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.

Papaya is a trioecious species with XX females, XY males, and XY^h hermaphrodites. Any combination of Y and Y^h chromosomes is lethal. The gene underlying YY lethality is pivotal in the transition from stage 2 to stage 3 in sex chromosome evolution. Here, we identify and functionally validate the YY lethality gene in papaya, CpYYL. Loss of CpYYL function accelerates glycolysis and reduces sucrose accumulation in embryo, causing embryo abortion. CpYYL interacts with CpAKRP and overexpression of both genes partially rescues seed abortion in the heterozygous emb506 and akrp mutants, indicating conserved role in plastid differentiation and embryo development. Viable YY^h and Y^hY^h plants are generated through genetic engineering and crossing, although both genotypes show high seedling mortality. Crossing YY^h male with XX female papaya shows 1:1 ratio of male to hermaphrodite plants without recombination between MSY and HSY. Identification of CpYYL fills a critical gap in papaya sex chromosome evolution, and provides new resources for genomic and epigenetic studies.

Papaya is a trioecious species, but a unified framework for comparing floral sex differentiation has been lacking. We combined stereomicroscopy, paraffin sectioning, and scanning electron microscopy to establish a continuous 12-stage developmental system from floral primordium initiation to anthesis. Male, female, and hermaphroditic flowers followed a common developmental trajectory during Stages 1–4 and diverged at Stage 5. Male flowers formed fertile stamens and a reduced pistillode, female flowers developed a complete gynoecium without stamens, and hermaphroditic flowers displayed normal, aborted, or transitional pistils and carpellodic stamens. RNA sequencing and weighted gene co-expression network analysis of morphologically defined tissues identified gene modules associated with normal pistil development, distinct modes of pistil abortion, and stamen carpellody. These modules were enriched in chromatin and RNA regulation, protein homeostasis, floral-organ identity, hormone and receptor-kinase signaling, pollen-wall formation, and stress-responsive pathways, yielding 30 representative candidate genes. This developmental framework integrates developmental morphology with transcriptional programs underlying reproductive-organ fate and plasticity in papaya.