Laminar patterning transcription factors orchestrate spatial metabolite partitioning in Capsicum fruit

Chili pepper (Capsicum annuum L.) produces specialized metabolites, notably the pungent capsaicin and the red capsanthin. Although their biosynthetic pathways are well characterized, the cellular architecture that underpins spatial regulation remains unclear. Here, we present a spatiotemporal single-nucleus atlas of pepper development, integrating single-nucleus RNA sequencing and spatial transcriptomics, profiling 263,076 high-quality cells from 56 samples spanning seedlings to mature fruits. This resource reveals a multilayered organization and precisely maps metabolic genes to defined cell types and spatial regions. We further identify laminar patterning transcription factors (LPTFs), including WRKY6, ZAT10, and BTF3, whose layer-specific expression correlates with localized capsanthin accumulation. Our work establishes a framework for dissecting laminar control of specialized metabolism and provides a valuable reference for comparative studies across species. The atlas is openly accessible at http://Pepper-Cell-Atlas.com.