Spatiotemporal Transcriptomic Atlas Reveals the Regulatory Mechanisms Underlying Early Inflorescence Development and Sex Differentiation in Spinach

Dioecious plant species produce male and female flowers on separate individuals. However, the molecular regulatory mechanisms underlying these processes remain poorly understood. In this study, the model dioecious spinach (Spinacia oleracea), a widely cultivated leafy vegetable, is investigated by analyzing spatial transcriptomes of the shoot apices at different developmental stages. The findings elucidate key regulatory pathways underlying both inflorescence development and sex differentiation. Through the characterization of dynamic transcriptional changes across four consecutive developmental phases, distinct cell types within the shoot apex and stage-specific marker genes are identified. Notably, pseudotime trajectory analysis reveals that spinach sex differentiation occurs at the four-leaf stage. Furthermore, the critical involvement of epigenetic mechanisms in this process is demonstrated. By integrating functional genetic validation, the epigenetic regulator MULTICOPY SUPPRESSOR OF IRA 1 (MSI1) together with histone deacetylase HDT2 is discovered to promote female differentiation. Collectively, this work provides comprehensive insights into the regulatory networks controlling inflorescence development and sexual differentiation in spinach and establishes a valuable resource for advancing sex differentiation research in dioecious plants.