Single-Nucleus Transcriptomics Reveals How Cell Type Shapes the Circadian Transcriptome of the Arabidopsis Leaf

Circadian regulation enables plants to coordinate cellular processes with daily environmental cycles, yet the dynamics and hierarchy of the clock across cell types remains poorly understood. To characterize circadian regulation across cell types in the mature Arabidopsis thaliana 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.