Effects of Aspartate and Other Compounds on Glyphosate Uptake and Growth Inhibition in Cultured Carrot Cells

Abstract

The strong correlation between glyphosate uptake and growth inhibition of cultured carrot (Daucus carota L. cv Danvers) cells incubated in the presence ofaspartate suggests that aspartate reverses glyphosate inhibition of growth primarily by reducing intracellular glyphosate concentration. Other compounds which reverse glyphosate inhibition of cell growth gave a range of effects on glyphosate uptake: succinate, a-ketoglutarate, glutamate, pyruvate, and malate at 10 millimolar and phenylalanine at 2 millimolar reduced uptake by 0,8,11,16, 27, and 34%, respectively. These results suggest that more than one mechanism of reversal may operate in these cels.Glyphosate and aspartate produced only minor effects on intracellular ammonia, media pH, and cell viability. This suggests that ammonia toxicity may not be an inportant mechanism of action of glyphosate in this system.The activity of the nonselective herbicide glyphosate [N-(phos-phonomethyl)glycinel in plants has been under investigation for more than 10 years. Early work (11) showed that glyphosateinduced growth inhibition of duckweed (Lemna gibba) and Rhizobium japonicum could be overcome by supplying aromatic amino acids. Similar results have been reported with Escherichia coli, Chlamydomonas reinhardii, Arabidopsis thaliana, and cultured cells of carrot (Daucus carota), soybean (Glycine max), and tobacco (Nicotiana tabacum) (6,8).Based on the reversal of glyphosate inhibition by aromatic amino acids, it was proposed that the herbicide may act by reducing the synthesis of these amino acids (11). Glyphosateinduced reductions in the levels of aromatic amino acids have been found in maize roots (9), soybean hypocotyls (4), wheat (16), and buckwheat hypocotyls (10). Recent work (1) has shown that the reduction in aromatic amino acid levels may result from the inhibition of an enzyme in the pathway from shikimate to the aromatic amino acids. Using a cell-free extract from Aerobacter aerogenes, the enzyme 5-enolpyruvylshikimic-3-P synthetase was identified as the specific site of glyphosate inhibition (17). Similar activity in higher plants was inferred from the accumulation of shikimate and reductions in levels of its metabolites (e.g. chorismate and anthocyanin) in cultured Galium mollugo cells and in buckwheat hypocotyls (1).In addition to the previously observed reversal of glyphosate inhibition by aromatic amino acids, it has been reported recently (12) that a variety of organic and amino acids-pyruvic, a-ketoglutaric succinic, malic, glutamic, and aspartic-reverse glyphos-'This work was carried out with funds from the Illinois Agricultural Experiment Station and National Science Foundation Grant PCM 80-10927.ate inhibition of the growth of cultured carrot cells. From work showing that organic acids can reduce ammonia toxicity in cultured cells (2), it was concluded that glyphosate may inhibit growth by causing ammonia to increase to toxic levels. Also, the tricarboxylic acid cycle indirectly feeds the shikimic acid pathway, and this cycle could be dep...

Glyphosate Uptake by Suspension-Cultured Potato (Solanum tuberosumandS. brevidens) Cells

“…The gly phosate taken up by Red Pontiac cells at 72-h uptake was 50 nmoles-g"" 1 by 72 h. This pattern of cellular uptake seems to be char acteristic for glyphosate, since it occurred in both UM and CM ( Figure 4). A similar pattern of uptake was reported in carrot cell-suspension cultures (18,26). Red Pontiac cells took up more glyphosate than S. brevidens.…”

“…Thus, the greater glyphosate uptake by Red Pontiac com pared to S. brevidens cells does not conclusively prove a greater capacity for the former to absorb glyphosate. These results with Solanum species (Figure 4) and those with carrot suspension cultures (18,26) indicate nonlinear uptake over time is a common pattern of glyphosate accumulation within plant cells.…”

Yeast of Eden: microbial resistance to glyphosate from a yeast perspective

“…Glyphosate acting as an amino acid mimic provides a mechanism of transport into the cell using D/E transporters. During the search for the mechanism of glyphosate, others research groups had proposed that glyphosate affected glutamate in plants (Killmer et al 1981 ; Nafziger et al 1983 ) but were overshadowed by the discovery of EPSPS as the TSR (Steinrücken and Amrhein 1980 ; Comai et al 1983 ). Given that glyphosate is transported as a D/E mimic, it is likely that other D/E-requiring enzymes also are negatively affected by glyphosate.…”

Selection and Characterization of a Carrot Cell Line Tolerant to Glyphosate

“…MATERIALS AND METHODS Adaptation of Cells. Garden carrot (Daucus carota L. cv Danvers) cells were cultured in a defined medium as previously described (14). Cells that grew slowly in the presence of0.25 mm glyphosate were transferred into medium containing progressively higher concentrations of the herbicide.…”