PagCOBL5 Integrates Transcriptional and Spatial Cues to Confer G-Layer Deposition in Poplar

Angiosperm trees reorient their stems by producing tension wood (TW), a specialised xylem that generates contractile force. TW fibres develop a gelatinous (G) layer rich in cellulose, yet the molecular mechanisms governing G-layer formation remain poorly understood. COBRA-like genes encode glycosylphosphatidylinositol (GPI)-anchored proteins known to regulate cellulose deposition. Here, we functionally characterised PagCOBL5 in hybrid poplar (Populus alba × P. glandulosa) by identifying its upstream regulator and interaction partner. PagCOBL5 was preferentially expressed in mature xylem fibres, with high abundance in TW. Under mechanical stress, cobl5 knockout mutants exhibited impaired negative gravitropism and significantly thinner G-layer-phenotypes that were reversed in PagCOBL5-overexpressing lines. Furthermore, we found that the transcription factors PagLBD21 and PagLBD25 participate in the upstream negative regulation of PagCOBL5 expression, with the lbd21 lbd25/+ mutant exhibiting an enhanced gravitropic response and altered xylem cell differentiation. At the protein level, PagCOBL5 directly interacted with the cellulose synthase subunits PagCESA8b. This interaction positions PagCOBL5 as a key component recruited and spatially organised by the directionally moving cellulose synthase complex (CSC). Together, these results reveal a sophisticated regulatory and protein-interaction framework centred on PagCOBL5, providing pivotal molecular insights into how transcriptional modulation and cellulose synthase complexes coordinate G-layer formation and mechanical force generation during the gravitropic response.