Single-Nucleus and Spatial Transcriptomics Reveal How Tissue Context Shapes the Circadian Transcriptome of the Arabidopsis Leaf

leaf, we performed a 24 hour single nucleus RNA-sequencing circadian time course. We captured ~30,000 nuclei across seven circadian time points, recovering all major leaf cell types. We identified over 7,400 genes with cluster-resolved circadian regulation, and used coexpression analysis to define five major temporal expression clades shared across all cell types. We leveraged these assignments to identify genes with cell-type-specific temporal shifts in expression. Single cell gene regulatory networks were generated for each cluster through GENIE3, identifying many shared and unique transcription factor target interactions across clusters. A close examination of core clock component targets identified complex light and hormone signaling associated networks, capturing both broad cross-cell type regulation and cell-type-specific target regulation. Our results demonstrate the extent to which circadian transcriptional regulation is present in the mature Arabidopsis leaf, and highlight the immense complexity in cell-type-specific regulation.