Decoding cellular transcriptional regulatory networks governing wheat inflorescence development

In wheat, inflorescence architecture critically determines yield potential, yet its structural complexity and asynchronous development have hindered cellular-resolution studies of spikelet and floret formation. Here, we integrate spatial transcriptomics, high-sensitivity multiplexed error-robust fluorescence in situ hybridization (MERFISH), and snRNA-seq across six developmental stages to generate a spatiotemporal atlas of the wheat inflorescence. We identified 20 cell types, spatially resolved into three categories: 1) proliferating cells within spikelet, marked by active division; 2) supporting cells along the central axis, including pith, cortex, and vasculature; and 3) developmental cells located both inside and at the base of the spikelets. The multi-omics approach enabled identification of the rare cell type ovary. Trajectory inference revealed that spikelets and florets originate from two temporally and spatially distinct sub-clusters of proliferating cells (R7), each defined by high expression of developmental regulators. These findings challenge the conventional model sequential meristem transitions (inflorescence-spikelet-floret) in wheat. Integration of time-series snATAC-seq and snRNA-seq delineated cellular transcriptional regulatory networks (cTRNs) governing spikelet formation, mediated by auxin and cytokinin signaling, and floret formation, driven by MADS-box transcription factors. Cell identity was maintained by cell type-specific accessible chromatin regions (csACRs), which are enriched for SNPs associated with spike-related traits. For instance, SNPs within csACRs of the WFZP and DUO1 promoters affect TaNAC30 binding, regulating supernumerary spikelet phenotypes. Our work provides a mechanistic framework for wheat inflorescence development and identifies csACRs and cTRN nodes as potential targets for optimizing yield-related inflorescence architecture.