Integrative Hormone Metabolomic, Transcriptomic, and miRNA Sequencing Analyses Reveal Brassinosteroid-Mediated Sex Differentiation in Walnut Flower Buds

Walnut (Juglans regia L.) is a monoecious species with far more male than female flowers, yet the molecular mechanisms regulating sex differentiation remain largely unexplored. In this study, we investigated the effects of exogenous brassinolide (BR) on the female-to-male flower bud ratio and its regulatory mechanisms. We identified 23 April to 13 May as the critical physiological differentiation period for the ‘Xin xin 2’ variety. Exogenous application of 0.05 mg/L BR during this period significantly increased the female-to-male flower bud ratio across three consecutive years (2023–2025). Hormone metabolomics revealed that BR treatment induced sex-specific changes in ethylene and auxin contents. Transcriptomic analysis showed that BR differentially regulated MADS-MIKC genes in female and male buds. Integrated miRNA-mRNA analysis identified sex-specific regulatory modules targeting MADS-MIKC genes, including four in female buds and two in male buds. RT-qPCR and dual-luciferase assays confirmed that jre-miR156g directly targets the 3’-untranslated region of JrSPL5, with reciprocal expression patterns. Collectively, our findings demonstrate that exogenous BR increases the female-to-male flower bud ratio in walnut through multi-layered regulation involving hormone signaling, sex-specific expression of MADS-MIKC genes, and miRNA-mRNA regulatory cascades. This study provides a theoretical basis for breeding high-yielding walnut varieties by manipulating sex expression.