Growth inhibition of tobacco protoplast-derived cells by methotrexate: Relationships with nitrate assimilation

Optimal conditions for the protection against growth inhibition induced by methotrexate (MTX) have been investigated. MTX was found to be much more toxic to protoplast-derived tobacco cells grown on nitrate as the sole nitrogen source, than to cells supplied with reduced nitrogen. In a reduced nitrogen-containing medium the growth inhibition was prevented by a mixture of methionine, adenine and thymidine. A valine-resistant tobacco mutant, affected in amino acid uptake, was not able to grow under these conditions, which are permissive for the wild type.

The effect of MTX on nitrate assimilation was also studied. Partial inhibition of the nitrate reductase (NR) activity of cells cultured in the presence of the drug was observed. When cells were cultured in a medium containing ammonium succinate, intracellular concentrations of the molybdenum cofactor (Mo-cofactor) of NR increased linearly during the culture, and decreased rapidly when MTX was added to the culture medium. A significant and reproducible protection was induced by exogenous xanthine oxidase (XDH) but not by the cofactor extracted from this enzyme.