia coli [8], Klebsiellu pneumoniae [9],

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Cultured carrot (Daucs carota L.) cells were adapted to growing in 25 millimolar glyphosate by transfer into progressively higher concentrations ofthe herbicide. Tolerance was increased 52-fold, and the adaptation was stable in the absence of glyphosate. The uptake of glyphosate was similar for adapted and nonadapted cells. Activity of the enzyme 5-enolpyruvylshikimic acid-3-phosphate synthase was 12-fold higher in the adapted line compared to nonadapted cells, while activities of shikimate dehydrogenase and anthranilate synthase were similr in the two cell types. The adapted cells had higher levels of free amino acids-especially threonine, methionine, tyrosine, phenylalanine, tryptophan, histidine, and arginine than did nonadapted cells. Glyphosate treatment caused decreases of 50 to 65% in the levels of serine, glycine, methionine, tyrosine, phenybaanine, and tryptophan in nonadapted cells, but caused little change in free amino acid levels in adapted cells.The adaptation reported here supports the growing body of evidence linking tolerance to glyphosate with increased levels of the enzyme 5-enolpyruvylshikimic acid-3-phosphate synthase. The elevated levels of aromatic amino acids, which may confer resistance in adapted cells, suggest that control of the shikimate pathway may be altered in these cells.The overproduction of enzymes required for growth has been observed in inhibitor-resistant lines of both mammalian (1, 10) and plant (3, 19) cells. In the case of the mammalian cells, the increases were due to gene amplification. The shikimic acid pathway enyme EPSP2 synthase has been found to be strongly inhibited by the broad-spectrum herbicide glyphosate (N-[phosphonomethyl]glycine) (15), and adaptation of plant cells to this herbicide has been accompanied by large increases in the extractable activity of this enzyme (3).While higher levels of EPSP synthase activity have been reported for cells growing in glyphosate, the effect of this change on levels of aromatic amino acids has not been reported. Glyphosate Uptake. Two g of cells in log phase were transferred into 100 ml medium containing 910 cpm/ml of (methyl-'4C) glyphosate and a total glyphosate concentration of0.25 mm.Three replicate flasks were used for each cell type. At 1, 2, 4, 8, 24, and 48 hr after transfer, 10-ml aliquots were taken and cells were collected on Miracloth. After washing three times with 5 ml of medium containing 0.25 mM unlabeled glyphosate, cells were weighed and dispersed in gel formed by adding 4 ml H20 and 15 ml Aquasol. Radioactivity was determined with a Packard scintillation spectrometer.Amino Acids. Five g of cells in early stationary phase (10 d after transfer) were inoculated into 100 ml fresh medium in 250-ml flasks. Four