Developmental and Stress-Mediated Transcriptional Shifts of Riboflavin Metabolism Pathway in Arabidopsis

Background: Flavin cofactors, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), are indispensable for plant metabolism, supporting photosynthesis, photorespiration, mitochondrial electron transport, nitrogen assimilation and cellular redox balance. Both cofactors derive from riboflavin (vitamin B₂), which plants synthesize de novo, unlike animals that rely on dietary intake. While the riboflavin biosynthetic pathway has been biochemically well characterized, its transcriptional regulation and cellular organization remain poorly understood. Methods: Here, using large-scale transcriptomic datasets, co-expression and cis-element analyses, we systematically investigated the expression dynamics of riboflavin metabolism genes in Arabidopsis thaliana. In addition, HPLC was employed to monitor flavin level fluctuations in plants under abiotic stresses. Results: Most genes displayed strong expression in photosynthetic and reproductive tissues, consistent with elevated metabolic demands for flavins in redox reactions and energy metabolism. Under osmotic stress, RIBA1, RIBA3, PYRD, PYRR, COS1/LS and RS, genes encoding enzymes involved in the early and intermediate steps of riboflavin biosynthesis were transcriptionally repressed. In contrast, RIBA2, FHY1/PYRP1 and FMN/FHY were upregulated, whereas FADS1 and NUDX23, genes encoding enzymes responsible for interconversion between FMN and FAD, were repressed. Gene expression responses are consistent with maintenance of flavin homeostasis and coincide with changes in flavin levels under abiotic stress. Conclusions: This study establishes a comprehensive framework for the transcriptional regulation of flavin biosynthesis in plants. The findings reveal stress-responsive reprogramming of flavin metabolism and identify promising strategies for engineering crop biofortification, metabolic efficiency, and stress resilience.