Effect of Glyphosate on Carrot and Tobacco Cells

Abstract

The growth of suspension-cultured carrot (Daucus carota L.) and tobacco (Nicotiana tabacum L. cv. Xanthi) cells was inhibited by glyphosate (N-[phosphonomethyllglycine). This inhibition was reversed by adding combinations of phenylalanine, tyrosine, and tryptophan or casein hydrolysate. Casein hydrolysate and phenylalanine + tyrosine + tryptophan were the most effective treatments. Reversal of glyphosateinduced inhibition occurred only if the aromatic amino acids were added during the first 8 days of glyphosate incubation. Glyphosate uptake was not reduced when the aromatic amino acids or casein hydrolysate were added.Even though phenylalanine biosynthesis is a suggested site for glyphosate action, inhibitory levels of glyphosate did not lower free phenylalanine concentrations in carrot cells within 10 days. "C-Phenylalanine studies indicated that the metabolic pool size was, likewise, not decreased.In carrot ceUs total free amino acids increased within 6 hours after glyphosate addition. Cell protein levels declined within 48 hours following glyphosate treatment.Studies on 14C-thymidine and 14C-uridine incorporation were complicated by rapid metabolism of these compounds to 14CO2.Glyphosate (N-[phosphonomethyl]glycine) is a nonselective herbicide with particular potential for perennial weed control due to excellent translocation (4,7,13).Initial studies on the mechanism of glyphosate action indicated that phenylalanine biosynthesis was inhibited (9). Later, glyphosate was shown to reduce photosynthesis in whole plants by 72 hr (13), affect chloroplast ultrastructure (2), and induce chromosome aberrations (1). Jaworski (9) found that the addition of phenylalanine with glyphosate partially reversed glyphosate inhibition of Lemna gibba L. Tyrosine and tryptophan did not prevent glyphosate inhibition, but when the aromatic amino acids, phenylalanine, tyrosine, and tryptophan, were combined, growth inhibition was reversed. Some phenylalanine precursors and derivatives also helped reduce growth inhibition. Other amino acids, some cofactors, and cinnamic acid did not reverse the inhibition. Similar results were noted for Rhizobium japonicum (9). Phenylalanine content decreased whereas the tyrosine level increased in glyphosate-treated Lemna (9). Because of these observations, an inhibition of phenylalanine biosynthesis at or near chorismate mutase or prephenate dehydratase was proposed as the primary mechanism of action. Roisch and Lingens (I 1) also found that Escherichia coli growth inhibition from glyphosate was prevented by all three aromatic amino acids. They found that glyphosate had no effect on the activities of I This work was supported by funds from the Illinois Agricultural Experiment Station. Research from Ph.D. thesis of senior author.2 Present address: Dept. of Agronomy, University of Nebraska, Lincoln, Neb. 68583. chorismate mutase, prephenate dehydrogenase, or prephenate dehydratase. However, glyphosate did inhibit 3-deoxy-2-oxo-Darabinoheptonic acid 7-phosphate synthetase and 5-dehydroquinic a...

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

“…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).…”

“…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).…”

“…By 2 d, these differences had largely disappeared, after which ammonia levels in the control and in both aspartate treatments declined. In contrast, ammonia level in cells treated with glyphosate alone rose between 2 and 4 d, and then declined by 8 The data presented in Figure 5 do not strongly support the suggestion (12) that glyphosate may cause ammonia toxicity. It has been found (2) that tobacco cells supplied with ammonia and organic acids grew very well with intracellular ammonia levels of 13 mol/g fresh weight, which is more than three times the maximum level observed in this study.…”

“…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.…”

The Site of the Inhibition of the Shikimate Pathway by Glyphosate

“…High concentrations of the herbicide inhibited the two initial enzymes of the shikimate pathway, but this inhibition was apparently too slight to account for growth inhibition caused by low glyphosate concentrations. Inhibition by glyphosate of the growth of carrot and tobacco cells was alleviated by the addition of the three aromatic amino acids, but Haderlie et al (15) hesitated to conclude that the synthesis of the aromatic amino acids was inhibited by glyphosate because glyphosate did not appreciably reduce the cellular concentrations of these amino acids. Just recently, Greshoff (14) extended the earlier studies of Jaworski (20) and Haderlie et al (15) with an investigation on glyphosate's growth inhibition of E. coli, Chlamydomonas reinhardtii, carrot and soybean cell cultures, and Arabidopsis thaliana seedlings.…”