Shibata, R.; Kuwata, H.; Sugimoto, T.; Kikuchi, M.; Maruta, T.; Ishikawa, T.; Ogawa, T.

Riboflavin (vitamin B2; RF) and its derivatives flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are indispensable cofactors for redox reactions in plants. While higher plants possess a conserved pathway for de novo riboflavin biosynthesis, how flavins are transported and spatially distributed between tissues remains unresolved. In particular, no genetically defined plasma membrane-localized flavin transporter has been identified in plants. Here, we identify Arabidopsis PURINE PERMEASE 5 (AtPUP5) as the first genetically defined plasma membrane-localized flavin transporter in plants. Using a riboflavin-auxotrophic yeast mutant, we show that AtPUP5 enhances cellular uptake of RF and, to a lesser extent, FMN, whereas FAD uptake is inefficient. In planta, AtPUP5 overexpression increases RF accumulation following external application. In contrast, loss-of-function mutants do not display defects in bulk RF uptake at the whole-plant level, indicating that AtPUP5 is not essential for global riboflavin acquisition. Notably, AtPUP5 deficiency results in RF overaccumulation in reproductive organs, including inflorescences, siliques, and seeds, irrespective of external RF supply. This organ-specific phenotype is fully suppressed by genetic complementation and coincides spatially with strong AtPUP5 promoter activity in reproductive tissues. These findings demonstrate that AtPUP5 functions at the plasma membrane to regulate localized riboflavin distribution in reproductive tissues. Together, our study establishes the first molecular framework for plasma membrane-mediated flavin distribution in plants and positions spatial regulation as a central component of riboflavin homeostasis in plants.