Functional Analysis of the Pyrimidine de Novo Synthesis Pathway in Solanaceous Species

Abstract

Pyrimidines are particularly important in dividing tissues as building blocks for nucleic acids, but they are equally important for many biochemical processes, including sucrose and cell wall polysaccharide metabolism. In recent years, the molecular organization of nucleotide biosynthesis in plants has been analyzed. Here, we present a functional analysis of the pyrimidine de novo synthesis pathway. Each step in the pathway was investigated using transgenic plants with reduced expression of the corresponding gene to identify controlling steps and gain insights into the phenotypic and metabolic consequences. Inhibition of expression of 80% based on steady-state mRNA level did not lead to visible phenotypes. Stepwise reduction of protein abundance of Asp transcarbamoylase or dihydro orotase resulted in a corresponding inhibition of growth. This was not accompanied by pleiotropic effects or by changes in the developmental program. A more detailed metabolite analysis revealed slightly different responses in roots and shoots of plants with decreased abundance of proteins involved in pyrimidine de novo synthesis. Whereas in leaves the nucleotide and amino acid levels were changed only in the very strong inhibited plants, the roots show a transient increase of these metabolites in intermediate plants followed by a decrease in the strong inhibited plants. Growth analysis revealed that elongation rates and number of organs per plant were reduced, without large changes in the average cell size. It is concluded that reduced pyrimidine de novo synthesis is compensated for by reduction in growth rates, and the remaining nucleotide pools are sufficient for running basic metabolic processes.Pyrimidine nucleotides are abundant molecules with essential functions in a multitude of biochemical processes. They are of particular importance in dividing and elongating tissues as building blocks for nucleic acid biosynthesis. In addition, as an energy source or precursors for the synthesis of primary and secondary products, they are participants in various metabolic processes. In particular, the pyrimidine nucleotides are directly involved in plant carbohydrate metabolism providing the energy-rich precursor UDP-Glc for many synthetic reactions, such as Suc and cell wall biosyntheses.In recent years, the basic processes of plant nucleotide de novo synthesis have been analyzed in some detail (Giermann et al., 2002;Moffatt and Ashihara, 2002;Boldt and Zrenner, 2003;Stasolla et al., 2003;Kafer et al., 2004). Plants utilize the same reactions as those found in other organisms. The so-called orotate pathway of pyrimidine de novo synthesis is defined as the formation of UMP from carbamoyl phosphate, Asp, and phosphoribosyl pyrophosphate. The pathway consists of the six enzymatic reactions as shown in Figure 1. The first reaction is conducted by the Gln hydrolyzing carbamoyl phosphate synthase. This enzyme is not unique to the pyrimidine pathway but is also involved in Arg biosynthesis. It is composed of two different subunits (carbamoyl phosp...

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