Gibberellin-mediated PtrLBD39–PtrHDA6– PtrHCT epigenetic path regulates lignin biosynthesis during tension wood formation in Populus trichocarpa

Abstract Tension wood (TW) is a specialized xylem tissue formed in angiosperm trees to counteract mechanical stresses. Increased phytohormones, particularly gibberellins (GAs), and decreased lignin deposition are two well-established features of TW, yet the molecular mechanisms linking GA signaling to lignin repression remain largely unknown. Here we revealed that TW formation in Populus trichocarpa elevates GA accumulation, which activates the expression of PtrLBD39, the most strongly induced transcription factor and a repressor of lignin biosynthesis in TW development. Inhibition of GA biosynthesis attenuates PtrLBD39 induction and restores lignin deposition in TW development. Consistently, CRISPR PtrLBD39 knockout increased lignin content and endonuclease-deficient Cas9-mediated loci-specific PtrLBD39 activation reduced lignin in TW-forming stems. ChIP–seq analysis and EMSA experiments demonstrated that PtrLBD39 binds directly to the promoters of two monolignol biosynthetic genes, PtrHCT1 and PtrHCT6, via a novel TCACGB motif, thereby repressing their transcription. The PtrLBD39–DNA binding allowed PtrLBD39 to recruit two histone deacetylases, PtrHDA6-A and PtrHDA6-B, from 16 known P. trichocarpa HDACs screened and validated by Y2H, BiFC and in vitro pull-down assays, and the specific recruitments to PtrHCT1 and PtrHCT6 were confirmed during TW development by ChIP–qPCR. PtrLBD39–PtrHDA6-A or PtrLBD39–PtrHDA6-B dimers were functional for deacetylating H3K9ac and H3K14ac on PtrHCT1 and PtrHCT6 leading to transcriptional repression and lignin reduction, as demonstrated in PtrHDA6-A overexpression lines under TW development. Consistently, PtrLBD39 overexpression lines under TW development also affected PtrHCT deacetylation for lignin reduction. Thus, we suggest that mechanical stresses transduce a GA-mediated LBD39–HDA6s–HCTs epigenetic regulatory path for effective causative regulations for TW specialization.