Uridine Ribohydrolase and the Balance between Nucleotide Degradation and Salvage

Nucleotides are ubiquitous in metabolism: in addition to being components of DNA and RNA, and the currency of energy metabolism in the cell, they also are formed into essential coenzymes, secondary metabolites, and plant hormones (reviewed in Zrenner et al., 2006). Given that nucleotides are essential to plant metabolism and that nucleotides contain a large reservoir of the plant's nitrogen and phosphorous, the regulation of nucleotide levels is both a complicated and an extremely important part of plant function. Nucleotides can be synthesized de novo, salvaged from the existing nucleotide pool, or broken down into their component parts. Salvaging nucleotides requires substantially less energy than synthesizing nucleotides, so the balance between salvage and degradation is important both for optimizing the energy economy of the plant and for maintaining levels of key elements. Thus, the regulation of nucleotide levels is highly balanced; for example, repression of a key enzyme in pyrimidine synthesis in potato caused a compensatory increase in the pyrimidine salvage pathways (Geigenberger et al., 2005).

This balance also encompasses nucleotide degradation, the first step of which is removal of the phosphates from nucleotides to form nucleosides. Nucleosidases are key enzymes in both salvage and breakdown pathways, cleaving the nucleobase from the sugar, thereby allowing the base to be recycled as a nucleotide monophosphate or further broken down. To examine nucleoside breakdown and salvage, Jung et al. (pages 876–891) focus on a nucleosidase that acts on uridine and affects this key balance. They clone and characterize URIDINE RIBOHYDROLASE1 (URH1) from Arabidopsis, initially using cell extracts to show that plants have a biochemical activity that breaks uridine into uracil. They next isolate URH1 by its sequence similarity to known nucleosidases and use the Arabidopsis cDNA to complement a yeast mutation that is defective in pyrimidine synthesis and salvage. URH1 protein produced in E. coli acted on uridine and, with lower activity, on inosine, adenosine, and the cytokinin riboside isopentenyladenine-riboside, but not on cytidine. URH1 was found to be a cytoplasmic enzyme expressed in root vasculature (see figure), root meristems, guard cells, and mature pollen cells.

Figure 1.

Histochemical localization of URH1 expression in an 8-d-old seedling shows expression in roots.

Moving to in planta experiments, plants with reduced URH1 activity were produced by targeted artificial microRNA lines and showed increased anthocyananin levels when grown under nitrogen stress, indicating possible difficulty in recycling nitrogen bound up in nucleotides. Plants with increased URH1 activity were produced by overexpression of either Arabidopsis or rice URH1 and showed resistance to uridine analogs 5-fluorouracil and 5-fluorouridine, which are metabolized into derivatives that are toxic when incorporated into DNA. This resistance, and an increase in uridine degradation in overexpressor plants, indicated an increase in pyrimidine breakdown and a decrease in the salvage pathways in these plants. Plants with either reduced or enhanced URH1 expression showed germination delays compared with the wild type, pointing to an overall alteration of pyrimidine nucleotide metabolism, since germination is a high-demand time for nucleotides. Thus, the balance between nucleotide breakdown and salvage can be shifted by changes in URH1 levels, and this balance is important for plant development.