Time-series transcriptome analysis of Populus roots reveals a key expression network involved in the response to...

As a critical determinant of nitrogen acquisition efficiency in perennial woody species, root systems profoundly influences productivity in plantation forestry. To investigate the molecular mechanisms underlying root responses to nitrate in forest trees, we employed RNA-seq technology, time-ordered gene coexpression networks (TO-GCNs), and weighted gene coexpression network analysis (WGCNA) in Populus '84 K'. Physiological profiling revealed that nitrate stress significantly increased root elongation in Populus, concurrently inducing a series of physiological responses: the net NO₃^- influx peaked at 6 h, nitrate reductase (NR) activity peaked at 12 h, and tissue nitrate/ammonium concentrations peaked at 36 h post-treatment. Temporal stratification of differentially expressed genes (DEGs) revealed eight hierarchical regulatory tiers through TO-GCNs. Integrated WGCNA revealed that bHLH, MYB and NAC transcription factor (TF)-mediated flavonoid biosynthesis was inhibited in the early stress phase. Concurrently, nitrogen metabolic processes driven by core regulators (ERFs, LBDs, and Nin-likes) were activated, among which the ERF2B-NRT2.1C module played a key role in the response of poplar roots to nitrate treatment. Subsequently, the Embden-Meyerhof-Parnas (EMP) pathway and the tricarboxylic acid (TCA) cycle, orchestrated by ERF64 A/B, were activated. WRKY-regulated ethylene signalling genes were significant upregulated after 72 h of nitrate treatment. This study provides a systematic framework for understanding plant nitrate response mechanisms and reveals potential regulatory hierarchies among TFs, providing novel insights for the precision breeding of nitrogen-use-efficient Populus cultivars through the targeted manipulation of core regulatory modules.