A high spatial resolution transcriptomics of tension wood reveals the genetic program of contractile G-layer formation.

Tension wood (TW) formation in Populus involves the differentiation of G-fibers that deposit a cellulose-rich, contractile gelatinous layer (G-layer). Despite extensive study, the biosynthetic programs underlying G-layer formation have remained incompletely resolved, in part due to imprecise sampling of developmental stages. Here, we generate a high spatial-resolution transcriptome atlas of developing Populus tremula wood by tangential cryo-sectioning of TW and normal wood (NW) forming tissue. We overlaid microscopically determined cell wall thickening profiles with whole-transcriptome data, resulting in seven distinguishable TW and three NW stages and a clear separation of early and late G-layer-specific transcriptional programs during TW formation. Across 22,363 expressed genes, 90 co-expression clusters captured major programs defining cell wall biosynthesis, signaling, cytoskeleton dynamics, and stress responses. Differential expression analysis identified 2,898 genes specifically regulated during early or late G-layer formation-indicating a massive transcriptional reorganization unique to TW. Genes involved in cellulose, rhamnogalacturonan I, arabinogalactan II, and callose metabolism showed distinct induction patterns, including CAZymes not previously implicated in G-layer formation. We also identified G-layer-specific genes, including XTH26 (PtXTH5), SCL18, AGL29, and genes encoding FLA12-like AGPs and LIM protein. Thirty transcription factors different than the secondary wall-inducing NACs and MYBs, and several genes involved in hormonal regulation specifically induced at the onset of G-layer formation represent novel putative regulators of the tertiary G-layer differentiation. Together, this spatially resolved transcriptome provides the most detailed map to date of the molecular program underlying TW formation, offering new insights into G-layer biosynthesis, signaling networks, and cell wall specialization in woody plants.