Glyphosate shikimate accumulation

Figure 13. Glyphosate effect on anthraquinone production and shikimate accumulation in G. mollugo cell cultures ( 84j. Cells were harvested 10 days after inoculation with 2 cm of packed cells into 25 mL medium containing glyphosate. Anthraquinone content of the cells was corrected for the amount present in the inoculum.

The ability of glyphosate to block phenylalanine synthesis, resulting in shikimate accumulation, makes this herbicide a useful tool for examining control of secondary metabolism. 

The effects of glyphosate on phenolic compound production are two-fold 1) accumulation of phenolic compounds that are derivatives of aromatic amino acids is reduced and 2) pools of phenolic compounds derived from constituents of the shikimate pathway prior to 5-enolpyruvylshikimate-3-phosphate become larger. Assays that do not distinguish between effects on these two groups, such as that for hydroxyphenolics of Singleton and Rossi (18), can lead to equivocal and difficult to interpret results (e.g. 3-5). 

All carbons are derived from either erythrose 4-phosphate (light purple) or phosphoenolpyruvate (pink). Note that the NAD+ required as a cofactor in step (3) is released unchanged it may be transiently reduced to NADH during the reaction, with formation of an oxidized reaction intermediate. Step (6) is competitively inhibited by glyphosate (COO—CH2—NH—CH2—PO ), the active ingredient in the widely used herbicide Roundup. The herbicide is relatively nontoxic to mammals, which lack this biosynthetic pathway. The chemical names quinate, shikimate, and chorismate are derived from the names of plants in which these intermediates have been found to accumulate. 

Alfalfa plants, derived from tissue culture and tolerant to Roundup, were evaluated to investigate their mechanism of resistance. No difference was found in the level of shikimate-3-biphosphate which accumulated in both resistant and susceptible lines upon treatment with glyphosate. 

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). 

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)... 

Tissues and cultured cells accumulate predominantly shikimate, rather than S-3-P, in the presence of glyphosate, and in cultured buckwheat cells shikimate was shown to accumulate in the vacuole.As neither S-3-P nor shikimate compete with glyphosate for binding sites on EPSP synthase, and as PEP does not accumulate in glyphosate-treated cells (Amrhein, unpublished), the inhibition of EPSP synthase is not overcome. This example illustrates that in order to effectively inhibit an enzyme vivo the inhibitor need not necessarily be of the tight-binding or suicide (Kcat active-site-directed irreversible) type. 

The pattern for glyphosate looks different again. One metabolite increases dramatically, namely shikimic acid. It is known that, after glyphosate treatment, plants accumulate shikimic acid as a consequence of inhibiting its further transformation (11). If we had not known of this mode of action, the response pattern would have given a clue to it. 

Endogenous levels of accumulated shikimate in horseweed 3 DAT with 1.9 kg ae/ha glyphosate applied as Roundup Ultramax herbicide. 

Table III. Accumulation of Endogenous Shikimate in Two (denoted East and West) Glyphosate-Resistant and Giyphosate-Susceptible (S)...

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