PalKCH1 and PalKCH2 are required for tension wood formation and gelatinous layer deposition in Populus.

Tension wood (TW) is a graviresponsive xylem tissue exclusive to angiosperm trees that counteracts gravitational stress. Its hallmark is a gelatinous layer (G-layer) in fiber secondary walls, rich in cellulose and low in lignin. Although kinesins mediate microtubule-microfilament crosstalk, their role in G-layer biogenesis during gravistimulation is unclear in woody angiosperms. We studied two kinesin paralogs, PalKCH1 and PalKCH2, in Populus alba var. pyramidalis during TW development. Both belong to the Group I KCH clade, possessing a kinesin motor domain and a calponin-homology (CH) domain. Multi-omic profiling showed preferential expression in xylem fibers and strong gravistimulation-induced transcription. CRISPR/Cas9 single and double knockout mutants showed normal xylem under upright growth, but responded to gravitational stress with reduced stem bending, narrower TW regions, and fewer G-layer fibers; double mutants were more affected, suggesting dosage dependence. Chemical assays showed that mutants retained the cellulose-rich, lignin-poor TW signature, but the changes were attenuated compared to wild-type. Transcriptomic analysis of double mutants revealed suppression of genes involved in microtubule dynamics, vesicular trafficking, cell wall remodeling, reduced PalWOX4A expression, and repressed cell cycle pathways. Thus, PalKCH1 and PalKCH2 are required for TW formation, likely via cytoskeletal signaling, and represent high-value targets for improving wood quality in Populus.