Single‐nucleus RNA sequencing reveals a key regulator RsHB8 affecting vascular cambium activity and xylem differentiation during radish taproot thickening

Radish taproot thickening is primarily driven by vascular cambium (VC) activity. However, the transcriptional regulatory mechanisms underlying taproot thickening at a single-cell resolution remain unclear. In this study, single-nucleus isolation coupled with fluorescence-activated cell sorting was used to construct a cell atlas of radish taproots using microdroplet-based single-nucleus RNA sequencing. Through canonical marker gene profiling and validation using laser capture microdissection transcriptome data, 12 cell types were identified, and the first single-nucleus transcriptomic atlas was constructed for radish taproots. VC cells exhibited heterogeneous transcriptional states with varying levels of expression of cell cycle-associated genes. Pseudotime analysis inferred a transcriptional trajectory from VC cells toward xylem parenchyma cells in radish taproots. Integrating pseudotime analysis with predicted protein-protein interaction networks highlighted HOMEOBOX 8 (RsHB8) as a candidate regulator of the inferred VC-to-xylem parenchyma (XP) trajectory. RsHB8m overexpression increased taproot width and promoted xylem differentiation, whereas RsHB8-silenced plants exhibited a reduced taproot width and narrower xylem region. Xyloglucan endotransglucosylase/hydrolase 22 (RsXTH22) was identified as a target gene of RsHB8. LOB domain-containing protein 21 (RsLBD21) interacted with RsHB8, inhibited its transcriptional activity, and attenuated the RsHB8-mediated transcriptional activation of RsXTH22. Silencing RsLBD21 significantly increased taproot and xylem width, whereas silencing RsXTH22 resulted in the opposite phenotypes. Together, these findings establish a single-nucleus transcriptomic atlas of radish taproot development and provide insights into the transcriptional regulation of VC differentiation during taproot thickening.