The metabolism of deoxypyrimidine nucleotides during germination

• Applicant: Dr. Marco Herde
• Subject Area: Plant Physiology
• Term: since 2016
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 342656586

Seed germination is a process unique to plants, and seed viability and seedling vigor are of great importance to agriculture. While other phenomena such as the remobilization of storage compounds during germination are well studied, less is known about the metabolism of deoxyribonucleotides (dNTs), in part due to technical challenges in analyzing these metabolites in plants. We have developed a method for the measurement and quantification of nucleotides in plants, and have used this to analyze dNTs and deoxyribonucleosides (dNs) over the time course of germination and seedling establishment. We focused on the synthesis of thymidylates (responsible for the 'T' building block in DNA) and, using mutants lacking enzymes for thymidylate synthesis, we evaluated the contribution of all processes leading to the formation of thymidylates. In particular, phosphorylation of the nucleoside thymidine in chloroplasts is crucial for the synthesis of thymidylates and a defect in the corresponding enzyme leads to reduced DNA synthesis in the chloroplast during seedling establishment. This finding is surprising since thymidine has no role in the previous model of the synthesis pathway. Different experimental strategies suggest that the synthesis of thymidylates proceeds via thymidine - and there is evidence that a dN intermediate may also be important for the synthesis of other dNTs. We have identified an enzyme (VENOSA4) that links thymidine to the known part of the synthesis pathway. Our data suggests that a similar enzymatic step enables the conversion of nucleotides released during DNA repair to thymidine. We have also identified possible candidate enzymes for this step with initial experiments. These will be fully characterized together with VENOSA4. We also propose, that an additional 'detour' pathway is involved in the supply of the substrate for 'T' biosynthesis suggesting that this is a more general concept with an impact on the current textbook knowledge on dNT synthesis. The 'detour' in the synthesis via thymidine described here consumes metabolic energy and appears disadvantageous at first glance. The project aims to show why this 'detour' may nevertheless be subject to positive selection. Two concepts are in the focus: (I) The 'detour' via thymidine is a metabolic proofreading mechanism that allows the removal of damaged thymidylates (e.g. by oxidation) and (II) thymidine represents a form of transport for thymidylates that facilitates exchange between chloroplasts and the cytosol. This project aims to provide a comprehensive model for complex dNT synthesis, to fully elucidate the metabolic pathway of thymidylate biosynthesis and to show how the plant is able to provide high quality dNTs in the correct amounts at all times.

• DFG Programme: Research Grants