Integrative analysis of transcriptome, proteome, and phosphoproteome reveals the complexity of early nitrogen responses in poplar roots.

Nitrogen (N) availability is a key factor in plant growth, but the molecular mechanisms underlying the early responses of poplar (Populus × xiaohei T. S. Hwang & Liang) roots to nitrogen are not well understood. The primary objective of this study was to elucidate these early molecular responses by integrating transcriptome, proteome, and phosphoproteome under low-nitrogen (LN, 0.2 mM NHNO) and high-nitrogen (HN, 2 mM NHNO) conditions. Specifically, the objectives of this study were: (i) to identify key metabolic pathways involved in nitrogen responses in poplar roots; (ii) to explore the relationship between differentially expressed genes (DEGs) and transcription factors (TFs) within these pathways; and (iii) to construct co-expression networks to uncover the regulatory mechanisms of nitrogen signaling. KEGG pathway enrichment analysis indicated that nitrogen metabolism and phenylpropanoid metabolism were key pathways in RNA-seq and proteome, while starch and sucrose metabolism were crucial in transcriptome and phosphoproteome. Plant hormone signal transduction was a key pathway in transcriptome, and gluconeogenesis/glycolysis was essential in proteome. WGCNA revealed three key modules (MEgreenyellow, MEblack, and MEblue) significantly associated with physiological indices, including NO, soluble sugar, and sucrose contents. Co-expression networks highlighted TFs as central regulators of nitrogen-responsive pathways, with distinct expression patterns between LN and HN treatments. These findings elucidate the complexity of nitrogen-regulated metabolic networks in poplar roots and reveal potential links between nitrogen signaling, carbohydrate metabolism, and secondary metabolism. This study provides a foundation for improving nitrogen-use efficiency in forest trees, with implications for sustainable forestry and ecosystem management.