Single-Nucleus RNA Sequencing Reveals WRKY12a Regulating Secondary Cell Wall Formation During Xylem Differentiation...

Secondary xylem differentiation determines wood structure and function in perennial plants, yet its regulatory mechanisms remain poorly understood in Hevea brasiliensis. Here, we generated a single-nucleus RNA sequencing (snRNA-seq) atlas from the cambium to mature xylem region in stems, resolving transcriptionally distinct cell populations corresponding to cambium, xylem mother cells, and fibre-vessel cells. Pseudotime analysis reconstructed the developmental trajectories from cambium to late xylem cell states and revealed stage-specific activation of secondary cell wall (SCW) biosynthesis programmes. Gene regulatory network analysis identified HbWRKY12a as a fibre-vessel enriched transcription factor functioning as a key regulator in SCW formation. Molecular assays demonstrated that HbWRKY12a directly binds and activates HbMYB1R1c, which subsequently modulates lignin-associated SCW deposition. Heterologous overexpression of either HbWRKY12a or HbMYB1R1c in Arabidopsis resulted in significant reductions in stem diameter and plant height. Both overexpression lines exhibited markedly decreased SCW thickness in fibre, vessels, and pith cells. These findings reveal a WRKY-MYB cascade that mediates SCW formation in H. brasiliensis, extending the classical NAC-MYB regulatory framework and providing molecular targets for improving xylem properties and stress resilience in tropical perennial crops.