Tangential single-cell spatial transcriptomics resolves early ray lineage trajectory in poplar.

Vascular cambium drives secondary growth in woody plants, yet its closely related stem-cell populations-fusiform and ray initials-are difficult to distinguish, limiting resolution of early ray fate specification. In Populus tremula × Populus alba clone 717, we integrated a 2-yr biweekly field survey of xylem ray anatomy with time-series stem transcriptomes and single-cell-resolution tangential spatial transcriptomics to resolve early ray-lineage states in situ. Across 2 growing seasons, ray cell number and size/area traits showed reproducible dynamics and were most strongly associated with daylength, radiation and temperature averaged over the preceding 1 to 4 wks. Co-expression analysis identified gene modules that covary with ray traits and these environmental indices. By enhancing recovery of ray cambial cells through tangential sectioning, we generated a single-cell-resolution spatial transcriptomic atlas that distinguishes fusiform initial cells (FICs) and ray initial cells (RIC_1/2), and reconstructs a continuous early ray trajectory from FICs to RICs and xylem ray precursor cells (XRP_1-3), supported by RNA in situ hybridization. Integrating field-linked candidates, public single-cell RNA sequencing resources, and spatial markers, we identified 227 ray-lineage candidates, enriched for chromatin/nuclear functions and ribosome biogenesis, with relatively few transcription factors. These results provide stage-resolved markers and candidates for dissecting ray development and its seasonal environmental covariation.