Molecular regulation of vegetative to reproductive transition in dioecious spinach

Background The vegetative to reproductive transition is a critical developmental process that determines plant reproductive success, yet its molecular regulation in dioecious spinach remains poorly understood. We integrate physiological characterization, transcriptome profiling, and microRNA analysis to investigate transition in male and female spinach. Results Physiological characterization of juvenile vegetative tissues (root, shoot and leaf) and three meristem stages (SAM, PTM, FAM) revealed progressive developmental changes and clear sex-specific differences, with male plants transitioning to flowering earlier and attaining greater height than females. Transcriptome profiling identified 25,495 differentially expressed genes, including 1,177 transcription factors from 56 families. SpbHLH , SpMYB , SpNAC and SpMADS -box genes showed prominent stage- and sex-specific expression pattern, while key regulators including SpSBP/SPL , SpLFY and SpAP2 , displayed dynamic expression associated with meristem identity transitions. Promoter analysis revealed stage-specific enrichment of transcription factor -binding sites and hormone-responsive elements, with ABA-, ethylene- and jasmonate-responsive motifs enriched at floral stages. Notably, the proposed SpmiR156–SpSBP/SPL–SpmiR172–SpAP2 regulatory module showed antagonistic dynamics during phase transition: SpmiR156 declined, whereas SpmiR172 increased 117-folds in males and 10.3-folds in females at the FAM stage. qRT-PCR further validated sex-biased expression of floral regulators, including male biased SpAP3/PI -class MADS-box genes. Gene Ontology enrichment revealed progressive functional shifts from transcriptional and developmental regulation to floral organ morphogenesis. Conclusions These results establish an integrated regulatory framework linking miRNA–transcription factor mediated regulation, hormone signaling, and sex-specific transcriptional programs during the vegetative to reproductive transition in dioecious spinach. The identify regulator modules and candidate genes provide potential targets for manipulating flowering time and reproductive development.