PALA-mediated pyrimidine starvation increases expression of aspartate transcarbamoylase (pyrB) in Arabidopsis seedlings

Aspartate transcarbamoylase (ATCase; EC 2.1.3.2) catalyzes the committed step in the de novo synthesis of pyrimidine nucleotides. We investigated the effects of N-(phosphonacetyl)-L-aspartate (PALA), a transition-state analog inhibitor of ATCase, on seedling growth and development, RNA and soluble protein contents, ATCase activity and enzyme protein levels, and pyrB gene expression in Arabidopsis thaliana L. cv. “Columbia”. In vitro, PALA was a potent inhibitor of ATCase, with an apparent Ki = 22 nM. After 5 d of treatment with 1 mM PALA, seedlings exhibited delayed germination, inhibition of cotyledon expansion, leaf development and root growth, and general chlorosis. Total RNA contents of these seedlings were decreased by 81% and total soluble protein contents decreased by 74%, compared with untreated control plants. Levels of pyrB mRNA increased about tenfold in PALA-treated plants, while ATCase activity and enzyme protein levels increased twofold. Plants grown on media containing a lower (0.1 mM) concentration of PALA did not exhibit significant inhibition of growth until after 9 d of treatment, but had markedly reduced RNA contents (40% of controls) and elevated pyrB mRNA levels (fourfold increase) after 12 d of treatment.