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 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.

The mechanism of nitrate inhibiting soybean (Glycine max L. Merr.) nodulation and nitrogen fixation is still controversial. The aim of this study was to evaluate whether increased carbon allocation could mitigate the inhibitory effect of indirectly supplied nitrate on nodule nitrogen fixation In this study, dual-root soybean plants were constructed with one side nodulated and the other side non-nodulated. Nitrate was applied to the non-nodulated lateral roots, either directly or following the girdling of the base of the non-nodulated root, to enhance carbon transport from the shoot o the nodulated side. After 2 days and 7 days of treatment, the nitrogen fixation capacity was assessed, nodule ultrastructure was examined, and the concentrations of sucrose, starch, and soluble sugars in roots and nodules on both sides were determined. Indirect nitrate supply significantly suppressed nodulation and nitrogen fixation, reduced sucrose and starch concentrations in nodules, and induced premature senescence of nodule structure. Enhancing carbon allocation to the nodules increase sucrose and starch levels, restored the integrity of the infected cell wall and peribacteroid membrane, and improved nitrogenase activity. Increasing carbon distribution to nodules can restore the structural integrity of infected cells and alleviate the negative impact of indirect nitrate supply on soybean nitrogen fixation.

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).

In this study, dual-root soybean (Glycine max L. Merr.) plants, with one side nodulated and the other nonnodulated, were used as experimental materials. The nonnodulated lateral roots were treated with excessive nitrate (200 mg·L⁻¹ nitrogen) for three days, followed by a three-day nitrate withdrawal, and then subjected to excessive nitrate again for another three days. Meanwhile, the nodulated side was continuously supplied with nitrogen-free nutrient solution. We measured the nitrogenase activity, nodule quantity, and concentrations of sucrose, starch, and soluble sugars, along with the microstructure of the nodules. By analyzing these data, we aim to provide theoretical insights into how indirect nitrate supply affects the nitrogen fixation capacity of nodules. The results demonstrated that indirect supply of nitrate to the soybean nodules reduced the nodule nitrogen fixation ability, which was manifested in the decrease in nodule dry weight, nodule number, and nitrogenase activity. The reason was found to be related to the decrease in carbon sources (sucrose, starch, and soluble sugar) allocated to the nodules. Further observation of the internal structure of the nodules found that the number of infected cells in the nodules decreased with the addition of nitrate, and increased with its withdrawal. However, the addition and withdrawal of nitrate did not change the effect of nitrate on the structure of infected cells around the nodules after the first addition of nitrate. These may be one of the important reasons why nitrate indirectly affects the activity of nitrogenase in nodules.