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Ultrastructural localisation by protein A-gold immunocytochemistry of 5-enolpyruvylshikimic acid 3-phosphate synthase in a plant cell culture which overproduces the enzyme

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Abstract

Recently we have shown that cultured cells of the higher plant Corydalis sempervirens Pers., adapted to growth in the presence of high concentrations of the herbicide glyphosate, a potent specific inhibitor of the shikimate pathway enzyme 5-enolpyruvylshikimic acid 3-phosphate (EPSP) synthase (EC 2.5.1.19, 3-phosphoshikimate 1-carboxyvinyltransferase) oversynthesize the EPSP synthase protein (Smart et al., 1985, J. Biol. Chem. 260, 16338–16346). We now report that the EPSP synthase protein can be detected in cells of the adapted as well as of the non-adapted strain by the use of protein A-colloidal gold immunocytochemistry. The overproduced EPSP synthase in the glyphosate-adapted cells is located exclusively in the plastid and we find no evidence for the existence of extra-plastidic EPSP synthase in either strain.

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Abbreviations

EPSP:

5-enolpyruvylshikimic acid 3-phosphate

References

  • Amrhein, N., Johänning, D., Schab, J., Schulz, A. (1983) Biochemical basis for glyphosate-tolerance in a bacterium and a plant tissue culture. FEBS Lett. 157, 191–196

    Google Scholar 

  • Amrhein, N., Schab, J., Steinrücken, H.C. (1980) The mode of action of the herbicide glyphosate. Naturwissenschaften 67, 356–357

    Google Scholar 

  • Bickel, H., Palme, L., Schultz, G. (1978) Incorporation of shikimate and other precursors into aromatic amino acids and phenylquinones of isolated spinach chloroplasts. Phytochemistry 17, 119–124

    Google Scholar 

  • Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein dye binding. Anal. Biochem. 72, 248–254

    Google Scholar 

  • Comai, L., Facciotti, D., Hiatt, W.R., Thompson, G., Rose, R.E., Stalker, D.M. (1985) Expression in plants of a mutant aro A gene from Salmonella typhimurium confers tolerance to glyphosate. Nature 317, 741–744

    Google Scholar 

  • Craig, S., Millerd, A. (1981) Pea seed storage proteins-immunocytochemical localisation with protein A-gold by electron microscopy. Protoplasma 105, 333–339

    Google Scholar 

  • d'Amato, T.A., Ganson, R.J., Gaines, C.G., Jensen, R.A. (1984) Subcellular localisation of chorismate-mutase isoenzymes in protoplasts from mesophyll suspension-cultured cells of Nicotiana silvestris. Planta 162, 104–108

    Google Scholar 

  • Ellis, R.J. (1981) Chloroplast proteins: synthesis, transport, and assembly. Annu. Rev. Plant Physiol. 32, 111–137

    Google Scholar 

  • Feierabend, J., Brassel, D. (1977) Subcellular localisation of shikimate dehydrogenase in higher plants. Z. Pflanzenphysiol. 82, 334–346

    Google Scholar 

  • Goers, S.K., Jensen, R.A. (1984) Separation and characterisation of two chorismate-mutase isoenzymes from Nicotiana silvestris. Planta 162, 109–116

    Google Scholar 

  • Holländer-Czytko, H., Amrhein, N. (1986) 5-Enolpyruvylshikimate 3-phosphate synthase, the target enzyme of the herbicide glyphosate, is synthesised as a precursor in a higher plant. Plant Physiol., in press

  • Horisberger, M., Rosset, J. (1977) Colloidal gold, a useful marker for transmission and scanning electron microscopy. J. Histochem. Cytochem. 25, 295–305

    Google Scholar 

  • Hurn, B.A.L., Chantler, S.M. (1980) Production of reagent antibodies. Methods Enzymol. 70, 124–125

    Google Scholar 

  • Mousdale, D.M., Coggins, J.R. (1985) Subcellular localisation of the common shikimate-pathway enzymes in Pisum sativum L. Planta 163, 241–249

    Google Scholar 

  • Neumann, P.R., Hart, G.E. (1983) Genetic control of shikimate dehydrogenase in hexaploid wheat. Biochem. Genet. 21, 963–968

    Google Scholar 

  • Olmsted, J.B. (1981) Affinity purification of antibodies from diazotised paper blots of heterogeneous protein samples. J. Biol. Chem. 256, 11955–11957

    Google Scholar 

  • Roth, J. (1982) The protein A-gold technique-A qualitative and quantitative approach for antigen localisation on thin sections. In: Techniques in immunocytochemistry, pp. 108–133, Bullock, G.P., Petrusz, P., eds. Academic Press, New York London

    Google Scholar 

  • Rothe, G.M., Hengst, G., Mildenberger, I., Scharer, H., Utesch, D. (1983) Evidence for an intra- and extraplastidic pre-chorismate pathway. Planta 157, 358–366

    Google Scholar 

  • Rubin, J.L., Gaines, C.G., Jensen, R.A. (1984) Glyphosate inhibition of 5-enolpyruvylshikimate-3-phosphate synthase from suspension-cultured cells of Nicotiana silvestris. Plant Physiol. 75, 839–845

    Google Scholar 

  • Smart, C.C., Johänning, D., Müller, G., Amrhein, N. (1985) Selective overproduction of 5-enolpyruvylshikimic acid 3-phosphate synthase in a plant cell culture which tolerates high doses of the herbicide glyphosate. J. Biol. Chem. 260, 16338–16346

    Google Scholar 

  • Stalker, D.M., Hiatt, W.R., Comai, L. (1985) A single amino acid substitution in the enzyme 5-enolpyruvylshikimate-3-phosphate synthase confers resistance to the herbicide glyphosate. J. Biol. Chem. 260, 4724–4728

    Google Scholar 

  • Weeden, N.F., Gottlieb, L.D. (1980) The genetics of chloroplast enzymes. J. Hered. 71, 392–396

    Google Scholar 

  • Weibel, E.R. (1973) Stereological techniques for electron microscopic cytology. In: Principles and techniques of electron microscopy, vol. 3: Biological applications. pp. 238–296, Hayat, M.A., ed. Van Nostrand Rheinhold, New York

    Google Scholar 

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Smart, C.C., Amrhein, N. Ultrastructural localisation by protein A-gold immunocytochemistry of 5-enolpyruvylshikimic acid 3-phosphate synthase in a plant cell culture which overproduces the enzyme. Planta 170, 1–6 (1987). https://doi.org/10.1007/BF00392373

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  • DOI: https://doi.org/10.1007/BF00392373

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