Glyphosate Inhibition

Figure 5. Inhibition of anti-glyphosate antibody by glyphosate. Inhibition curves for polyclonal anti-glyphosate antibodies raised in rabbits were conducted in 50 mM TRIS buffer at pH 5.8 and pH 7.3. The curves show an increased affinity between antibody and glyphosate at the lower pH buffer, illustrating that for some compounds, optimization for pH is critical. Careful optimization of assay conditions is especially important as the molecule becomes smaller, for zwitterionic materials, and for analytes where hydrogen bonding may play a major role in antibody binding.

Jaworski (4) reported that growth inhibition of both plant and microbes by glyphosate could be reversed by aromatic amino acids. Further work of Amrhein and his coworkers revealed that glyphosate inhibits the shikimate pathway enzyme, 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase (5). This enzyme catalyzes the reaction shown in Figure 1. Glyphosate-treated plant and bacterial cultures accumulate shikimate and/or shikimate 3-phosphate (S3P), confirming that inhibition of EPSPS is at least a part of the in vivo mechanism of action of this herbicide (6, 7). 

Based on the knowledge that glyphosate inhibits EPSPS, two mechanisms were evaluated for genetic engineering of glyphosate... 

The mode of action of glyphosate is not yet fully understood. Jaworski (1972) found in investigations on Lemna gibba and Rhizobitun Japonicum bacteria that glyphosate inhibits the biosynthesis of aromatic amino acids. 

For example, glyphosate inhibits the enzyme, EPSP (5-enolpyruvylshikimate 3-phosphate) synthase, that catalyzes a step in the synthesis of the aromatic amino acids. Similarly, both the imidazolinones and sulfonylureas inhibit acetolactate synthase (ALS), the enzyme that catalyzes the first step in the formation of branched-chain amino acids (11). Triazine herbicides act by binding to a specific protein in the thylakoid membranes of the chloroplasts, preventing the flow of electrons and inhibiting photosynthesis (12). 

Figure 4. Enzymes of Rhizobium (a) and Lemna (b) proposed as sites of glyphosate inhibition of aromatic amino acid synthesis. Abbreviations CM, chorismate mutase PDH, prephenate dehydrogenase and PD, prephenate dehydratase.

Although phenylalanine alone was able to alleviate glyphosate inhibition of anthocyanin formation, feeding tyrosine in addition to phenylalanine was required to achieve partial alleviation of the inhibition of chlorophyll formation. Additional tryptophan did not further increase the chlorophyll content of cotyledons. Further work with buckwheat indicated that a 24-h light or dark incubation of untreated excised hypocotyls had little effect on endogenous shikimate content... 

Figure 14. Conversion of shikimate-3-phosphate to 5-enolpyruvylshikimate-3-phosphate blocked by glyphosate inhibition of 5-enolpyruvylshikimate-3-phosphate synthase (%1).

The following experiments enabled us to reduce considerably the number of putative enzyme target sites of the herbicide glyphosate inhibited the incorporation of radioactively labelled shikimate into all three aromatic amino acids, and the labelled precursor was metabolized to only a minor extent. Following this lead, we found that buckwheat hypocotyls, as well as intact plants and cultured plant cells, accumulate large amounts of shikimic acid in the presence of the herbicide. In buckwheat hypocotyls, there was an excellent correlation between the accumulation of shikimic acid and the depletion of anthocyanin, and a correlation between the accumulation of shikimic acid and the reduced growth of cultured plant cells (in the absence of amino acids in the nutrient mediijm)... 

Glyphosate inhibition of 5-enolpyruvylshikimate 3-phosphate synthase from suspension-cultured cells of Nicotiana silvestris. Plant Physiol. 75 829-845. 

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