Integrated scRNA‐Seq and ATAC‐Seq Decipher the Xylem Development Atlas and Regulatory Landscape in Rubber Tree ( <i>Hevea brasiliensis</i> )

Rubber tree is an economically important woody plant, whereas the cellular heterogeneity and regulatory networks underlying its xylem differentiation remain poorly characterised. Here, we built a protocol to isolate differentiating xylem protoplasts and performed scRNA-seq on two rubber tree cultivars (RK525 and RY73397) with distinct rates of girth growth. The first single-cell transcriptional atlas of differentiating xylem in the rubber tree was established, providing a cellular basis for growth variations. Ten distinct cell clusters were identified, with cell types-including vessel element, libriform fibre, and ray parenchyma-determined by established marker genes. Pseudotime analysis revealed two clearly developmental trajectories, underscoring fusiform lineages (fibres and vessels) being crucial for growth differences. Specifically, the fast-growing cultivar RK525 exhibited a cluster of upregulated genes in fibre and vessel cells. Subsequently, differentially expressed genes (DEGs) in fusiform cell clusters between the two cultivars were intersected with subcluster-upregulated genes and pseudotime-core DEGs in RK525, highlighting secondary cell wall (SCW) biosynthesis genes that regulate xylem development and promote rapid growth. WGCNA further identified a specific module significantly correlated with fibre cells, which includes six key transcription factors. Moreover, ATAC-seq revealed variety-specific chromatin accessibility patterns and transcription factor binding motifs associated with xylem development. Functional validation using Arabidopsis mutants of rubber tree homologues confirmed their crucial roles in vascular development. These findings reveal intricate regulatory programmes in differentiating xylem, and provide valuable resources and targets for genetic improvement of the rubber tree.