Purine and pyrimidine metabolism in cultured white spruce (Picea glauca) cells: Metabolic fate of ¹⁴C‐labeled precursors and activity of key enzymes

Abstract

In order to examine the biosynthesis, interconversion, and degradation of purine and pyrimidine nucleotides in white spruce cells, radiolabeled adenine, adenosine, inosine, uracil, uridine, and orotic acid were supplied exogenously to the cells and the overall metabolism of these compounds was monitored. [8‐14C]adenine and [8‐14C]adenosine were metabolized to adenylates and part of the adenylates were converted to guanylates and incorporated into both adenine and guanine bases of nucleic acids. A small amount of [8‐14C]inosine was converted into nucleotides and incorporated into both adenine and guanine bases of nucleic acids. High adenosine kinase and adenine phosphoribosyltransferase activities in the extract suggested that adenosine and adenine were converted to AMP by these enzymes. No adenosine nucleosidase activity was detected. Inosine was apparently converted to AMP by inosine kinase and/or a non‐specific nucleoside phosphotransferase. The radioactivity of [8‐14C]adenosine, [8‐14C]adenine, and [8‐14C]inosine was also detected in ureide, especially allantoic acid, and CO2. Among these 3 precursors, the radioactivity from [8‐14C]inosine was predominantly incorporated into CO2. These results suggest the operation of a conventional degradation pathway. Both [2‐14C]uracil and [2‐14C]uridine were converted to uridine nucleotides and incorporated into uracil and cytosine bases of nucleic acids. The salvage enzymes, uridine kinase and uracil phosphoribosyltransferase, were detected in white spruce extracts. [6‐14C]orotic acid, an intermediate of the de novo pyrimidine biosynthesis, was efficiently converted into uridine nucleotides and also incorporated into uracil and cytosine bases of nucleic acids. High activity of orotate phosphoribosyltransferase was observed in the extracts. A large proportion of radioactivity from [2‐14C]uracil was recovered as CO2 and β‐ureidopropionate. Thus, a reductive pathway of uracil degradation is functional in these cells. Therefore, white spruce cells in culture demonstrate both the de novo and salvage pathways of purine and pyrimidine metabolism, as well as some degradation of the substrates into CO2.

Purine and pyrimidine metabolism during the partial drying treatment of white spruce (Picea glauca) somatic embryos

“…This was performed as described by Kanamori‐Fukuda et al (1981), Hirose and Ashihara (1983), and Ashihara et al (2000) Briefly, mature and partially dried embryos (approximatively 40 embryos) were incubated in 2 ml of hormone‐free (AE) medium in a 25‐ml Erlenmeyer flask with a center well. A filter paper moistened with 100 μl of 20% KOH was placed in a glass tube in the well in order to absorb 14 CO 2 .…”

“…The enzyme assays were carried out as described by Ashihara et al (1981), Hirose and Ashihara (1984), and Ashihara et al (2000). Somatic embryos (approximatively 500 mg fresh weight) were homogenized in 50 m M HEPES‐NaOH buffer (pH 7.6), 2 m M NaEDTA, 2 m M dithiothreitol (DTT), and 0.5% sodium ascorbate.…”

“…The main objective of this study is to determine whether the imposition of a PDT on white spruce somatic embryos results in changes of purine and pyrimidine metabolism, which are presumably necessary for successful embryo conversion. Based on previous results (Ashihara et al 2000), purine metabolism was investigated by following the metabolic fate of 14 C‐labeled adenine and adenosine, substrates of the salvage pathway, and inosine, an intermediate of the degradation pathway (Fig. 1).…”

Arabidopsis thaliana nucleosidase mutants provide new insights into nucleoside degradation

“…Nucleoside kinase activity and nucleobase phosphoribosyltransferase activity have been demonstrated for a variety of plants (Ashihara et al , 2000; Katahira & Ashihara, 2002; Loukanina et al , 2008). Mutants deficient in adenine phosphoribosyltransferase are male sterile (Gaillard et al , 1998), while A. thaliana mutants affected in different uracil phosphoribosyltransferase genes display phenotypes of varying severity (Islam et al , 2007; Mainguet et al , 2009).…”

Nucleoside transport and associated metabolism*

“…Nucleoside uptake into intact plant tissues has been demonstrated several times. The tissues that have been analysed cover seedlings from black gram ( Phaseolus mungo ) and white spruce ( Picea abies ) (Ashihara & Nobusawa 1981; Ashihara et al. 2000), potato ( Solanum tuberosum ) tubers (Katahira & Ashihara 2002), petunia ( Petunia hybrida ) pollen (Kamboj & Jackson 1985, 1987), ricinus ( Ricinus communis ) seedlings (Kombrink & Beevers 1983; Flörchinger et al.…”