Transcriptomics and comparative physiological analyses reveal hormone-mediated reprogramming during adventitious root induction in American chestnut (Castanea dentata)

Adventitious root (AR) formation is a critical regenerative process in woody plant propagation, but remains inefficient in recalcitrant species such as the American chestnut ( Castanea dentata ). The physiological and molecular bases underlying chestnut cuttings' limited rooting capacity remain poorly understood. To address this gap, we integrated anatomical, hormonal, metabolic, and transcriptomic analyses, using the readily rooting hybrid poplar as a comparative model, to identify key constraints on AR formation in American chestnut. Chestnut cuttings exhibited structural constraints, including a continuous sclerenchyma ring and dense vascular cambium, likely limiting early cell proliferation. Hormonal profiling indicated lower initial indole-3-acetic acid levels (only 50% of those in poplar) and an unfavorable auxin-cytokinin balance. Metabolomic profiles revealed enrichment of sugars and phenolic acids, but depletion of rooting-related amino acids and flavonoids. Transcriptomic and RT-qPCR analyses identified stage-specific regulation of genes involved in meristem activation, auxin transport, and stress responses, with delayed or reduced expression in chestnut. Key genes such as CdABCG37 were upregulated at later stages, whereas CdPID and CdMYB12 were downregulated, highlighting coordinated spatial and temporal regulation during rooting formation. • Cuttings from American chestnut adult trees have high recalcitrance to adventitious root (AR) formation. • Metabolite profiles and hormone dynamics in American chestnut cuttings are unfavorable to AR induction. • American chestnut cuttings have higher levels of lignin and H 2 O 2 compared to easy-to-root poplar. • Time-course transcriptomics revealed potential AR development genes in American chestnut.