Assessing the Systematic Effects of the Concentration of Nitrogen Supplied to Dual-Root Systems of Soybean Plants on Nodulation 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 15 NH 415 NO 3 or NH 4 NO 3 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 15 N 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 15 N calculations), and N accumulation nodules (based on 15 N calculations), it was found that the ARA reliably reflected the Ndfa% but not the N accumulation of nodules.


Introduction
The severe global climate changes have a serious impact on the development of global agriculture [1]. China occupies 9% of the world's arable land, but it accounts for 30% of the global fertilizer use [2]. Excessive fertilizer application leads to serious negative impacts on the ecosystem [3]. To address these impacts, more attention has been given to research on crop biological nitrogen fixation in order to foster sustainable agriculture [4,5]. Soybean (Glycine max (L.) Merr.) is an important nitrogen-fixing crop. The amount of nitrogen fixed by soybean plants during their lifetime accounts for 25-85% of the total aboveground nitrogen [6][7][8]. Overly high concentrations of inorganic nitrogen can inhibit nodulation and nitrogen fixation [9][10][11][12][13][14][15], whereas low concentrations of nitrogen supplied can promote these processes [16][17][18].
To better understand the regulation of nodulation and nitrogen fixation by nitrogen in leguminous plants, split-root and dual-root experiments were performed. When treating one soybean root system with high nitrogen (using either NO3 − or NH4 + ), the nodule number and weight on the nitrogen-treated side are significantly suppressed, whereas those of the nitrogen-untreated root system do not show significant changes [19,20]. The nodule number, nodule weight, and nitrogenase activity of the nitrogen-treated side are also significantly lower than those of the nitrogen-untreated side [21]. When NO3 − is supplied to one side of a dual-root system of soybean plants under hydroponic conditions, the weight and nitrogenase activity of root nodules on the untreated side are not significantly affected as long as the nitrogen concentration is below 100 mg/L, but they are adversely affected when the nitrogen concentration is 200 mg/L [22]. In split-root systems of Trifolium repens (white clover), Casuarina cunninghamiana Miq. (Casuarina equisetifolia), and Medicago truncatula (barrel medic), in which one side was experimentally treated with nitrogen and the other was not, the weight and number of root nodules of the nitrogen-treated side were significantly inhibited with increasing concentrations of nitrogen supplied, but those of the untreated side did not change significantly [10,23,24]. These studies indicate that the concentration of nitrogen supplied can locally inhibit the growth and nitrogenase activity of root nodules of leguminous crop plants. In a split-root system of peanut (Arachis hypogaea L.) one side was treated with 4 mM NO3 -for 30 days, and the nitrogenase activity of root nodules on both sides were inhibited [25]. Kosslak and Bohlool [26] used a split-root system of soybean plants cultured in sand medium to perform rhizobium inoculation of one side first and then the other side 10 days later and found that the root nodulation of the side with delayed rhizobium inoculation was inhibited. Van Brussel et al. [27] obtained the same results when using pea (Pisum sativum L.) as the test material, indicating the systematic regulation of nodulation by the other side of the split-root system.
There have been studies on biological nitrogen fixation by leguminous crops [28][29][30]. Since the innovation of the 15 N2 exposure method by Burris and Miller [31] for studying the effects of nitrogenfixing bacteria on nitrogen gas (N2) fixation, 15 N2 tracer technology has been widely used to study the root nodule nitrogen fixation of leguminous crops [32][33][34]. Similarly, in a split-root system of soybean plants with 15 NO3 -provided to one side of the root system, 15 N was detected in the root and root nodules of the untreated side, and the amount of 15 N increased with the concentration of nitrogen supplied [22]. Reynolds, et al. [35] added 13 NH4 + to one side of the split-root system of soybean plants and detected 13 N-labeled amino acids in the shoot and the untreated side. These findings indicate that nitrogen can systematically affect the growth of the other side of the root system through some aboveground plant parts. Split-root and dual-root systems (both sides were nodulated) are useful for studying the effects of nitrogen on the local inhibition of nodulation and nitrogen fixation of leguminous crops.
When we used the split-root and dual-root system (both sides were nodulated) methods, when the nitrogen concentration of the nitrogen-treated side increases, there is no effect on the nodulation and nitrogen fixation on the other side [19][20][21][22][23]36]. Nodulation and nitrogen fixation of nitrogentreated side are first affected, buffering the ability of the root system to transport nitrogen to the rest of the plant and influencing the results in systemic effect of nitrogen supply. Therefore constructing a reasonable method to study the systemic effect of nitrogen on soybean nodules and nitrogen fixation is necessary. There have been separate studies on the effects of long-term or short-term nitrogen supply on soybean nodulation and nitrogen fixation in different reports [20][21][22][23][25][26][27]; it is impossible to compare the results of two or more researches, due to different experimental conditions (varieties, cultivation methods, and environmental factors). Therefore, it is meaningful to compare the effects of indirect long-term and short-term nitrogen supply on soybean nodule nitrogen fixation under the same experimental conditions.
In this study, based on the dual-root systems of Xia et al. [20], we innovatively constructed a soybean dual-root system with a single nodulated side to study the systematic regulation of nitrogen supply on soybean nodulation and nitrogen fixation. The objective of this study was to explore the mechanisms underlying the short and long term systematic regulation of nitrogen uptake from fixation and fertilizer in soybean, thereby providing new insights for the rational and proper use of nitrogen fertilizer in soybean. The long-term nitrogen treatments were performed at the V3-R3 growth stages (for 30 days), and the short-term nitrogen treatments were performed at the V3-V4 growth stages (for 5 days). Different concentrations of 15 N-labeled nitrogen were added to the nonnodulated root system. The nodule number, nodule dry weight and nitrogenase activity of the nodulated side were determined, and the 15 N abundance, percentage of nitrogen derived from the atmosphere (Ndfa%), and nitrogen accumulation (Naccumulation) were measured. In this study, we proposed the following hypotheses: (1) Both long-term and short-term nitrogen treatments systematically inhibit the nitrogen fixation ability of root nodules of the non-nitrogen supply side, but different nitrogen supply times lead to different underlying mechanisms (short-term nitrogen supply systematically regulates the specific nitrogenase activity (SNA), thereby inhibiting the acetylene reduction activity (ARA); under long-term nitrogen supply, the concentration of nitrogen supplied systematically regulates the growth of root nodules, thereby inhibiting ARA). (2) Under an increasing nitrogen concentration, the ARA, Ndfa%, and Naccumulation of nodules did not change synchronously.

Materials and Methods
The study was performed at the Northeast Agricultural University experimental station (Harbin, Heilongjiang Province, China, 126°43′ E, 45°44′ N) in 2018 and 2019. The seeds were nodulated soybeans (HeiNong40 Glycine max L. cv.) and non-nodulated soybeans (WDD01795, L8-4858 Glycine max L. cv. obtained from the Academy of Agricultural Sciences in China, Beijing).

Experimental Design and Treatment
Based on the method described by Xia et al. [20], we constructed a soybean dual-root system with a single nodulated side, which was cultured in sand medium in pots. Five pots were used for each treatment. A detailed description of the construction and preparation of the dual-root system is provided in Figure S1.
Before the VC (unfolded cotyledons) stage, the seedlings were irrigated with distilled water once daily. From the VC stage to the V3 (third trifoliate leaf) stage, the prepared nutrient solution (nitrogen concentration: 25 mg/L, NH4NO3) was added once daily to culture the soybean seedlings. Next, experiments I and II were treated as follows: Experiment I (long-term nitrogen treatment): From the V3 stage to the R3 (initial seed filling) stage (for 30 days), nutrient solution (nitrogen concentration: 0 mg/L, 25 mg/L, 50 mg/L, 75 mg/L, and 100 mg/L; 15 NH4 15 NO3, 15 N abundance: 3.36%) was supplied to the non-nodulated root system (the N+ side); nitrogen-free nutrient solution was supplied to the nodulated root system (the N-side). Conducted in 2018 and 2019.
Experiment II (short-term nitrogen treatment): From the V3 stage to the V4 (fourth trifoliate leaf) stage (for 5 days), nutrient solution (nitrogen concentration: 0 mg/L, 25 mg/L, 50 mg/L, 75 mg/L, and 100 mg/L; NH4NO3) was supplied to the non-nodulated root system (the N+ side); nitrogen-free nutrient solution was supplied to the nodulated root system (the N-side). Conducted in 2019.
The study is a single factor experimental design, and the nutrient solution ingredients are listed in Table S1. Nutrient solution (250 mL) was poured on each side (irrigated slowly to make sure the nutrient solution did not flow into the opposite side). After the R1 (initial flowering) stage, the nutrient solution was irrigated twice a day. At the VC stage, both sides of the roots were inoculated with rhizobia as follows: the soybean nodules harvested in the previous year were ground and added to the nutrient solution. The nodule-containing nutrient solutions were applied to the corresponding root systems for five consecutive days.

Sampling and Parameter Analysis
In both treatments, basic samples were taken at the V3 stage, which continued until the R3 stage for experiment I and the V4 stage for experiment II. The aboveground part of the plant along the grafting site was cut, the underground double root system was washed with distilled water, and then dried with filter paper. The nodules were removed and counted. After drying at 65 °C, the 15 N abundance, weight, and nitrogen concentration of each part were measured. The V3 stage is the initial stage of soybean nodule growth. From the V3 stage, long-term and short-term experimental treatments are carried out. Nitrogen has a strong effect on young nodules. The R3 stage is the most prosperous period of nodule growth, and it is more representative to study the effect of nitrogen on nodules.
The acetylene reduction assay uses the method described in Lyu et al. [36]. The soybean roots were rinsed and dried with filter paper, and all roots and nodules were placed in a wide-mouth amber glass bottle. Fifty milliliters of air were drawn with a syringe, and then fifty milliliters of acetylene was injected. After 2 h of reaction, the ethylene concentration in the bottle was determined by a gas chromatograph (Model GC7900, Shanghai Techcomp Scientific Instrument Co., Ltd., China).
Plant nitrogen content assay: The plant nitrogen content was determined by an automatic Kjeldahl analyzer (Model B324, Shanghai Shengsheng Automatic Analysis Instrument Co., Ltd., China). 15 N abundance assay: The plant nitrogen concentration was first determined using the Kjeldahl method. 15 N abundance was determined by a mass spectrophotometer (Thermo-Fisher Delta V Advantage IRMS, Waltham, MA, USA) in dual-inlet (DI) mode.
Where Ndfa% is the percentage of nitrogen derived from the atmosphere, 1 − Ndfa% is the percent of nitrogen derived from fertilizer, fnature is the natural 15 N abundance, ffertilizer is the 15 N abundance of the fertilizer, NR3 is nitrogen accumulation at the R3 growth stage, NV3 is nitrogen accumulation at the V3 growth stage, and Naccumulation is nitrogen accumulation at the V3-R3 growth stages.
The accumulation of nitrogen derived from atmosphere at the V3-R3 stages was calculated as: Naccumulation of nodules = Naccumulation × Ndfa% (4) The accumulation nitrogen derived from fertilizer at the V3-R3 stages was calculated as:

Statistical Analyses
All statistical analyses were performed using SPSS 21.0 (SPSS Inc., Chicago, IL, USA). Before performing one-way analysis of variance (ANOVA) on the data, all data were tested for normality and Duncan's multirange test was used with a significance level of p < 0.05. Table 1 shows the nodule dry weight and nodule number of a dual-root system with a single nodulated side at the R3 stage under experiment I. During the two-year experiment, the concentrations of nitrogen was 0 mg/L (N0) had the highest number of nodules in all treatments, and the number of nodules in the five treatments gradually decreased with increasing nitrogen supply. In 2018, the N0 treatment root nodule dry weight was also the highest, significantly higher than all other treatments, and the root nodule dry weight in the five treatments decreased with increasing nitrogen supply concentration. In 2019, although the N0 treatment had the highest dry weight of root nodules, there was no significant difference between the N0 and the concentrations of nitrogen was 25 mg/L (N25) treatments. In general, the nitrogen supply to the non-nodulated side significantly affected the number and weight of root nodules of the nodulated side, which decreased with increasing nitrogen supplied in both years of the experiment.   Table 2 shows the long-term effects of changing concentrations of nitrogen supplied on the ARA and SNA in soybean plants. In 2018, the ARA of the N0 treatment was higher than that of the other treatments. The ARA of the five treatments decreased as the nitrogen supply increased. In 2019, ARA at the N-side in the N0 treatment was significantly higher than those in all other treatments. During the two-year experiment, ARA decreased linearly as the concentration of nitrogen supplied increased. In 2018, the SNA in the N0 treatment was higher than that in the other treatments, and the SNA of the five treatments linearly decreased with increasing nitrogen supply. In 2019, the SNA in the N0 treatment was highest among all treatments, and those in N25, the concentrations of nitrogen was 50 mg/L (N50), the concentrations of nitrogen was 75 mg/L (N75), and the concentrations of nitrogen was 100 mg/L (N100) did not differ significantly. During the two-year experiment, the SNA decreased as the concentration of nitrogen supplied increased, though to a lesser extent than the reduction in ARA. In combination with changes in the soybean root nodules, these data show that ARA decreased due to the reduction in the number and dry weight of root nodules. Table 3 shows the short-term effects of changing concentrations of nitrogen supplied on the nodulation and nitrogen fixation of the dual-root systems of soybean plants. On day five, changing concentrations of nitrogen supplied had no significant effect on the root nodules, but the SNA changed with increasing concentration. The SNA in the N0 treatment was significantly higher than that in all other treatments, and the SNA of the other four treatments decreased by 6.1%, 11.3%, 34.2%, and 43.9%, respectively. The effects of short-term nitrogen treatment on ARA and SNA were similar. The ARA in the N25 treatment was significantly higher than that in all other treatments, and the ARA of the other four treatments decreased by 8.9%, 12.1%, 27.8%, and 38.2%, respectively. During the short-term nitrogen supply, both SNA and ARA decreased as the concentration of nitrogen supplied increased, and the root nodules did not change significantly. This indicates that, during the shortterm treatment, the decrease in ARA was due to the reduction in SNA.   Table 4 shows the 15 N abundance of the shoots, two sides of the roots, and root nodules of the soybean plants at the R3 stage for experiment I. During the two-year experiment, the 15 N abundance of shoots, root system on both sides, and root nodules increased with increasing concentrations of nitrogen supplied. For the two-year experiment, the 15 N abundance of the root system and root nodules in the N25, N50, N75, and N100 treatments were higher than those in N0. This indicates that fertilizer nitrogen absorbed by the root system at the N+ side was transferred to the roots and root nodules at the N-side, and the extent of nitrogen transfer increased with the concentration of nitrogen supplied to the N+ side.  Table 5 shows the changes in the Ndfa% of the shoots, two sides of the roots, and root nodules of the soybean plants at the V3-R3 stages. The Ndfa% of the shoots at the V3-R3 stages decreased with increasing concentrations of nitrogen supplied, which was consistent across the two years of the experiment. In 2018, the Ndfa% in the N0 treatment was significantly higher than that in all other treatments, and the Ndfa% of the other four treatments decreased by 20.3%, 23.3%, 33.4%, and 43.6%, respectively. In 2019, the Ndfa% of the shoots at the V3-R3 stages differed significantly between treatments with increasing concentrations of nitrogen supplied. In the N25, N50, N75, and N100 treatments compared with the N0, the Ndfa% decreased by 15.2%, 21.8%, 48.0%, and 61.6%, respectively. During the two-year experiment, the Ndfa% of the root system on the N+ side decreased with increasing concentrations of nitrogen supplied. In 2018, the Ndfa% value of the root system on the N+ side in N0 was significantly higher than those in all other treatments. The Ndfa% of the other four treatments decreased by 50.2%, 55.3%, 55.4%, and 62.6%, respectively. The Ndfa% of the root system on the N+ side in the N25, N50, and N75 treatments did not differ significantly from each other but were significantly higher than that in N100. In 2019, the Ndfa% of the root system on the N+ side also decreased with increasing concentrations of nitrogen supplied and was significantly different among the five treatments. The Ndfa% of the other four treatments decreased by 42.9%, 56.6%, 67.3%, and 75.1%, respectively. During the two-year experiment, the Ndfa% of the root system on the N-side showed the same pattern between treatments, whereas the Ndfa% of the root system on the N-side in the N0 treatments was significantly higher than that in all other treatments. In 2018, the Ndfa% of the other four treatments decreased by 5.4%, 6.6%, 13.7%, and 17.6%, respectively. In 2019, the Ndfa% of the other four treatments decreased by 5.3%, 7.6%, 15.8%, and 24.6%, respectively. During the two-year experiment, the Ndfa% of the root nodules on the N-side all decreased with increasing concentrations of nitrogen supplied. In 2018, the nitrogen treatments compared with N0, the Ndfa% of the other four treatments decreased by 2.4%, 2.6%, 5.9%, and 8.8%, respectively. In 2019, the Ndfa% of the other four treatments decreased by 1.9%, 2.7%, 6.0%, and 8.5%, respectively. It can be seen from the two-year experimental data that the root system directly exposed to nitrogen supply underwent significant changes. The roots and root nodules on the other side also experienced some changes, the extent of which was far less than that of the side directly exposed to nitrogen.  Table 6 shows the changes in the Naccumulation of all organs in soybean plants at the V3-R3 stages under experiment I. During the two-year experiment, the Naccumulation of shoots in the N100 treatments was higher than that in the other four treatments. In 2018, the Naccumulation of the other treatments decreased by 52.2%, 33.9%, 26.5%, and 19.1%, respectively. In 2019, the Naccumulation of the other treatments decreased by 70.0%, 59.5%, 49.4%, and 28.1%, respectively. During the two-year experiment, the Naccumulation of the N+ side roots in the N100 treatments was higher than that in the other four treatments. In 2018, the Naccumulation of the other treatments decreased by 80.9%, 58.6%, 229.0%, and 1.6%, respectively. In 2019, the Naccumulation of the other treatments decreased by 64.8%, 29.7%, 19.8%, and 15.9%, respectively. During the two-year experiment, the Naccumulation of the N-side roots in the N100 treatments was higher than that in the other four treatments. In 2018, the Naccumulation of the other treatments decreased by 55.9%, 37.9%, 25.9%, and 5.5%, respectively. In 2019, the Naccumulation of the other treatments decreased by 57.8%, 32.6%, 21.2%, and 9.2%, respectively. During the two-year experiment, the Naccumulation of root nodules in the N0 treatments was higher than that in the other four treatments. In 2018, the Naccumulation of the other treatments decreased by 3.7%, 8.2%, 38.2%, and 46.6%, respectively. In 2019, the Naccumulation of the other treatments decreased by 11.5%, 22.0%, 26.7%, and 33.8%, respectively. The data are represented as the mean values ± standard error and independent measurements with four replicates. Values with the same letters are not significantly different at the 5% level. Table 7 shows the accumulation of nitrogen derived from fertilizer and atmosphere of the whole soybean plants at the V3-R3 stages under different concentrations of nitrogen supplied, from which a consistent trend across the two years of experiments can be observed. During the two-year experiment, the total Naccumulation increased with increasing concentrations of nitrogen supplied. In 2018, total Naccumulation in N100 was significantly higher than that in all other treatments. The total Naccumulation in N75 and N50 did not differ from each other, but the former was significantly higher than that in N25 and N0. The total Naccumulation did not differ between N25 and N0. In 2019, the differences in total Naccumulation were significantly different among all five treatments. During the twoyear experiment, the accumulation of nitrogen derived from fertilizer increased with the concentration of nitrogen supplied and differed significantly between treatments. In 2018, the accumulation of nitrogen derived from the atmosphere in N100, N75, N50, and N25 did not differ significantly, but they were all significantly higher than that in N0. In 2019, the accumulation of nitrogen derived from the atmosphere in N100, N75, and N50 did not differ significantly, and they were all slightly higher than that in N25 and significantly higher than that in N0. From Table 7, it can be seen that increasing the concentration of nitrogen supplied significantly affected the accumulation of nitrogen derived from fertilizer but not the amount of nitrogen derived from the atmosphere. The data are represented as the mean values ± standard error and independent measurements with four replicates. Values with the same letters are not significantly different at the 5% level. Figure 1 shows the effects of the concentration of nitrogen supplied on ARA, whole-plant Ndfa%, and accumulation of nitrogen fixed by root nodules. The pattern of differences across treatments was similar between the ARA and Ndfa% at the R3 stage. The Ndfa% decreased with increasing concentrations of nitrogen supplied in N25, N50, N75, and N100, with a smaller extent of reduction at higher concentrations. The extent of reduction in the ARA did not differ significantly between the five treatments. In 2018, the pattern of differences across treatments varied between the ARA and whole-plant accumulation of nitrogen fixed by root nodules in response to increasing concentrations of nitrogen supplied. The accumulation of nitrogen fixed by root nodules at the V3-R3 stages in the N0 treatment was lower than that in all other treatments and increased with concentration of nitrogen supplied in N25, N50, N75, and N100, with a pattern of differences across treatments similar to that of the ARA. However, the extent of the reduction in whole-plant accumulation of nitrogen fixed by root nodules across treatments was smaller than that in ARA. As shown in Figure 1, changes in the ARA and whole-plant Ndfa% across treatments were similar in 2019, and their extent of reduction differed only in N50. The changes in the ARA measured by the acetylene reduction method and those in the Ndfa% measured by the 15N tracer method were similar. In 2019, changes in the ARA and whole-plant Naccumulation of nodules differed in N0 and N25 and were similar in N50, N75, and N100, which decreased with increasing concentrations of nitrogen supplied. This indicates that the whole-plant amount of nitrogen fixed by root nodules was not fully reflected by the ARA in the same way as the Ndfa%. This is because the ARA only measures the nitrogen fixation ability of root nodules at a specific time and cannot reflect the amount of nitrogen fixed by root nodules over a period of time.

Discussion
We compared two soybeans systems in Table S2, the regular dual-root systems (both sides were nodulated) and dual-root systems (a single side nodulated); these data show the advantages of using a soybean dual-root system with a single side nodulated. The concentration of nitrogen supplied can locally inhibit the nodule growth and the nitrogenase activity of leguminous crop plants [10,[19][20][21]37,38], which are systemic in nature [22,25]. In this study, by supplying nitrogen at different concentrations (experiment I) to the soybean plants for a relatively long time, the root nodules decreased significantly with increasing concentrations of nitrogen supplied to the N+ side. This indicates that, for dual-root systems, supplying nitrogen to non-nodulated strains can indirectly and significantly affect the nodulation and growth of root nodules. Long-term nitrogen supply systematically regulated the growth of root nodules, which was inconsistent with the findings of some studies [10,19,36]. This is likely because a non-nodulated strain was designated as the N+ side in this study, which removed the moderating effects of nitrogen fixation by nodules. In this study, changes in the number and weight of nodules were consistent with the observations by Tanaka et al. [22] and Daimon and Yoshioka [25]. During the long-term nitrogen treatment (experiment I), the ARA decreased significantly with increasing concentrations of nitrogen supplied. This indicates that nitrogen supply to the non-nodulated side could systematically affect the ARA of the nodulated side. The SNA of nodules changed in response to the concentration of nitrogen supplied to the N+ side, though less significantly than ARA. This suggests that long-term nitrogen treatment could systematically regulate ARA but does not significantly affect SNA. Changes in the regulation of ARA by long-term nitrogen supply mainly depend on the regulation of the nodule number and nodule dry weight, but not on SNA. Skrdleta, et al. [39] cultivated pea using perlite as the test material. With a 1-day treatment of NO3--N at a concentration of 20 mM, the nitrogenase activity of pea root nodules was rapidly inhibited. With a 3-day treatment at a concentration of 20 mM, a reduction of the dry weight of root nodules began to show. Streeter and Wong [9] used sand culture to grow soybean plants. With a 1-day treatment of NO3--N at a concentration of 15 mM, the nitrogenase activity of root nodules was significantly inhibited, and with the increase in time of nitrogen treatment, it was approximately 80% lower than that of the control. Daimon and Yoshioka [25] suggested that NO3could inhibit the formation of root nodules and nitrogen fixation of peanut plants. The short-term addition of NO3 -to one side did not affect the dry weight or nitrogen content of roots or the number or fresh weight of root nodules but significantly reduced the nitrogenase activity on the nitrogentreated side. However, the short-term addition of NO3 -possibly led to the regulation of nitrogen fixation by exogenous nitrogen, although it might not be systematic. In this study, the short-term nitrogen supply to the non-nodulated side (experiment II) did not significantly affect the dry weight or number of root nodules of the nodulated side, and both the SNA and ARA decreased significantly with increasing concentrations of nitrogen supplied to the N+ side. This indicates that the reduction in ARA under a short-term treatment was due to the decrease in SNA. Under both long-term and short-term experiments, the nitrogenase activity was systematically regulated but by different mechanisms, which need to be studied further.
Studies using 15 N and 13 N have found that nitrogen can be transported from the absorption site to other organs [22,35,[40][41][42]. In experiment I, following the 15 N supply to the non-nodulated side, 15 N was detected in the shoots and roots on both sides and in the root nodules. The 15 N abundance of various parts increased with the concentration of nitrogen treatment, which is consistent with the findings of previous studies. This suggests that after the transport of fertilizer nitrogen absorbed from the roots on one side, a part of it is redistributed to the other side's roots and nodules. The differences in the amount of nitrogen transported and distributed between nitrogen treatments were related to the concentrations of nitrogen supplied. In experiment I, the Ndfa% of various parts decreased as the concentration of nitrogen supplied increased, and the decrease was significant for the shoots and N+ side's roots. The Ndfa% of the nodules was higher than those of the nitrogen-supplied roots and shoots, suggesting that nitrogen fixed by the root nodules was mainly supplied to the root system and root nodules to which they were attached. In experiment I, the Naccumulation in the shoots and both sides of the roots increased with the concentration of nitrogen supplied, which differed significantly between treatments. The Naccumulation in the root nodules at the V3-R3 stages decreased with increasing concentrations of nitrogen supplied to the N+ side. This indicates that nitrogen absorbed by the root system and fixed by the root nodules was mainly transported to the shoots for growth, while only a small part was supplied to the growth of the root system and root nodules. The reduction in the Naccumulation in the root nodules was related to the effects of the concentration of nitrogen supplied to the N+ side on the growth of nodules.
Nitrogenase activity was first determined by the acetylene reduction method in the 1960s. This method is widely used to evaluate the nitrogen fixation ability of root nodules [43]. However, the ratio of C2H2 and N2 assimilated by root nodules needs to be calibrated [44][45][46][47][48][49]. A theoretical C2H2:N2 ratio of 3:1 can be used to obtain the amount of nitrogen fixed. In fact, various ratios ranging from 1.7 to 26.5 have been reported [45,[50][51][52], mainly due to differences in measurement conditions. Schubert [53] introduced the removed soybean root nodules separately to C2H2 and 15 N2 for 30 min to measure 15 N2 fixed and C2H2 reduced, which increased by the same percentage in different treatments. Therefore, the results of the acetylene reduction method are thought to be similar to those of the 15 N tracer method. As the acetylene reduction method is highly sensitive, relatively simple, fast, and inexpensive to operate, it should be used to provide reliable estimates of the amount of nitrogen fixed after the calibration of 15 N2 fixed. In this study, variable concentrations of nitrogen supplied to the non-nodulated roots over the long term systematically affected the ARA of root nodules, and the nitrogenase activity of root nodules decreased with increasing concentrations of nitrogen supplied. At the same time, changes in the 15 N abundance of soybean plants were used to calculate the Ndfa% by root nodules per plant, which decreased with increasing concentrations of nitrogen supplied. The responses the ARA and Ndfa% to the concentration of nitrogen supplied showed the same pattern. We think that the ARA can reflect the nitrogen fixation ability of root nodules at a certain time.
Although it was found that the Ndfa% per plant decreased with increasing nitrogen concentrations, the whole-plant amount of nitrogen fixed by root nodules did not vary much between different concentrations of nitrogen supplied. Changes in the Naccumulation in soybean plants depended on the Naccumulation derived from fertilizer and not much on the Naccumulation derived from the atmosphere by root nodules. As changes in the amount of nitrogen in root nodules and ARA responded differently to the concentration of nitrogen supplied, we think the whole-plant amount of nitrogen fixed by root nodules cannot be fully reflected by ARA in the same way as Ndfa%. This is because ARA measures the nitrogen fixation ability of root nodules at a specific time but not the amount of nitrogen fixed by root nodules over a period of time. The acetylene reduction method is suitable for comparing nitrogen fixation between treatments in the short term but not for the quantitative determination of long-term symbiotic nitrogen fixation in the field. As variations in light intensity, temperature, and humidity (weekly, daily, or seasonal) can lead to changes in the activity of the test system, it is difficult to obtain meaningful results from a series of short-term measurements.
The amount of nitrogen fixed by soybean root nodules can be expressed as the product of the Naccumulation and Ndfa% [54]. Therefore, changes in the Ndfa% and Naccumulation can affect the amount of nitrogen fixed by soybean root nodules. Nitrogen fertilizer application can inhibit the number and weight of root nodules, thereby lowering Ndfa%. However, the amount of nitrogen fixed by root nodules is compensated for by the relatively large accumulation of nitrogen [9,55,56]. Gan et al. [18] found that although the Ndfa% of soybean plants with nitrogen supply was smaller than that without nitrogen supply under hydroponic conditions, the amount of nitrogen fixed by the root nodules of soybean plants with nitrogen supply was smaller than that of soybean plants without nitrogen supply. In experiment I, the Ndfa% decreased significantly with increasing concentrations of nitrogen supplied, but changes in the amount of nitrogen fixed by the root nodules were not significant. We suggest that interactions between the concentration of fertilizer nitrogen, the duration of nitrogen supply, and the growth stage of soybean plants determine the total amount of nitrogen fixed by root nodules.

Conclusions
1. In dual-root systems of soybean plants with a nodulated side, we revealed that short-term nitrogen supply systematically regulated SNA, thereby inhibiting nitrogen fixation capacity. Under long-term nitrogen supply, the concentration of nitrogen systematically regulated the growth of root nodules, thereby inhibiting nitrogen fixation capacity. Although an increasing concentration of nitrogen supplied significantly reduced the Ndfa%, it did not significantly influence the amount of nitrogen fixed by root nodules.
2. By establishing the relationships between the ARA, Ndfa% and Naccumulation of nodules, we found that the ARA reliably reflected the Ndfa% but not the whole-plant Naccumulation of nodules.
3. This experiment studied the indirect effects of the concentration of nitrogen supplied to soybean plants on nodulation and nitrogen fixation, providing new insights for the rational and proper use of nitrogen fertilizer in soybean plants.
Supplementary Materials: The following are available online at www.mdpi.com/2073-4395/10/6/763/s1, Figure  S1: Preparation of plant materials with a dual-root system; Table S1: Ingredients of universal nutrient solution for sand culture; Table S2: Change in nodules of two soybeans root systems.