Isolation and Characterization of UMP Synthase Mutants from Haploid Cell Suspensions of Nicotiana tabacum

Abstract

Uridine 5'-monophosphate (UMP) synthase mutants of tobacco have been produced from haploid cell-suspension cultures of a transgenic Nicotiana tabacum line, Tr25. The mutants were induced by incubating the suspension-cultured cells with 1 mm N-nitroso-N-methylurea for either 5 or 12 hours. Twenty mutant calli were isolated on selection medium containing 20 milligrams per liter of 5-fluoroorotic acid. Of those tested, most had reduced regeneration capacity. Characterization of UMP synthase activities in the isolated calli showed that UMP synthase activity varied from 8 to nearly 100% of the wild-type activity. The growth of the calli on the media containing different levels of 5-fluoroorotic acid correlated with decreasing UMP synthase activity. Because the UMP synthase enzyme has two separate enzymic activities (orotate phosphoribosyl transferase and orotidine-5'-monophosphate decarboxylase), several mutants were further characterized to determine how the mutations affected each of the two enzymic activities. In each case, the enzymic activity affected was the orotate phosphoribosyl transferase and not the orotidine-5'-monophosphate decarboxylase. The wound-inducible phenotype of the Tr25 plants as measured by the activation of the pin2-CAT gene remained unchanged by introduction of the UMP synthase mutations. studying wound-inducible genes in the Solanaceae. In this paper we describe the first step in such a selection scheme, the production and characterization of mutants that have reduced levels of UMP synthase. These mutants will be used in the future for selection of mutants that are blocked in the wound-induction pathway. In addition, these mutants should also be useful to understand the biosynthesis of pyrimidines in plants.

Purine and Pyrimidine Nucleotide Synthesis and Metabolism

“…In fact, recent results demonstrate that OPRT and ODCase reside in a single polypeptide that is termed UMP synthase. This bifunctional enzyme catalyzes the last two steps of the de novo pyrimidine pathway in plants as well as mammals (Santoso and Thornburg, 1992). This structure improves the efficiency of these reactions by channeling the product of the first reaction to the second enzyme without dissociation from the complex.…”

Inhibition of de Novo Pyrimidine Synthesis in Growing Potato Tubers Leads to a Compensatory Stimulation of the Pyrimidine Salvage Pathway and a Subsequent Increase in Biosynthetic Performance

“…Furthermore, molecular studies are lacking to assess the importance of changes in uridine nucleotide pool levels for various aspects of metabolic performance (Moffat and Ashihara, 2002;Stasolla et al, 2003). In one study, UMPS mutants were isolated from haploid cell suspensions of Nicotiana tabacum (Santoso and Thornburg, 1992). There was a reduced ability to regenerate plants from these mutant cells; however, given the somewhat cursory nature of that study, it is unclear whether this was a direct or a pleiotropic effect of the mutation.…”

“…In plants, the first three steps are catalyzed by separate enzymes-carbamoylphosphate synthase (CPS), aspartyl transcarbamoylase, and dihydroorotasewhereas the last two steps, converting orotate to UMP, are catalyzed by a single protein (UMP synthase [UMPS]) having two enzymatic activities, orotate phosphoribosyltransferase and orotidylate decarboxylase. UMPS is one of the key enzymes of de novo pyrimidine synthesis and is often regarded, both in mammalian and plant systems, as a key regulatory step of the pathway (Santoso and Thornburg, 1992;Traut and Jones, 1996).…”

Characterization of the de novo pyrimidine biosynthetic pathway in Arabidopsis thaliana

“…UMP synthase is the rate limiting and final step of pyrimidine biosynthesis in plants (Santoso and Thornburg, 1992). It is transcriptionally up-regulated in response to thymine 78 starvation (Santoso and Thornburg, 1998 The results of these studies confirm in Arabidopsis.…”

“…1998. Santoso andThornburg, 1992). UMP synthase is also the rate limiting step in pyrimidine biosynthesis in plants (Santoso and Thornburg, 1992).…”