Purified phaseolotoxin inhibits the growth of carrot cells. Such inhibitions can be reversed completely by citrufline but not by arginine. This toxin inhibits ornithine transcarbamylase activity in vitro, which leads to an accumulation of ornithine and a decrease in argnine levels intracellularly. In carrot cells, 5-fluorouracil (5-FU) toxicity can be reduced by the addition of purified toxin and citrulline, or ornithine. The toxin also decreases the incorporation of I'4Cluracil and '4C15-FU into trichloroacetic acid precipitable material by 50%. Finally, a 5-FU-resistant line, F5 (Sung ZR, Jacques S 1980 Planta 148: 389-396), was found to be more sensitive to the toxin than were 5-FU-sensitive cells. One millimolar 5-FU roughly doubled the ability of F5 to tolerate phaseolotoxin. These results demonstrate a close regulation between the pyrimidine and arginine pathways in carrots.toxin, N8-(phosphosulfamyl)ornithylalanylhomoarginine (12), which inhibits OTC (18). Blockage of citrulline synthesis by phaseolotoxin may cause ornithine accumulation in carrots, as has been shown in bean (17). Ornithine is shown to increase in vitro CPS enzyme activity and to alleviate in vitro uridine monophosphate inhibition of CPS in Phaseolus aureus (16). The inhibition of OTC for citrulline synthesis, and the accumulation of ornithine, may allow more pyrimidine synthesis and, hence, increase resistance to 5-FU toxicity.Here, we use two inhibitors, one in each of the pyrimidine and arginine pathways, in studying the regulation of their biosynthesis. Purified phaseolotoxin is used to confirm previous results of culture filtrate's effect in reversing 5-FU toxicity on carrot culture. The 5-FU-resistant carrot variant F5 is used to study the effect of 5-FU in counteracting phaseolotoxin toxicity on carrots. We also compare the effect of phaseolotoxin on the growth of F5 and a 5-FU-sensitive carrot line, WOO IC.The pyrimidine and arginine biosynthetic pathways interact via their common precursor CP2 [see review by O'Donovan and Neuhard (14)1. In plants, the utilization of CP for pyrimidine nucleotide biosynthesis follows the orotic acid pathway (10), which begins with the conversion of CP and aspartate to carbamyl aspartate by aspartate transcarbamylase. OTC, on the other hand, converts ornithine and CP to citrulline, which eventually leads to arginine synthesis. The concentration of CP must be carefully regulated to produce adequate amounts of both pyrimidine and arginine, and a disturbance in one pathway may affect the synthesis of the other metabolite. In mammals, lesions in OTC result in overproduction and excretion of orotic acid (5).Metabolite analogs and enzyme inhibitors that interfere with a biosynthetic pathway or inhibit a specific enzyme are useful in studying metabolic consequences in related pathways. Hoogenraad et al. (7) showed that N-(phosphonacetyl)-L-ornithine, an inhibitor of OTC, not only inhibited the conversion of CP to citrulline, but also stimuilated the synthesis of orotate and uridine nucleotides i...

Physiological studies of 5-fluorouracil (5-FU)-resistant cell line of wild carrot (Daucus carota L.), F5, showed that this variant is also resistant to 5-fluorouridine, but is as sensitive to 6-azauracil as the 5-FU-sensitive parent line, WOO1C. High levels of exogenous uracil, uridine, and thymine are slightly toxic to F5, but not to WOO1C. 5-FU sensitivity in WOO1C cannot be reversed by bases and nucleosides; bases like uracil and thymine even increase 5-FU toxicity. No substantial differences were found in the uptake, incorporation and degradation of WOO1C and F5. Carrot cultures seem to take up 5-FU by rapid diffusion, the kinetics being characteristic of non-saturable uptake, with infinite Km and zero Vmax. The rapid uptake of 5-FU and extensive degradation of bases and nucleosides are probably responsible for the inability of uracil and uridine to reverse the growth inhibition caused by 5-FU in carrot cells while, as shown earlier, phaseolotoxin ((N-phosphosulfamyl)ornithinylalanylhomoarginine), an inhibitor of the arginine biosynthetic enzyme, ornithine transcarbamylase was capable of reducing 5-FU toxicity. F5 callus contained less histidine and arginine than WOO1C. 5-FU increased the endogenous levels of arginine, histidine and aspartate in both lines. The aspartate transcarbamylase of F5 appears to be normal; it is as sensitive to uridine-monophosphate inhibition as that of WOO1C. The 5-FU resistance of F5 was stable in undifferentiated cells, but only 8 out of 50 calli reinitiated from the regenerated plantlets remained resistant to 5-FU. F5 is an aneuploid culture. Five 5-FU-sensitive reinitiated calli that were examined were all diploid whereas of the eight 5-FU-resistant reinitiated calli two became diploid and six remained as aneuploid.