Age-dependent DNA labeling and deoxyribonucleotide synthesis in wheat seeds

Utilisation of ribonucleosides as precursors of DNA biosynthesis was studied in germinating wheat embryos because the reductive pathway leading to deoxyribonucleotides is very difficult to demonstrate in extracts of higher plants in vitro. [5-3H]Cytidine and [6-3H]uridine are incorporated into wheat DNA (RNA-free) via ribonucleotide reduction without intermediate scission of the glycosidic bond. This reaction is observed at 20-30 h after the onset of germination only in aged (2-4-year-old) seeds while the embryos isolated from fresh grains show very little cytidine incorporation; in contrast, thymidine incorporation into DNA between 10 and 18 h of germination is not age dependent. Fresh wheat contains a soluble, heat-stable inhibitor fraction, most probably a modified oligonucleotide, which efficiently prevents cytidine incorporation when added to old embryos together with the labeled nucleoside. This material also inhibits purified Escherichia coli ribonucleotide reductase and is thought to be part of the control system for ribonucleotide reduction in wheat; it may gradually decay during storage of the seeds. Dry wheat embryos do not contain deoxyribonucleoside triphosphates. Pool sizes of dATP and dTTP in germinating embryos were found to reach 1 pmol/microgram DNA at 10-15 h of germination (i.e. before ribonucleotide reduction) and were independent of the age of seeds. These data suggest that wheat contains other preformed dexoyribonucleoside derivatives which are phosphorylated at an early time and can initially sustain DNA synthesis. Induction of measurable ribonucleotide reductase activity in fresh winter wheat was for the first time accomplished by 15 days of vernalization of the seeds at +2 degrees C.