Integrative transcriptomic and metabolomic analyses reveal mechanisms underlying calcium-induced enhancement of female flower production in Cucumis sativus L.

Cucumber sex differentiation is a complex process regulated by multiple factors, including calcium (Ca²⁺) signaling. Although Ca²⁺ has been implicated in flower sex determination, its precise regulatory mechanisms remain unclear. In this study, the androecious cucumber line Banna 31-10 was treated with CaCl₂ at the shoot apices at seven-day intervals for three applications (Ca-1W, Ca-2W, Ca-3W). CaCl₂ treatment significantly increased the number of female flowers, with the effect becoming more pronounced with prolonged application. Integrated transcriptomic and metabolomic analyses revealed extensive molecular and metabolic reprogramming in response to CaCl₂. Transcription factor (TF) analysis identified 347 TFs from 56 families, with ERF, bHLH, MYB, C2H2, and MADS-box families playing key roles. Integrated transcriptome-metabolome correlation highlighted the involvement of flavonoids and plant hormone pathways, including ethylene, abscisic acid, gibberellin, cytokinin, auxin, jasmonic acid, salicylic acid, and brassinosteroid. Notably, CsACS2 and multiple calmodulin-like (CML) genes were strongly associated with hormone biosynthesis and signaling, suggesting a central role of Ca²⁺-CML-mediated regulation in female flower differentiation. Weighted gene co-expression network analysis (WGCNA) identified hub genes in co-expression modules linked to CaCl₂ response, further supporting the regulatory network underlying sex differentiation. This study provides new insights into the role of the Ca²⁺ signaling system in cucumber sex differentiation.