Single-nucleus RNA sequencing reveals a key regulator RsHB8 affecting vascular cambium activity and xylem...

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.