The mechanism by which nitrate inhibits nitrogen fixation in soybean (Glycine max L.) is not fully understood. Accumulation of ureide in soybean plant tissues may regulate the nitrogen fixation capacity through a feedback pathway. In this study, unilaterally nodulated dual-root soybeans prepared by grafting were grown in sand culture. They were subjected to the removal of the nodulated side roots, and were given either nitrate supply or no supply to the non-nodulated side roots for 3 days (experiment I). Additionally, they received nitrate supply to the non-nodulated side roots for 1–14 days (experiment II). The results showed that nitrate supply increased the levels of asparagine and ureide in soybean shoots (Experiment I). In Experiment II, nodule dry weight, nodule number, nodule nitrogenase activity, and nodule urate oxidase activity decreased significantly after 3, 7, and 14 days of nitrate supply. Ureide content in the shoots and nodules increased after 1, 3, and 7 days of nitrate supply, but decreased after 14 days of nitrate supply. There was a significant positive correlation between urate oxidase activity and nitrogenase activity. Hence, we deduced that nitrate supply increased the asparagine content in soybean shoots, likely inhibiting ureide degradation, which induced the accumulation of ureide in soybean shoots and nodules, and, in turn, feedback inhibited the nodule nitrogen fixation. In addition, urate oxidase activity can be used to assess the nitrogen fixation capacity of nodules.

In this study, the systematic analysis of nitrate assimilation and transport in soybean roots was further improved by analyzing the concentrations of nitrate assimilates, asparagine and glutamine, in soybean roots and the related enzyme activities. This provided a theoretical basis for the efficient utilization of nitrogen fertilizer in soybean farming. A dual-root soybean system with both sides being nodulated was used to provide nitrate withdrawal and resupply in three phases on one side, while the other side received nutrients without nitrogen under sand culture conditions. Measurements were taken of the root’s nitrate reductase and glutamine synthetase activities, as well as the concentrations of asparagine. Measurements were also taken of the asparagine concentration in the basal root peeled skin (where the primary transport pathway is the phloem) and the woody parts (where the primary transport pathway is the xylem). Furthermore, the concentration of glutamine in the roots was also assessed. The findings indicated a positive correlation between the nitrate concentration in the roots and the activity of glutamine synthetase in the roots on both sides. The levels of asparagine in the roots, specifically those in the basal root peeled skin and woody part on both sides, rose when nitrate was added and declined when nitrate was removed. There was no significant change in glutamine concentration within the roots of soybeans. Hence, we deduced that the local provision of nitrate to the dual-root soybeans might enhance the absorption of nitrate into the roots on both sides. Additionally, the assimilated substances were predominantly carried as asparagine through the peeled skin and woody sections of the basal root, facilitating transportation in both directions within the soybean plants (from shoot to root and from root to shoot).

Soybean (Glycine max) nitrogen fixation is inhibited by nitrate, which has been linked to a reduction in carbon allocation and metabolism within nodules. However, the underlying mechanisms remain unclear. In this study, we tested the hypothesis that the nitrate-induced suppression of nitrogen fixation is mediated through altered sucrose allocation and catabolism in nodules. Using unilaterally nodulated dual-root soybean plants in sand-based systems, we applied 200 mg·L⁻¹ nitrate exclusively to the non-nodulated roots for 14 days. Nitrate supply enhanced the proportion of dry weight in leaves but reduced it in nodules at 3, 7, and 14 days. Similarly, nodule dry weight, nodule number, acetylene reduction activity (ARA), and specific nodule activity (SNA) all declined significantly during the same intervals. Notably, sucrose content in the nodules decreased significantly by 20.4% after 3 days but recovered at 7 and 14 days. In contrast, sucrose synthase (SuSy) cleavage activity and malate content in nodules decreased significantly following nitrate treatment, with reductions of 27.8% and 30.7% observed at 7 days, and further decreased to 38.5% and 39.2% at 14 days, respectively. These results suggest that transient sucrose scarcity may drive the initial decline in nitrogen fixation capacity, while restricted sucrose catabolism and decreased malate levels may be a consequence rather than a cause.