Responses of dioecious Populus euphratica seedlings to foliar and soil nitrogen application: core regulation of phenylpropanoid biosynthesis

The escalating global increase in reactive nitrogen (Nr) poses profound implications for human well-being and biosphere ecological processes. This study systematically investigated the distinct response strategies of male and female Populus euphratica seedlings to simulated canopy nitrogen (CN) and soil nitrogen (SN) application. We integrated comprehensive analyses of leaf and root physicochemical indicators, transcriptomics, Weighted Gene Co-expression Network Analysis (WGCNA), and protein-protein interaction (PPI) networks. The results showed that among the two factors of sex and nitrogen application method, the latter dominated the physiological difference and had a greater impact on the leaves (leaf: R 2 = 32.0%; root: R 2 = 28.3%). Transcriptomic analyses further revealed that leaf gene expression was primarily differentiated by sex, while root gene expression was primarily segregated by nitrogen application method. Notably, male plants exhibited more extensive transcriptional reprogramming in both organs in response to nitrogen treatment variations. KEGG pathway analysis highlighted phenylpropanoid biosynthesis as a central responsive pathway, with lignin synthesis-related genes significantly upregulated in roots under SN treatment. Furthermore, ammonium/nitrate transporters and aquaporins displayed organ- and sex-specific transcriptional regulation. In leaves, Hub genes associated with N application methods activated nutrient metabolism processes, whereas sex-related Hub genes exhibited suppressed phenylpropanoid biosynthesis under soil N application. These findings demonstrate that foliar N addition triggers physiological and transcriptional responses distinct from those of conventional soil N application. Therefore, incorporating canopy N processes into future studies is essential for more realistic and accurate assessments of the ecological impacts of atmospheric nitrogen deposition on desert ecosystems. • N addition method predominantly influenced physiological changes in Populus seedlings. • Male seedlings showed more robust transcriptional reprogramming. • Phenylpropanoid biosynthesis pathway constituted a core molecular basis. • Hub gene mediates the response of dioecious Populus euphratica to N addition method.