Chromatin accessibility dynamics and a hierarchical transcriptional regulatory network for shoot apex cold stress in Eucalyptus grandis

Subtropical perennials lack formal winter dormancy mechanisms, rendering them more vulnerable to cold stress than temperate or boreal trees. While cold-response pathways are well characterized in model plants, the regulatory landscape in woody species remains elusive. Here, we demonstrate that shoot apices of the fast-growing subtropical tree Eucalyptus grandis respond to cold stress within 0.5 h through rapid chromatin accessibility remodeling and transcriptomic reprogramming. Time-series ATAC-seq and RNA-seq analyses revealed a hierarchical regulatory architecture over a 24 h period; this architecture is initiated by hormone- and circadian-related genes, followed by metabolic, cell-cycle, and physiological adjustments. Transcriptomic reprogramming was concentrated in cold- and red-light-responsive genes (e.g., CBF1 and CBF4), which were induced as early as 0.5–2 h post-treatment. Functional validation confirmed that four rapid cold-responsive motifs, including a CBF-binding site, exhibited elevated luciferase activity at 4 °C. While over 50% of dynamic chromatin accessibility regions correlated with differential gene expression, chromatin opening did not always coincide with immediate transcriptional activation. Notably, canonical auxin signaling components exhibited sustained induction and synchronized chromatin-expression dynamics. De novo motif analysis indicated that constitutively expressed AGL42 and ERF transcription factors in shoot apices may directly activate CBF4, initiating a 'development-to-stress' switch via downstream chromatin remodeling. Direct activation of the CBF4 promoter by two Eucalyptus grandis AGL42 orthologs was confirmed, establishing a novel regulatory relationship in the cold response. These findings provide a high-resolution map of how chromatin accessibility integrates transcription factors into a hierarchical regulatory network to modulate cold adaptation in subtropical woody plants.