Selective overproduction of 5-enol-pyruvylshikimic acid 3-phosphate synthase in a plant cell culture which tolerates high doses of the herbicide glyphosate.

C C Smart,D Johänning,G Müller,N Amrhein

doi:10.1016/s0021-9258(17)36242-7

Abstract

Cultured cells of the higher plant Corydalis sempervirens Pers. which had been adapted to growing in the presence of 5 mM glyphosate (N-[phosphonomethyl]-glycine), a herbicide and a potent specific inhibitor of the shikimate pathway enzyme 5-enol-pyruvylshikimate-3-phosphate (EPSP) synthase, had a nearly 40-fold increased level of the extractable activity of EPSP synthase. Activities of five other shikimate pathway enzymes were, however, similar in the adapted and nonadapted cells, and the concentrations of the free aromatic amino acids in the two cell lines were also similar. EPSP synthases purified from glyphosate-adapted, as well as nonadapted cells, had identical physical, kinetic, and immunological properties, which indicated that the glyphosate-sensitive enzyme was overproduced in the adapted culture. Overproduction of EPSP synthase in the adapted culture was unequivocally established by two-dimensional polyacrylamide gel electrophoresis, as well as by one-dimensional sodium dodecyl sulfate-gradient gel electrophoresis and quantitation of EPSP protein by immunoassay after transfer to nitrocellulose membranes. While about 0.06% of the total soluble protein from nonadapted cells was EPSP synthase protein, the proportion was 2.6% in the adapted cells. In vivo pulse-labeling experiments with [35S]methionine established that the adapted cells have an increased rate of EPSP synthase protein synthesis.

Highlights

Cultured cells of the higher planCt orydalis semper- 1982, 1984S;tark and Wahl, 1984).in at least one virens Pers. which had been adapted to growing in tchaese, overproduction of argininosuccinate synthetase in culpresenceof5 mM glyphosate (N-[phosphonomethyl]- tured human cells, the basis for resistance is an alteration of glycine), a herbicide and a potent specific inhibitor of transcriptional regulation (Su et al, 1981)
We have recently shown in our laboratory (Amrhein et al, adapted cells have an increasedrate of EPSP synthase 1983)that cultured cells of the higher plant Corydalis semprotein synthesis
5-enol-Pyruvylshikirnate-3-pOShvyoensrtphhraaostdeeuction plants, in particular, produce in addition a multitude of nat- C. sempervirens, andlittle, if any, growth is observed at ural productswhich are biosynthesized from intermediates or concentrations of the inhibitor exceeding 0.5 mM (Fig. 1).In end productsof the shikimate pathway (Haslam, 1974).Prior agreement with results obtained with other plantcell cultures to the identification of EPSP synthase as the target of gly- (Haderlieet al., 1977;Gresshoff, 1979),the effect of glyphosate phosatein microorganisms andplants, it had been shown can be fully reversed by a combination of phenylalanine, that glyphosate inhibition of cell growth could be reversed by tyrosine, and tryptophanin the culture medium (Table I)

Summary

Present address

Max-Planck-Institut fur Ernahrungsphysiologie, D 4600 Dortmund, Federal Republic of Germany. 7 To whom correspondence should be addressed. 5-enol-Pyruvylshikirnate-3-pOShvyoensrtphhraaostdeeuction plants, in particular, produce in addition a multitude of nat- C. sempervirens, andlittle, if any, growth is observed at ural productswhich are biosynthesized from intermediates or concentrations of the inhibitor exceeding 0.5 mM (Fig. 1).In end productsof the shikimate pathway (Haslam, 1974).Prior agreement with results obtained with other plantcell cultures to the identification of EPSP synthase as the target of gly- (Haderlieet al., 1977;Gresshoff, 1979),the effect of glyphosate phosatein microorganisms andplants, it had been shown can be fully reversed by a combination of phenylalanine, that glyphosate inhibition of cell growth could be reversed by tyrosine, and tryptophanin the culture medium (Table I). Due to tissues,’ and metabolic resistance by accumulation of a sub- the fact that for some experimental purposes cultures from strate which effectively competes with the inhibitor (Chris- both the adapted and the nonadapted strains were required topherson and Duggleby, 1983) does not, develop with roughly the same fresh weight in thelogarithmic phase, in the case of glyphosate.

RESULTS

Method I

DISCUSSION