The nucleotide metabolome of germinating Arabidopsis thaliana seeds reveals a central role for thymidine phosphorylation in chloroplast development

Thymidylates are generated by several partially overlapping metabolic pathways in different subcellular locations. This interconnectedness complicates an understanding of how thymidylates are formed in vivo. Analyzing a comprehensive collection of mutants and double mutants on the phenotypic and metabolic level, we report the effect of de novo thymidylate synthesis, salvage of thymidine, and conversion of cytidylates to thymidylates on thymidylate homeostasis during seed germination and seedling establishment in Arabidopsis (Arabidopsis thaliana). During germination, the salvage of thymidine in organelles contributes predominantly to the thymidylate pools and a mutant lacking organellar (mitochondrial and plastidic) thymidine kinase has severely altered deoxyribonucleotide levels, less chloroplast DNA, and chlorotic cotyledons. This phenotype is aggravated when mitochondrial thymidylate de novo synthesis is additionally compromised. We also discovered an organellar deoxyuridine-triphosphate pyrophosphatase and show that its main function is not thymidylate synthesis but probably the removal of noncanonical nucleotide triphosphates. Interestingly, cytosolic thymidylate synthesis can only compensate defective organellar thymidine salvage in seedlings but not during germination. This study provides a comprehensive insight into the nucleotide metabolome of germinating seeds and demonstrates the unique role of enzymes that seem redundant at first glance. The nucleotide metabolome of seeds reveals a crucial role for thymidine phosphorylation in chloroplast genome replication during germination.

Automated summary

This study comprehensively analyzes the thymidylate metabolism during seed germination and seedling establishment in Arabidopsis. It reveals the important role of organelle thymidine salvage in thymidylate pool formation and the compensatory effect of cytosolic thymidylate synthesis in seedlings. The study also discovers a novel function of an organelle enzyme in removing noncanonical nucleotide triphosphates.