Overproduction of 5-enolpyruvylshikimate-3-phosphate synthase in a glyphosate-tolerant Petunia hybrida cell line

Abstract

Analysis of a Petunia hybrida cell culture (MP4-G) resistant to 1 m m glyphosate revealed a 15- to 20-fold increased level of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase in the herbicide-tolerant strain. Immunoblotting and enzyme kinetic measurements established that the increased EPSP synthase activity resulted from overproduction of a herbicide-sensitive form of the enzyme. Homogenous enzyme preparations were obtained from the herbicide-tolerant cell line by sequential ion-exchange, hydroxyapatite, hydrophobic-interaction, and molecular sieve chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and molecular sieve chromatography established the Petunia enzyme to be a monomeric protein with M r 49,000–55,800. K m values for phosphoenolpyruvate and shikimate 3-phosphate were about 14 and 8 μ m, respectively. Glyphosate inhibited the enzyme competitively with phosphoenolpyruvate ( K i = 0.17 μ m). These experiments provide further evidence that EPSP synthase is a major site of glyphosate action in plant cells.