In the context of global climate change, atmospheric nitrogen deposition is increasing, and precipitation patterns are becoming more variable. This study examines the impact of these changes on nitrogen (N) allocation mechanisms in semi-arid region tree species using one-year-old Mongolian pine (Pinus sylvestris var. mongolica) seedlings. The seedlings were planted in soil collected from the Daqinggou Sandy Ecological Experiment Station (42°54′ N, 122°25′ E). Three moisture treatments were applied (WC (normal moisture, approximately 65% ± 2.5% of the field capacity), WI (30% increased moisture), and WD (30% decreased moisture)), as well as three nitrogen treatments (NC (no nitrogen), NL (5 g·m−2·y−1 nitrogen), and NH (10 g·m−2·y−1 nitrogen)). The seedlings were sprayed with a 15N-labeled CH4N2O solution (46% N, 15N abundance 10.14%) in a pot trial, with samples taken in August and October to measure N content and 15N abundance in the seedling organs and the soil. Parameters such as Ndff (%) (the percentage of nitrogen derived from fertilizer), nitrogen content of organs, 15N absorption in organs, and 15N distribution ratio were calculated. The results showed that 15N allocation in seedlings followed the trend leaves > stems > roots. Under moisture treatments, 15N allocation ratios in leaves, stems, and roots were 63.63–71.42%, 14.89–24.14%, and 12.23–14.88% under low nitrogen, and 62.63–77.83%, 13.35–22.90%, and 7.31–19.18% under high nitrogen. Significant correlations were found in 15N abundance among the seedling organs, with coefficients ranging from 0.97 to 1.00. The main effects of moisture and nitrogen, as well as their interaction, significantly impacted 15N abundance in the seedling organs. Changes in moisture levels affected the nitrogen absorption capacity of Mongolian pine. Increased moisture significantly enhanced 15N absorption in all organs, leading to 62.63–71.42% of 15N being allocated to the leaves, maintaining an appropriate proportion with the roots and stems. Nitrogen deposition altered the nitrogen allocation strategy among different organs of Mongolian pine. Under conditions of reduced moisture and low nitrogen, a greater proportion of nitrogen was captured by the roots and stems, with an allocation increase of approximately 4.98–5.77% compared to the control group, thereby mitigating the adverse effects of water deficiency. In conditions of reduced moisture and high nitrogen, the leaves, being active organs, accumulated more limiting elements, with an increase in nitrogen allocation of 2.03–8.07% compared to the control group. To achieve an optimal allocation strategy, moderate nitrogen deposition combined with increased moisture enhanced nitrogen uptake in Mongolian pine seedlings. This study provides scientific evidence for ecological restoration, wind erosion control, and agricultural and forestry management in semi-arid regions under the context of global climate change.
