Nitrogen (N) inhibits soybean (Glycine max L.) nodulation and N₂ fixation. Isoflavones secreted by soybean roots can stimulate signal transduction for symbiotic nodules, thus playing a key role in root nodule development and N₂ fixation. The relationship between the inhibition of soybean nodulation, N₂ fixation and isoflavones by N is still unclear. In this study, dual-root soybean plants were prepared by grafting, and N or isoflavones were supplied to unilateral roots. The number and dry weight of the soybean nodules, nitrogenase activity, isoflavone concentrations and relative changes in the level of expression of nodulation-related genes were measured to study the response relationship between the N systemic regulation the soybean nodule N₂ fixation and changes in the concentrations of isoflavones in its roots. The results showed that N supply to one side of the dual-root soybeans systematically affected the N₂ fixation of root nodules on both sides, and this effect began in the early stage of nodulation. Moreover, a unilateral supply of N systematically affected the concentrations of daidzein and genistein on both sides of the roots. The concentrations of isoflavones were consistent with the change trend of soybean root nodule and nodulation-related gene expression level. Treatment with unilateral N or isoflavones affected the soybean nodule N₂ fixation and its nodulation-related genes, which had the same response to the changes in concentrations of root isoflavones. N regulates soybean nodulation and N₂ fixation by systematically affecting the concentrations of isoflavones in the roots.

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.

The specific mechanisms by which nitrogen affects the nodulation and nitrogen fixation of soybean plants are unclear. Investigating the relationship between nitrogen, nodulation and nitrogen fixation can provide new insights for the rational and proper use of nitrogen fertilizer in soybean plants. In this study, we grafted soybean roots to construct a dual-root system with a single nodulated side. Experiment I was performed at the third trifoliate leaf to initial seed filling (V3-R3) growth stages (for 30 days) for long-term nitrogen supply, and Experiment II was performed at the third trifoliate leaf to fourth trifoliate leaf (V3-V4) growth stages (for 5 days) for short-term nitrogen supply. For the two experiments, a nutrient solution providing ¹⁵NH₄¹⁵NO₃ or NH₄NO₃ as the nitrogen source was added to the non-nodulated side, while a nitrogen-free nutrient solution was added to the nodulated side. The concentrations of nitrogen supplied were 0 mg/L, 25 mg/L, 50 mg/L, 75 mg/L, and 100 mg/L. The results showed the following: (1) Short-term nitrogen supply systematically regulated the specific nitrogenase activity (SNA), thereby inhibiting the acetylene reduction activity (ARA). Under long-term nitrogen supply, the recovery of SNA was generally consistent across treatments, and the concentration of nitrogen supplied systematically regulated the growth of root nodules, thereby inhibiting the ARA. (2) Using the ¹⁵N tracer method, the concentration of fertilizer nitrogen was positively correlated with the amount of nitrogen redistributed to other organs. Although the percentage of nitrogen derived from the atmosphere (Ndfa%) decreased significantly with increasing concentrations of nitrogen supplied, the effect on the accumulation of nitrogen fixed by nodules (N_accumulation of nodules) was not significant. By establishing the relationships between the ARA (measured by the acetylene reduction method), Ndfa% (based on ¹⁵N calculations), and N_accumulation nodules (based on ¹⁵N calculations), it was found that the ARA reliably reflected the Ndfa% but not the N_accumulation of nodules.

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 regulatory effects of uneven phosphorus supplies on phosphorus transport in soybean roots are still unclear. To further analyze the regulatory effects of low-phosphorus stress on phosphorus transport in soybean roots and the effects of uneven phosphorus application on the physiological mechanism of phosphorus transport in soybean roots, dual-root soybean plants were prepared via grafting, and a sand culture experiment was performed. From the unfolded cotyledon stage to the initial flowering stage, one side of each dual-root soybean system was irrigated with a low-phosphorus-concentration solution (phosphorus-application [P+] side), and the other side was irrigated with a phosphorus-free nutrient solution (phosphorus-free [P-] side); this setup allowed the study of the effects of different phosphorus supply levels on the expression of genes and proteins and the accumulation of metabolites in soybean roots on the P- side to clarify the method through which phosphorus transport is regulated in soybean roots and to provide a theoretical basis for improving the use rate of phosphorus fertilizer. The results revealed that the unilateral supply of low-concentration phosphorus promoted the uptake of phosphorus by soybean roots and the transport of phosphorus from the P+ side to the P- side. Compared with the normal concentration of phosphorus supply and the phosphorus-free supply, the low concentration phosphorus supply affected the regulation of the metabolic pathways involved in starch and sucrose metabolism, glycolysis, fructose, and mannose metabolism, etc., thereby affecting soybean root phosphorus transport. The low-phosphorus stress inhibited fructose synthesis and sucrose synthase synthesis in the soybean roots and the synthesis of hexokinase (HK) and fructose kinase, which catalyzes the conversion of fructose to fructose-6-phosphate. Low-phosphorus stress promoted the synthesis of sucrose invertase and the conversion of sucrose into maltose by the activity of starch synthase (StS) and stimulated the synthesis of UDPG pyrophosphorylase (UGP) and phosphoglucose isomerase (GP1), which is involved in the conversion of UDP-glucose to glucose-6-phosphate. The phosphorus transport pathway of soybean roots was then affected, which promoted phosphorus allocation to UTP and glucose-6-phosphate. Additionally, low-phosphorus stress hastened glycolysis in the soybean roots and inhibited the synthesis of malic acid, thereby promoting the transport of phosphorus in the roots. In addition, low-phosphorus stress inhibited the synthesis of fructose, mannose, and mannose-1-phosphate and the synthesis of other enzymes involved in phosphorus transport as well as invertase, thereby inhibiting the transport and synthesis of several organic phosphorus-containing compounds.

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.