Changes in deoxyribonucleotide biosynthesis during carrot somatic embryogenesis

The activities of the de novo and salvage pathways of deoxynucleotide biosynthesis were investigated during the different stages of development in carrot (Daucus carota) somatic embryos by following the metabolic fate of [2-14C]cytidine, which after conversion to CDP, is an intermediate of the de novo pathway of deoxypyrimidine nucleotides, and [2-14C]deoxycytidine and [2-14C]thymidine, substrates of the salvage pathway. Increasing de novo activity, estimated by cytidine (CR) incorporation into DNA, was observed as maturation progressed. This increase was parallel to a decrease in CR degradation to CO2. Utilization of CR for CMP synthesis was mainly due to the activity of cytidine kinase (CK), as low levels of non-specific phosphotransferases (NPT) measured with AMP as a phosphate donor and CR as a substrate, were detected throughout the course of the experiment. CR was also utilized for RNA synthesis, either directly, after conversion to CTP, or indirectly, after deamination to uridine and then converted to UTP. Salvage of supplied deoxycytidine (CdR) and thymidine (TdR) were also observed during embryo development. Turnover of CdR was a rapid event, as a large percentage of radioactivity from this precursor was recovered as nucleotides after 2 h and nucleic acids, mostly RNA, after 18 h. Salvage of CdR was presumably due to the activity of non-specific phosphotransferases (NPT), as low levels of deoxycytidine kinase (dCK) were observed at all stages of embryo development. Compared to embryogenic (M) cells, a lower utilization of CdR for RNA synthesis was observed in non-embryogenic, habituated (H) cells. A large fraction of supplied TdR was degraded to CO2 at all stages of embryo development. Salvage of this precursor to nucleotides and nucleic acids, mainly DNA, was due to the activity of NPTs, as the activity of the other salvage enzyme, thymidine kinase (TK) was low throughout the course of the experiment. The activity of TdR salvage in habituated (H) cells was lower compared to that of embryogenic (M) cells. Collectively, specific changes in deoxyribonucleotides occur during the initiation and development of carrot somatic embryos.