Nucleoside Diphosphate Kinase Family: Evolutionary Analysis and Protective Role in Mitochondrial ROS Production.

Nucleoside diphosphate kinase (NDPK) is a ubiquitous enzyme that maintains cellular nucleotide balance by catalyzing the transfer of phosphate groups between nucleoside diphosphates and triphosphates. Although the evolutionary conservation of NDPK is well established, several aspects of its diversification and functional adaptation remain unclear. The central question of this work is how NDPK evolved across plant species, focusing on the Solanaceae family and how its evolutionary history relates to the diversification of its cellular functions. Phylogenetic and molecular dating analyses showed that the division between NDPK groups 1 and 2 predates the divergence of plants and animals, whereas plant-specific NDPK types (I-IV) originated early in streptophyte evolution. Solanaceae species retain a conserved set of NDPK genes, including a type III isoform with features consistent with mitochondrial targeting. Functional assays in isolated potato tuber mitochondria revealed high NDPK activity in the intermembrane space, sustaining ADP supply to oxidative phosphorylation. Activation of mitochondrial NDPK induced a phosphorylative respiratory state, which partially dissipated the mitochondrial membrane potential and significantly reduced reactive oxygen species (ROS) production. GDP and UDP were preferentially phosphorylated, conferring a stronger antioxidant effect than other nucleotides. Consistently, the mitochondrial isoform StNDPK3 was upregulated during tuber development. Together, our results demonstrate that NDPKs are evolutionarily conserved yet functionally diversified enzymes in plants and identify mitochondrial NDPK as a key modulator of mitochondrial redox homeostasis. By linking nucleotide metabolism to Δψm control and ROS suppression, this study highlights a previously underappreciated antioxidant mechanism that integrates mitochondrial energy metabolism with developmental and stress-related processes in plants.