An ectopic xylem cell induction system to unravel the molecular mechanisms of secondary cell wall formation in the coniferous tree, Cryptomeria japonica.

Wood is a vital renewable resource for energy, construction, and pulp production. Understanding the molecular mechanisms governing wood formation is therefore crucial for both basic research and applied forestry. The xylem, a major component of wood, plays a crucial role in water transport and mechanical support in trees, requiring a robust secondary cell wall to endure water pressure and support the tree's weight. Gaining deeper insight into xylem cell differentiation is therefore important for both fundamental biological research and industrial applications. In vitro systems for inducing xylem cell differentiation have been developed in various plants, including Arabidopsis thaliana where key regulators such as the VASCULAR-RELATED NAC DOMAIN (VND) transcription factor (TF) have been identified. However, research on coniferous trees remain limited, with most studies focusing on morphological aspects with limited molecular analysis. In this study, we developed an efficient xylem cell induction system for Cryptomeria japonica using bikinin, a glycogen synthase kinase 3 (GSK3) inhibitor, in combination with cytokinin, auxin, and brassinolide. This system induced ectopic xylem cells in the somatic embryos and cotyledons of seedlings within 2 weeks, significantly faster than methods reported in previous studies. We conducted a comprehensive time-series transcriptome analysis during xylem cell induction in somatic embryos and identified genes expressed throughout the course of xylem cell formation. Our analysis revealed a sequential upregulation of key regulatory genes, including VND- and MYB-like TFs, followed by genes involved in cellulose biosynthesis, suggesting their role in tracheary element formation. These findings suggest that the molecular mechanisms regulating xylem cell formation in the gymnosperm C. japonica are fundamentally conserved with the NAC-MYB transcriptional network known in angiosperms.