Regulation of symbiotic nitrogen fixation in soybean by exogenous nitrogen concentrations: Construction of a multi-dimensional evaluation system

Soybean growth is fundamentally constrained by nitrogen availability. Although part of the required nitrogen can be supplied through symbiotic nitrogen fixation (SNF) with rhizobia, exogenous nitrogen application is needed in production systems for optimal yield. However, exogenous nitrogen exerts complex regulatory effects on SNF remains insufficiently resolved. In this study, ammonium and nitrate were supplied at different concentrations, and samples were collected at the peak nodule development stage (full flowering stage, R2) and the nodule senescence stage (beginning seed stage, R5). The ¹⁵N isotope labeling method was employed to precisely assess the percent of nitrogen derived from air (Ndfa%). In addition, a series of indicators were systematically determined, including nodule traits, acetylene reduction activity (ARA), specific nitrogenase activity (SNA), leghemoglobin content, and the activities of key enzymes involved in ureide metabolism, namely uricase and xanthine dehydrogenase (XDH). High concentrations of exogenous nitrogen strongly suppressed nodule development and nitrogen fixation function, as reflected by marked declines in nodule number, nodule dry weight, ARA, SNA, leghemoglobin content, and the Ndfa%. As nitrogen supply increased, soybean nitrogen acquisition shifted from symbiotic nitrogen fixation toward exogenous nitrogen uptake. High nitrogen concentrations suppressed uricase activity while inducing XDH activity, leading to ureide accumulation in nodules and impaired ureide translocation to the shoots, thereby establishing a negative feedback regulatory mechanism. With Ndfa% as the reference standard, a multi-dimensional, stage-specific evaluation system for nodule nitrogen fixation capacity was established. XDH activity was identified as a reliable inverse indicator of nitrogen fixation efficiency, whereas leghemoglobin content and uricase activity were effective auxiliary indicators. ARA functioned as a stage-specific indicator during the early growth stage, whereas nodule dry weight and nodule number were suitable for assessing nitrogen fixation potential at the later stage.