Gibberellin Disrupts Hormonal Homeostasis and Anther Integrity to Trigger Sex Reversal in Spinach

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 <i>SpAMS</i> or <i>SpPGIP</i> caused extensive carpelization, and in situ hybridization localized their expression to developing anthers. Dual-luciferase assays confirmed that <i>SpAMS</i> directly activates the B-class gene <i>SpPI</i>, and genomic mapping placed <i>SpAMS</i> in the pseudo-autosomal region of the Y chromosome. These results indicate that GA disrupts hormonal homeostasis and anther wall integrity, while the <i>SpAMS-SpPI</i> pathway regulates tapetal development to maintain male identity. Our findings identify <i>SpAMS</i> as a key male-promoting factor in spinach and provide a framework for elucidating sex determination mechanisms in dioecious plants.