Crop yields heavily depend on nitrogen (N) fertilizer supply. Owing to the low cost and high N content of urea, farmers often apply N fertilizers excessively and indiscriminately in pursuit of higher yields, leading to yield stagnation, significant declines in nitrogen use efficiency (NUE), and exacerbated environmental pollution through energy-intensive production and application (Liu et al., 2021; Bodirsky et al., 2014; Camargo et al., 2005). Consequently, enhancing NUE is of crucial importance for sustainable agriculture. In Xinjiang, under mulched drip irrigation combined with integrated water-fertilizer management has repeatedly achieved record-high soybean yields in China. However, the total N supply during the growing season remains excessively high (240∼310 kg ha^-1), resulting in high yields but low NUE. This inefficiency stems from farmers adopting cotton field management practices for soybeans, ignoring the unique role of symbiotic nitrogen fixation (SNF) in soybean nitrogen acquisition, which differs fundamentally from graminaceous crops like cotton and rice. While soybean nitrogen sources include soil N, fertilizer N, and SNF, relying solely on SNF cannot achieve high yields; thus, targeted exogenous N supplementation based on soil fertility remains essential (Thorburn et al., 2024). Coordinating SNF, N fertilization, and yield by optimizing exogenous N supply to maximize SNF potential, establish an efficient soybean SNF system, and achieve high yield with resource efficiency holds significant practical importance (Goyal et al., 2021; Coba de la Peña et al., 2018).

Soybean plants acquire N through root uptake and SNF in root nodules. However, excessive N supply is well-documented to suppress root nodulation. When soil N availability increases significantly, plants prioritize direct root N absorption over SNF due to its lower energy cost, leading to reduced nodule number, nodule biomass, nitrogenase activity, leghemoglobin content, impaired glutamine synthetase (GS)/glutamate synthase (GOGAT) and glutamate dehydrogenase(GDH) assimilation pathways, and accelerated nodule senescence (Lyu et al., 2020; Albornoz, 2016; Wang et al., 2015). It is suggested by most studies that high N inputs inhibit root hair infection and nodule initiation (Mendes et al., 2018; Santachiara et al., 2018). Conversely, some researchers argue that low-level N fertilization does not compromise nodulation, as nodules may outcompete roots for resources; moderate N application during reproductive growth slightly reduces nodule weight but maintains nitrogenase activity (Ke et al., 2023; Pampana et al., 2018; Carter and Tegeder, 2016). Notably, Nguyen et al. (2020) demonstrated that novel rhizobial strains exhibit robust nodulation and SNF capacity even under high nitrate conditions. Proposed mechanisms for N-mediated suppression of SNF include carbon starvation (Lu et al., 2022; Parvin et al., 2020), nitrogen metabolite feedback regulation (Nazari et al., 2019; Castañeda et al., 2018), and internal oxygen limitation (Larrainzar et al., 2020; Gourion et al., 2015). Studies indicate that 50∼60% of soybean N demand is met via SNF (Salvagiotti et al., 2008), with some cases exceeding 90% (Mastrodomenico and Purcell, 2012). Strong positive correlations exist between nodule number, nodule dry weight, SNF efficiency, and seed yield, underscoring the critical role of nodule health in N fixation and productivity (Ferguson et al., 2019; Burias and Planchon, 1990). To optimize high-yield soybean N demand while minimizing exogenous N input, strategies such as deep placement of slow-release N fertilizers in nodulation zones (Yaseen et al., 2021), early sowing (Kurdali, 1996; Sprent and Thomas, 1984), and split N application after the beginning pod stage (R₃) (Zapata et al., 1987) can sustain nodule activity and enhance yield efficiency.

Ureides (allantoin and allantoic acid), the primary SNF products exported from soybean root nodules to stems, are temporarily stored in stems (Atkins and Smith, 2007; Smith and Atkins, 2002). Enhancing ureide export from nodules positively impacts soybean reproductive growth by increasing SNF capacity and nodule number (Carter and Tegeder, 2016). In the enzymatic synthesis of ureides, urate oxidase (UO) activity catalyzes the oxidation of uric acid to allantoate, playing a critical role in nodule development and SNF. Ureide synthesis helps plants avoid ammonia toxicity by reducing glutamine and asparagine accumulation in nodules (Britto and Kronzucker, 2002; Engvild, 1987). Excessive N fertilization suppresses nodule uricase activity, elevates ammonia toxicity risks, and reduces stem ureide content (Wang et al., 2024). Under low N conditions, ureide export from nodules to stems is enhanced, whereas high N supply inhibits ureide translocation. Accumulated ureides in nodules directly suppress nitrogenase activity and indirectly impair SNF via feedback regulation mediated by stem ureide levels (Gil-Quintana et al., 2013; Serraj et al., 1999).

Previous studies have demonstrated that excessive exogenous N supply significantly suppresses soybean root nodulation and SNF (Thorburn et al., 2024). Under the mulched drip irrigation, this study systematically investigates the response mechanisms of root nodule developmental dynamics and nitrogen fixation metabolism in high-yield soybean to varying N levels. We evaluate the adaptability of soybean nodules under different N regimes from the perspectives of N fixation, assimilation, and translocation. We hypothesize that under mulched drip irrigation, excessive N application at the beginning of pod formation will impede nodule formation, downregulate nitrogenase activity, inhibit carbohydrate allocation to nodules, and reduce long-distance transport of ureides (allantoin and allantoic acid) to aboveground tissues.

In well-managed soybean fields, SNF can supply 70∼85% of the crop’s N demand (Alves et al., 2003). Accumulating evidence from numerous studies indicates that high levels of N in the soil strongly inhibit rhizobial infection efficiency, nodule organogenesis, and nitrogen fixation efficiency, manifesting as reduced nodule number, decreased nodule biomass, inhibited nitrogenase activity, and accelerated nodule senescence or degradation (Pampana et al., 2018; Carter and Tegeder, 2016; Albornoz, 2016; Wang et al., 2015). Conversely, low levels of N enhance nodulation and nitrogen fixation activity (Ke et al., 2023). Given evidence that R₃ stage N application significantly impacts grain yield (Mourtzinis et al., 2018; Zapata et al., 1987), this study applied N treatments at R₃ stage, resulting in no significant differences in nodule morpho-physiological traits across treatments at the R₂ stage. In our experiment, the application of 180 kg ha^-1 nitrogen increased nodule biomass and number, while the application of N at rates exceeding 180 kg ha^-1 caused significant declines in both parameters. These findings partially contrast with those of Gan et al. (2002) and Nanjareddy et al. (2014), likely due to the lower initial soil N levels in our experimental plots, where N was not the primary limiting factor for nodulation. While nodulation is foundational to SNF, high nodule numbers do not necessarily correlate with high nitrogen fixation capacity (Herridge and Rose, 2000; Song et al., 1995). Extensive research demonstrates that nitrogenase activity strongly links to SNF efficiency and yield, with elevated soil mineral suppressing nitrogenase activity and leghemoglobin content (Liu et al., 2022; Lyu et al., 2020). Our results corroborate this: higher soil N concentrations reduced nodule nitrogenase activity, likely due to diminished leghemoglobin content, which compromises oxygen regulation in infected nodule cells. Since nitrogenase is highly oxygen-sensitive, this dysregulation leads to marked declines in enzymatic activity (Larrainzar et al., 2020; Kawashima et al., 2001). Consistently, our data confirm that N fertilization reduces nodule leghemoglobin content.

Nitrogen availability significantly impacts ammonium assimilation in soybean nodules: elevated soil N enhances GS and GOGAT activities in roots but suppresses these enzymes in nodules, particularly GOGAT (Sawhney et al., 1985). This study also reached the same conclusion, increased N application reduced both GS and GOGAT activities, with GOGAT showing greater sensitivity, consistent with Do Amarante et al. (2022).To prevent excessive ammonium accumulation from inhibiting plant growth, the GDH pathway is activated to promote ammonium assimilation. In this study, nodule GDH activity increased with higher N application. Although GDH activity did not differ significantly among treatments in 2022, these trends still highlight the critical role of GDH in reducing NH₃ toxicity in nodules, suggesting a potential shift in ammonium assimilation from the GS/GOGAT pathway to the GDH pathway. While high soil N concentrations transiently affect the nodule GDH pathway, this effect is not persistent. As plants absorb soil N, the inhibitory effect on the GDH pathway is alleviated, likely due to nonsignificant differences in GDH activity among treatments at R₆ stage. This phenomenon may also be attributed to accelerated root and nodule senescence under low N conditions during the late growth stages (Nishida and Suzaki, 2018).

Besides ammonium assimilation, the synthesis and transport of ureide compounds in nodules also play crucial roles in the nitrogen metabolism of soybean. High N application elevates ureide content in nodules (Yamashita et al., 2019). In this study, under N-free conditions, nodule ureide export to stems and their allocation were significantly increased compared to other treatments, supported by reduced nodule ureide content and elevated stem ureide content. When N was applied, we observed significant reductions in nodule ureide synthase activities (e.g., UO), yet nodule ureide content remained higher than in N-free treatments, with the opposite trend in stems. This indicates that N application impairs ureide synthesis and transport in nodules, leading to ureide accumulation. Accumulated ureides likely exert feedback inhibition on nitrogenase activity, as evidenced by significantly reduced nitrogenase activity under high N conditions, consistent with findings by Serraj et al. (1999) and Gil-Quintana et al. (2013). Nodules act as strong carbon sinks, requiring energy for ureide synthesis. Thus, insufficient carbon transport to nodules limits SNF and related processes (Lu et al., 2022; Parvin et al., 2020). As the primary form of carbon transport, sucrose accumulation in nodules is closely linked to nitrogen fixation and assimilation. In this study, nodule sucrose and starch contents were lower in N-free and high-N treatments compared to other treatments, particularly in high-N plots. Reduced nodule carbon availability may be associated with decreased ureide export (Rawsthorne et al., 1980), resulting in elevated nodule ureide content, suppressed nitrogen fixation, accelerated nodule senescence, and reduced nitrogen use efficiency.

Although low N levels restrict soybean growth, high N application only marginally enhances yield, while excessive N supply reduces both yield and NUE (Thorburn et al., 2024).In this study, soybean yield reached its maximum at a N application rate of 180 kg ha^-1, primarily attributed to increased pod and seed numbers. During the reproductive growth stage, reliance solely on SNF cannot fully meet plant N demand, necessitating supplemental N fertilization. Moderate N application compensates for N deficits, balances vegetative and reproductive growth, and provides energy for pod formation and grain filling, while simultaneously maximizing NUE. To evaluate relationships among nodule growth, stem ureide content, and %Ndfa at the R₄ stage, correlation and structural equation modeling revealed significant positive correlations between nitrogenase activity, leghemoglobin content, UO activity, stem ureide content, and %Ndfa, consistent with van Berkum et al. (1985). Conversely, nodule ureide content showed a significant negative correlation with %Ndfa, further confirming that high N inhibits ureide translocation from nodules. Nitrogenase activity and leghemoglobin content positively regulated nodule ureide levels, whereas UO activity exhibited negative regulation, suggesting feedback control of UO activity by ureide accumulation. Nodule ureide content inversely influenced stem ureide content, while stem ureide content strongly and positively affected %Ndfa (path coefficient = 0.95). This explains why stem ureide content serves as a reliable proxy for nitrogenase activity, given that ureides account for approximately 80% of total stem N, aligning with findings by Mcclure and Israel (1979).

Achieving simultaneous improvements in NUE and yield stability in future soybean production represents a core challenge for sustainable agriculture. Across the 2022 and 2023 growing seasons, our data demonstrate that under mulched drip irrigation, the application of 180 kg ha^-1 nitrogen significantly enhanced root nodulation capacity (95.5% increase in nodule number and 45.6% increase in nodule dry weight at the R₅ stage) and optimized nodule nitrogenase activity (5.8 µmol g^-1 h^-1 at the R₅ stage) and leghemoglobin synthesis (7.5 mg g^-1 at the R₅ stage). These improvements elevated NUE to 8.2 kg kg^-1 and increased yield by 22.1%. However, the application of N exceeding 180 kg ha^-1 suppressed SNF (35.5% reduction in SNF) by impairing root nodulation capacity, inhibiting the GS/GOGAT metabolic pathway, and reducing ureide compound translocation from nodules to stems, leading to significant ureide accumulation in nodules. SEM revealed the stem ureide content exhibited a strong direct positive effect on %Ndfa (path coefficient=0.95), establishing stem ureide content as an effective physiological indicator for evaluating SNF capacity. Given the strong dependence of soybean nodule nitrogen fixation capacity on climatic conditions and agronomic practices, future research should focus on understanding interactions between SNF and environmental conditions to optimize adaptability, thereby achieving synergistic improvements in yield and NUE for future cultivars.