Spinach is a dioecious vegetable and an excellent model for investigating plant sex differentiation. Exogenous gibberellin treatment induced sepal hypoplasia and sex reversal, converting 42% of stamens into pistils in male plants. Transcriptome analysis identified 112 male-biased genes enriched in stamen and pollen development, while hormone profiling revealed coordinated changes in GA, cytokinins, auxin, jasmonic acid, and abscisic acid. Functional assays demonstrated that silencing SpAMS or SpPGIP caused extensive carpelization, and in situ hybridization localized their expression to developing anthers. Dual-luciferase assays confirmed that SpAMS directly activates the B-class gene SpPI, and genomic mapping placed SpAMS in the pseudo-autosomal region of the Y chromosome. These results indicate that GA disrupts hormonal homeostasis and anther wall integrity, while the SpAMS–SpPI pathway regulates tapetal development to maintain male identity. Our findings identify SpAMS as a key male-promoting factor in spinach and provide a framework for elucidating sex determination mechanisms in dioecious plants.

Vegetative to reproductive phase transition is an important developmental process in flowering plants, regulated in part by microRNAs that repress target genes post-transcriptionally. However, the role of miR156 and its target Squamosa Promoter Binding Protein-Like (SPL) transcription factors remains poorly understood. In this study, we identified 14 SPL gene members in spinach and analyzed their expression during phase transition. Genome-wide identification and transcriptome-based analysis revealed that 11 of these genes are likely direct targets of SpmiR156a/b/c/d, with binding sites confirmed by sequence-based interaction prediction. Expression profiling showed that SpSPL3 and SpSPL8, which are strongly repressed during vegetative growth, were significantly regulated during the transition phase. Gene Ontology (GO) and promoter cis-element analyses support that SPL genes are involved in hormonal pathways and floral development Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) further validated the transcriptome expression patterns of key SPL genes. Together, these findings outline a regulatory framework in which SpmiR156 modulates SpSPL gene activity to control developmental phase change in spinach, highlighting both the expansion and functional diversification of the SPL gene and the central role of SpmiR156 in vegetative to reproductive transition.