Glutathione-induced growth of embryogenic tissue of white spruce correlates with changes in pyrimidine nucleotide metabolism

Exogenous applications of reduced glutathione (GSH) and oxidized glutathione (GSSG) promote growth of embryogenic tissue of white spruce during a 7-day subculture period. Compared to control tissue, a statistically significant increase in fresh weight, as well as RNA and DNA content, was observed in the presence of GSH and GSSG during the last days in culture. The effects of these two metabolites on pyrimidine nucleotide metabolism was investigated by following the metabolic fate of 14C-orotic acid, a precursor of the de novo synthesis, and 14C-uridine and 14C-uracil, intermediates of the respective salvage and degradation pathways. Compared to control embryos, GSH-treated tissue was able to utilize a larger fraction of supplied orotic acid for UMP production, possibly due to the increased activity of orotate phosphoribosyltransferase (OPRT). The activity of this enzyme increased markedly in tissue cultured with GSH during the last days in culture. Salvage of uridine for nucleotide and nucleic acid synthesis was observed in all treatments, especially in GSSG-treated tissue at day 7. The increased salvage activity in this tissue correlated with the increase in activities of the two uridine salvage enzymes, uridine kinase (URK) and nucleoside phosphotransferase measured with uridine (NPT(uridine)). Compared to control tissue, tissue treated with either GSH or GSSG was able to utilize a large fraction of uracil for nucleotide synthesis, denoting a better ability to divert this precursor from degradation. Nucleotide and nucleic acid analyses revealed that in both GSH and GSSG-treated tissue, the endogenous levels of UTP, CTP, as well as those of RNA and DNA, were increased compared to those of control tissue. Overall, the results from this study suggest that GSH and GSSG can induce growth of embryogenic tissue of white spruce through distinct metabolic changes of pyrimidine nucleotides.