The specific mechanisms by which nitrogen affects nodulation and nitrogen fixation in leguminous crops are still unclear. To study the relationship between nitrogen, nodulation and nitrogen fixation in soybeans, dual-root soybean plants with unilateral nodulation were prepared by grafting. At the third trifoliate leaf (V3) to fourth trifoliate leaf (V4) growth stages (for 5 days), nitrogen nutrient solution was added to the non-nodulated side, while nitrogen-free nutrient solution was added to the nodulated side. The experiment was designed to study the effects of exogenous nitrogen on proteins and metabolites in root nodules and provide a theoretical reference for analyzing the physiological mechanisms of the interaction between nitrogen application and nitrogen fixation in soybean root nodules. Compared with no nitrogen treatment, exogenous nitrogen regulated the metabolic pathways of starch and sucrose metabolism, organic acid metabolism, nitrogen metabolism, and amino acid metabolism, among others. Additionally, exogenous nitrogen promoted the synthesis of signaling molecules, including putrescine, nitric oxide, and asparagine in root nodules, and inhibited the transformation of sucrose to malic acid; consequently, the rhizobia lacked energy for nitrogen fixation. In addition, exogenous nitrogen reduced cell wall synthesis in the root nodules, thus inhibiting root nodule growth and nitrogen fixation.

Phosphorus (P) is an important nutrient affecting nodulation and nitrogen fixation in soybeans. To further investigate the relationship of phosphorus with soybean nodulation and nitrogen fixation, the seedling grafting technique was applied in this study to prepare dual-root soybean systems for a sand culture experiment. From the unfolded cotyledon stage to the initial flowering stage, one side of each dual-root soybean system was irrigated with nutrient solution containing 1 mg/L, 31 mg/L, or 61 mg/L of phosphorus (phosphorus-application side), and the other side was irrigated with a phosphorus-free nutrient solution (phosphorus-free side), to study the effect of local phosphorus supply on nodulation and nitrogen fixation in soybean. The results are described as follows: (1) Increasing the phosphorus supply increased the nodules weight, nitrogenase activity, ureide content, number of bacteroids, number of infected cells, and relative expression levels of nodule nitrogen fixation key genes (GmEXPB2, GmSPX5, nifH, nifD, nifK, GmALN1, GmACP1, GmUR5, GmPUR5, and GmHIUH5) in root nodules on the phosphorus-application side. Although the phosphorus-application and phosphorus-free sides demonstrated similar changing trends, the phosphorus-induced increases were more prominent on the phosphorus-application side, which indicated that phosphorus supply systematically regulates nodulation and nitrogen fixation in soybean. (2) When the level of phosphorus supply was increased from 1 mg/L to 31 mg/L, the increase on the P– side root was significant, and nodule phosphorus content increased by 57.14–85.71% and 68.75–75.00%, respectively; ARA and SNA were 218.64–383.33% and 11.41–16.11%, respectively, while ureide content was 118.18–156.44%. When the level of phosphorus supply was increased from 31mg/L to 61mg/L, the increase in the regulation ability of root and nodule phosphorus content, ARA, SNA, and ureide content were low for roots, and the value for nodules was lower than when the phosphorus level increased from 1 mg/L to 31 mg/L. (3) A high-concentration phosphorus supply on one side of a dual-root soybean plant significantly increased the phosphorus content in the aboveground tissues, as well as the roots and nodules on both sides. In the roots on the phosphorus-free side, the nodules were prioritized for receiving the phosphorus transported from the aboveground tissues to maintain their phosphorus content and functionality.

The specific mechanism by which phosphorus affects nodule nitrogen fixation and nitrogen absorption in soybeans remains inconclusive. To further quantitatively analyze the effect of phosphorus on nodule nitrogen fixation and nitrogen accumulation in soybeans, this experiment was carried out under sand culture conditions. The experiment consisted of six phosphorus supply levels (1 mg/L, 11 mg/L, 21 mg/L, 31 mg/L, 41 mg/L, 51 mg/L). The acetylene reduction method and ¹⁵N tracer method (50 mg/L (NH₄)₂SO₄) were used to determine and analyze the nodule growth status, nodule nitrogenase activity, nitrogen content, and nodule nitrogen fixation rate at initial flowering (R1 stage), initial pod (R3 stage), seed filling (R5 stage) and maturity stages (R8 stage). The results are described as follows: 1. The nitrogen fixation of soybean nodules at different growth stages has different requirements for phosphorus supply levels. The initial flowering stage and seed-filling stage were 31 mg/L–41 mg/L, and the initial pod stage was 51 mg/L. 2. The nitrogen source in different parts of soybean showed different trends with different growth periods and phosphorus supply concentrations. Among them, from the initial flowering stage to the seed filling stage, the main body of the nitrogen supply of soybean shoots in the low phosphorus treatment (1 mg/L–31 mg/L) gradually changed from fertilizer nitrogen to nodule nitrogen fixation, while the main body of the nitrogen supply of soybean shoots in the high phosphorus treatment (41 mg/L–51 mg/L) always showed nodule nitrogen fixation and was transformed into fertilizer nitrogen at the mature stage. The main nitrogen supply to the roots of soybean at different levels of phosphorus supply from the initial flowering to the initial pods and maturity stage was fertilizer nitrogen, and the main nitrogen supply at the seed filling stage was nodule nitrogen fixation. The nitrogen supply to the main body of soybean nodules was constantly nodule nitrogen fixation. 3. Different phosphorus supply levels significantly affected the nitrogen fixation of soybean nodules (R² ≥ 0.803), and both the acetylene reduction method and the ¹⁵N tracer method could be used to determine the nitrogen fixation capacity of soybean nodules. This study indicated the optimal phosphorus supply level of nodules in different growth stages of soybean and clarified the main body of phosphorus supply in different parts of soybean at different growth stages, which pointed out the direction for further improving the utilization efficiency of soybean nitrogen and phosphorus fertilizer.

Global climate warming and increased climate variability may increase the number of annual freeze–thaw cycles (FTCs) in temperate zones. The occurrence of more frequent FTCs is predicted to influence soil carbon and nitrogen cycles and increase nitrogen leaching. Biochar has the potential to increase soil organic carbon storage and decrease nitrogen leaching. This study aims to investigate the impact of freeze–thaw cycles (FTCs) on soil nitrogen leaching in temperate zones, considering the potential exacerbation of FTCs due to global climate warming and increased climate variability. This study focuses on how biochar, a carbon-rich material produced from biomass, might mitigate nitrogen leaching by influencing soil characteristics. This study explores the interactions between different laboratory-simulated FTC frequencies (ranging from 0 to 12 cycles) and various biochar addition ratios (0%, 2%, 4%, and 6% w/w) on soil nitrogen leaching based on a total of 60 soil columns. Pearson correlations between the soil quality indicators and nitrogen leaching characteristics were detected, and partial least squares path modeling (PLS-PM) was used to assess the effects of the FTCs, biochar addition ratios, and soil quality indicators on the nitrogen leaching content. The results showed that the amount of leached soil NH₄⁺-N and NO₃⁻-N reached 0.129–1.726 mg and 2.90–7.90 mg, respectively. NH₄⁺-N and NO₃⁻-N first increased and then decreased under the FTCs, with the highest values being observed after the 6th FTC. As the biochar addition ratio increased, the NH₄⁺-N and NO₃⁻-N contents decreased. Correlation analysis showed that the nitrogen leaching content was significantly related to the soil pH, soil organic matter (SOM), NH₄⁺-N content, and microbial biomass carbon content (MBC) (p < 0.01). The results of the conceptual path model revealed that nitrogen leaching characteristics were significantly affected by the pH, SOM, soil nitrogen content, and biochar addition ratio. Our results suggest that biochar addition can help reduce nitrogen leaching in farmland soil in areas with black soil and seasonal freeze–thaw cycles.