The deoxypyrimidine nucleotide metabolism in plant mitochondria

Abstract

The synthesis of deoxythymidine triphosphate (dTTP), as one of the four building blocks of DNA, plays a crucial role for the plant. Each cell compartment with its own sub genome therefore has a subset of enzymes necessary for the formation of thymidylates. However, the respective compartments are not purely self-sufficient systems, but are in constant exchange and can support each other depending on the developmental stage. Furthermore, the contribution of individual steps of thymidylate metabolism to the thymidylate pools varies in importance over time. According to current knowledge, the essential enzymes involved in the synthesis of dTTP in Arabidopsis thaliana are Dihydrofolate Reductase-Thymidilate Synthase (DHFR-TS), Thymidine Kinase (TK) and Deoxythymidine Monophosphate Kinase (TMPK, ZEU1). Complete loss of these enzymes is lethal for the plant, or in the case of TK, leads to a plant that can no longer reproduce. However, in the case of DHFR-TS and TK, only one of the two homologues is required for the development of a viable plant. This work shows that at germination, de novo biosynthesis, which proceeds via DHFR-TS, plays a minor role compared to salvage. However, the exclusively mitochondrial DHFR-TS2 isoenzyme appears to be more important than its cytosolic counterpart DHFR-TS1. A similar picture emerges for salvage. TK1b, present in chloroplasts and mitochondria, plays a more important role than its cytosolic representative TK1a. Particularly the chloroplastic TK1b has a greater impact in supplying the cell with thymidylates, which coincides with the synthesis of cpDNA being also the greatest sink during this time. A TK1b mutant shows a severe arrest in cpDNA synthesis during germination. The synthesis of cpDNA is partially restored in the established seedling and thymidylate pools are then also affected in a mutant lacking cytosolic TK1a suggesting that the supply of thymidylates is then secured by both cytosolic and organellar salvage. Among the less important enzymes, appear to be dUTP Pyrophosphatase 1 localized in chloroplasts and mitochondria (DUT1org) and dCMP Deaminase (DCD) localized in mitochondria. Both enzymes play a minor role in providing dUMP for DHFR-TS. In neither of the two associated mutants a serious disruption of thymidylate formation is observable. This may be due to an involvement of both enzymes in the formation of dUMP, as indicated by preliminary data suggesting that a DCD DUT1org mutant is not viable.