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

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Abstract
Photoautotrophic cells of Euglena gracilis can be adapted to N‐(phosphonomethyl)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‐(phosphonomethyl)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‐(phosphonomethyl)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‐(phosphonomethyl)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‐(phosphonomethyl)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‐(phosphonomethyl)glycine.