N‐(Phosphonomethyl)glycine (glyphosate) tolerance in Euglena gracilis acquired by either overproduced or resistant 5‐enolpyruvylshikimate‐3‐phosphate synthase

Abstract

Photoautotrophic cells of Euglena gracilis can be adapted to N-(phosphonomethy1)glycine (glyphosate) by cultivation in media with progressively higher concentrations of the herbicide. Two different mechanisms of tolerance to the herbicide were observed. One is characterized by the overproduction and 40-fold accumulation of the target enzyme, 5-enolpyruvylshikimate-3-phosphate synthase, in cells adapted to 6 mM N-(phosphonomethy1)glycine. The other is connected with a herbicide-insensitive enzyme. No evidence was obtained for the involvement of the putative multifunctional arom protein previously reported to be involved in the biosynthesis of aromatic amino acids in Euglena. Cells adapted to N-(phosphonomethy1)glycine excreted shikimate and shikimate 3-phosphate into the medium; the amounts depended on the actual concentration of the herbicide. Two-dimensional gel electrophoresis and determination of 5-enolpyruvylshikimate-3-phosphate synthase activity in crude extracts, as well as after separation by non-denaturing gel electrophoresis, revealed that the overproduction of the enzyme in adapted cells correlates with the accumulation of a 59-kDa protein. Overproduction of this 59-kDa protein resulted from a selectively increased level of a mRNA coding for a 64.5-kDa polypeptide which appeared in adapted cells, as shown by cell-free translation in the wheat germ system.In contrast to this quantitative, adaptive type of tolerance, the second mechanism causing tolerance to N-(phosphonomethy1)glycine in the Euglena cell line NR 6/50 was probably related to a qualitatively altered 5-enolpyruvylshikimate-3-phosphate synthase, which could not be inhibited by even 2 mM N-(phosphonomethy1)glycine in vitro. In agreement with this observation, the putatively mutated cell line excreted neither shikimate nor shikimate 3-phosphate into the growth medium containing N-(phosphonomethyl)glycine, even if cultivated in the presence of 20 mM or 50 mM N-(phosphonomethy1)glycine. synthases characterized so far are monomeric plastid proteins of a relatively uniform molecular mass between 46-kDa and 47.6-kDa.In contrast to higher plants and bacteria, S-enolpyruvylshikimate-3-phosphate synthases of higher, as well as several lower fungi, e. g. from Neurospora crassa [l 1, 121, Aspergillus nidulans [13] and Schizosaccharomycespombe [14,15], are part of the multifunctional arom protein. A similar organization of enzyme activities, which convert 3-deoxy-~-arabinoheptulosonate 7-phosphate to 5-enolpyruvylshikimate 3-phosphate by steps 2 -6 of the pre-chorismate pathway, has been proposed for the unicellular phytoflagellate Euglena gracilis [16, 171. Since it is known that the 5-enolpyruvylshikimate-3-phosphate synthase domain of the pentafunctional arom protein of N. crassa is sensitive to N-(phosphonomethy1)glycine [18], this should be an excellent tool for probing the nature of the arom protein in E. gracilis.In this paper we demonstrate that Euglena can adapt to N-(phosphonomethy1)glycine when the herbicide concentration of the medium is p...

The Shikimate Pathway and Its Branches in Apicomplexan Parasites

“…This increase in activity may be the result of increased gene expression, as in C. sempervirens [97], or by gene amplification, as has been demonstrated in petunia [98], carrot [99], and tobacco [100]. While the adaptive mechanism appears to be the more common in plants and fungi, studies of glyphosate resistance in E. gracilis have demonstrated that either mechanism may occur [101].…”

Molecular Characterization of Photomixotrophic Tobacco Cells Resistant to Protoporphyrinogen Oxidase-Inhibiting Herbicides

“…Glyphosate inhibits the conversion of shikimate 3-phosphate to EPSP, which is catalyzed by EPSP synthase (Reinbothe et al, 1991). The mRNA levels of EPSP synthase in two lines of resistant carrot cells, CAR and CI, subjected to stepwise selection were 59 and 32 times the level of their respective wild-type cells (Shyr et al, 1992).…”

In Vitro Reconstitution of Light-harvesting POR-Protochlorophyllide Complex with Protochlorophyllides a and b

“…For the experiment described in the legend to Fig. 8, two parallel samples were prepared, of which one was exposed to flash light as above, whereas the other was kept in darkness before nondenaturing electrophoresis (25).…”

“…These and an original etioplast sample were analyzed with respect to the PORA and PORB protein abundances as well as Pchlide a and Pchlide b levels. Moreover, we used nondenaturing, analytical PAGE (25) to directly visualize POR-pigment complexes in etioplasts prior to fractionation. Table II shows the determined PORA and PORB as well as Pchlide a and Pchlide b levels.…”