Accumulation and Distribution of Fertilizer Nitrogen and Nodule-Fixed Nitrogen in Soybeans with Dual Root Systems

: The soybean ( Glycine max L. Merr. ) is a crop with a high demand for nitrogen (N). The root nodules that form in soybeans can ﬁx atmospheric N e ﬀ ectively, yet the goal of achieving high yields cannot be met by relying solely on nodule-ﬁxed N. Nonetheless, the application of N fertilizer may inhibit nodule formation and biological N ﬁxation (BNF), but the underpinning mechanisms are still unclear. In this study, we grafted the roots of non-nodulated soybeans onto nodulated soybeans to generate plants with dual root system. The experiment included three treatments conducted under sand culture conditions with NO − 3 and NH + 4 as N sources. Treatment I: The non-nodulated roots on one side received 50 mg · L − 1 15 NO − 3 or 15 NH 4 + , and the nodulated roots on the other side were not treated. Treatment II: The non-nodulated roots received 50 mg · L − 1 15 NO − 3 or 15 NH + 4 , and the nodulated roots received 50 mg · L − 1 14 NO − 3 or 14 NH + 4 . Treatment III: Both non-nodulated and nodulated roots received 50 mg · L − 1 15 NO − 3 or 15 NH + 4 . The results showed the following: (1) Up to 81.5%–87.1% of the N absorbed by the soybean roots and ﬁxed by the root nodules was allocated to shoot growth, leaving 12.9%–18.5% for root and nodule growth. Soybeans preferentially used fertilizer N in the presence of a NO − 3 or NH + 4 supply. After the absorbed fertilizer N and nodule-ﬁxed N was transported to the shoots, a portion of it was redistributed to the roots and nodules. The N required for root growth was primarily derived from the NO − 3 or NH + 4 assimilated by the roots and the N ﬁxed by the nodules, with a small portion translocated from the shoots. The N required for nodule growth was primarily contributed by nodule-ﬁxed N with a small portion translocated from the shoots, whereas the NO − 3 or NH + 4 that was assimilated by the roots was not directly supplied to the nodules. the the roots 9.4%–16.6% of the N accumulation in Nodule n of soybeans. Through the study of this experiment, the absorption, distribution and redistribution characteristics of fertilizer N and root nodule N ﬁxation in soybean can be clariﬁed, providing a theoretical reference for analyzing the mechanisms of the interaction between fertilizer N and nodule-ﬁxed N.


Preparation of Plant Materials with a Dual Root System
The soybean plants with dual root system was prepared based on the method in Xia et al. [25]. The treatments were conducted using plastic pots with a diameter of 0.3 m and a height of 0.3 m. Each pot was divided into two equal, independent spaces by vertically inserting a custom-made polycarbonate plate that was fitted for the inner shape of the pot and sealed with glue in the middle of the pot. The top of the partition plate was 2 cm below the rim of the pot. For each partitioned space, a drainage hole with a 1 cm diameter was drilled in the bottom of each pot. The hole was capped with a piece of gauze to prevent clogging by river sand. Each pot was filled with 20 kg of washed sand for cultivating the soybean plants with dual root.
Seeds of nodulated soybeans (Glycine max L. cv. Kenfeng 16) and non-nodulated soybeans (Glycine max L. cv. WDD01795, L8-4858, provided by the Crop Research Institute, Chinese Academy of Agricultural Sciences) were drilled into a fine sand medium at a depth of 2 cm and incubated in a growth chamber at 30 • C for 3 days. When the distance between the growing point of the cotyledon and the tip of the root was 7 to 10 cm, the roots of the soybean seedlings were rinsed with water and then used for grafting. Two seedlings of nodulated and non-nodulated soybeans were chosen and an incision of 0.5-1.0 cm (without cutting off) was made with a sterilized blade, which extended upward or downward slightly above the middle point of the hypocotyl. The non-nodulated seedling was cut from the cotyledon toward the root ( Figure 1A), whereas the nodulated seedling was cut from the root toward the cotyledon ( Figure 1B). The two seedlings were cross-inserted into their cuts ( Figure 1C) and clipped with a grafting clip. The root system of the two seedlings were planted separately into the fine sand medium on both sides of the partition plate in the pot, with the grafting site exactly on the top of the partition plate. The grafted seedlings were allowed to grow inside a weather-tight enclosure for a week. The grafting clip was then removed and the upper part of each non-nodulated seedling was cut from the grafting site, leaving its combined site and lower parts. This procedure generated seedlings with dual root system (nodulated and non-nodulated) and the shoots of a nodulated cultivar. The plants were grown under farmland conditions and treated experimentally. ( Figure 1D) shows the roots of a soybean plant with a dual root system at the time of sampling, with non-nodulated roots (Root non ) on the left and nodulated roots (Root n ) on the right side.
on the top of the partition plate. The grafted seedlings were allowed to grow inside a weather-tight enclosure for a week. The grafting clip was then removed and the upper part of each non-nodulated seedling was cut from the grafting site, leaving its combined site and lower parts. This procedure generated seedlings with dual root system (nodulated and non-nodulated) and the shoots of a nodulated cultivar. The plants were grown under farmland conditions and treated experimentally. ( Figure 1D) shows the roots of a soybean plant with a dual root system at the time of sampling, with non-nodulated roots (Rootnon) on the left and nodulated roots (Rootn) on the right side.  Before the full expansion of the opposite true leaves, the soybean seedlings were irrigated once a day with 250 mL of distilled water on each side of the root system [25]. Following the full expansion of the opposite true leaves, the seedlings were irrigated once a day with 250 mL of the corresponding nutrient solution on each side of the root system until the R 1 stage. From the R 1 stage on, the seedlings were irrigated twice a day, once in the morning and once in the evening, with 250 mL of the corresponding nutrient solution for each side of the root system until the end of the experiment. When the opposite true leaves were fully expanded, all the roots were inoculated with rhizobia as follows: Soybean root nodules harvested from the field during the previous year and stored in a freezer were washed, ground, and then added to the nutrient solutions at 5 g·L −1 . This method was used to inoculate soybean with soil rhizobia continuously for 5 days. Previous experiments showed that this method was feasible for soybean inoculation, and there would be spontaneous nodulation of rhizobia in the soil community [25,32]. The main N-fixing rhizobia in soil belonged to Bradyrhizobium, Bradyrhizobium japonicum, and B. liaoningense were the dominant bacteria [33].

Sampling and Parameter Analysis
Samples were taken at the R 1 and R 5 stages [34]. The plants were separated into different parts, deactivated at 105 • C for 30 min, and then dried at 85 • C. Dry samples were used to analyze the 15 N abundance, dry weight, and N content of each part.
Plant N content analysis: The plant N content was determined using a B324 automatic Kjeldahl analyzer after digestion with concentrated H 2 SO 4 (K 2 SO 4 and CuSO 4 as catalysts). 15 N abundance analysis: After plant N content analysis by the Kjeldahl method, the titrated samples were concentrated and allowed to react with lithium hypobromite to produce N 2 under freezing-vacuum conditions. The 15 N abundance was determined using a mass spectrometer (Thermo-Fisher Delta V Advantage IRMS) equipped with a dual-inlet system.

Data Calculations
The percent of 15 N-labeled N derived from fertilizer ( 15 Ndff%) in plants was calculated as: where f nature is the natural 15 N abundance, f fertilizer is the 15 N abundance of the fertilizer, and f treatment is the 15 N abundance of the treatment. The percent of N derived from atmosphere (Ndfa%) in plants was calculated as: The percent of 14 Nitrogen derived from fertilizer ( 14 Ndff%) plus the percent of N derived from atmosphere (Ndfa%) was calculated as: 14 Ndff% + Ndfa% = 1-15 Ndff% Based on the 15 N abundance of each organ, the ratio of N sources from 15 N, 14 N, and N-fixing root nodules in each organ can be calculated; if these values are then multiplied by the N accumulation, the N accumulation from 15 N, 14 N, and root nodules in each organ can be obtained.

Statistical Analyses
Descriptive statistics, one-way ANOVA, and correlation tests were performed on the data by IBM SPSS Software version 17.0. The results were mean ± standard deviation (SD) of three replicates. Duncan test was used for comparison between treatments (α = 0.05). All data were tested for normality and homogeneity of variance.  Table 2 shows the 15 N abundance in the various parts of soybean plants with dual root system for Treatments I, II, and III. The 15 N abundance in the vegetative organs of soybean plants differed significantly among the three treatments, indicating that the different treatments resulted in considerable differences in the 15 N abundance in various organs.

Ratio of Fertilizer N and Root Nodule N Fixation in Soybean Plants
In the dual root system, Root non represents non-nodulated roots, Root n represents nodulated roots, and Nodule n represents nodules on the same side of nodulated roots. The values are the means ± standard error (n = 3). Different lowercase letters indicate significant differences between treatments at the 5% level. Treatments I, II, and III are compared horizontally.
In Treatment I, under NO − 3 and NH + 4 N sources, the 15 N abundance in Root non at the R 1 and R 5 stages was lower than that of the N fertilizer (3.63%). However, the 15 N abundance in Root n and Nodule n at the R 1 and R 5 stages remained higher than the natural 15 N abundance (0.365%). The 15 N abundance in the soybean Shoot at the R 1 stage was higher than the natural 15 N abundance (0.365%) and lower than the 15 N abundance of fertilizer N (3.63%). In Treatments II and III, under NO − 3 and NH + 4 N sources, the 15 N abundance at the R 1 and R 5 stages in Root non was lower than that of fertilizer N (3.63%) and higher than the natural 15 N abundance (0.365%). There were no significant differences in the 15 N abundance between the NO − 3 and NH + 4 N sources for Treatments I, II, and III. However, during three treatments, the 15 N abundance in all the soybean organs under different treatments at R 5 was lower than it was at the R 1 stage. Table 3 shows that for Treatment I under NO − 3 and NH + 4 sources at the R 1 and R 5 stages, most of the N in Root non came from the fertilizer N that was self-absorbed by the roots. However, these results illustrate that at the R 1 and R 5 stages, most of the N in Root n and Nodule n came from the nodule-fixed N in this root system. At the R 1 and R 5 stages, nodule-fixed N contributed a larger proportion to the supply for the Shoot. In the dual root system, Root non represents non-nodulated roots, Root n represents nodulated roots, and Nodule n represents nodules on the same side as the nodulated roots. 15 Ndff% represents the proportion of 15 N-labeled fertilizer N, Ndfa% represents the proportion of nodule-fixed N, and 14 Ndff%+Ndfa% represents the proportion of nodule-fixed N plus unlabeled fertilizer N. The values are the means ± standard error (n = 3). Different lowercase letters indicate significant differences between treatments at the 5% level. The two N sources are compared horizontally.

Ratio of N Absorbed from Different Sources in Dual Root Soybeans
In Treatment II under NO − 3 and NH + 4 N sources, the results indicate that when N was added to both sides of the roots, the N in Root non primarily came from fertilizer N that was self-absorbed by the roots with small proportions from absorbed fertilizer N and nodule-fixed Root n N (translocated from the Shoot). A comparison with Treatment I revealed that there was little difference in the nutrient proportions of different N sources in Root non as contributed by the two roots of the dual root system with an addition to both sides versus one side. Furthermore, the proportions of different N sources in various parts of the soybean plants in Treatments I and II were compared. Similarly, the nutrient proportions contributed by the two roots of the dual root system did not change markedly in the Root n , Nodule n , or Shoot. These results indicate that the soybean plants contributed similarly to the plant N with or without N addition, showing the integrity of fertilizer N absorption and nodule N fixation.
No significant differences were detected in the proportions of various N sources between the NO − 3 and NH + 4 N sources among Treatments I, II, and III, indicating that the nutritional effect of NO − 3 vs. NH + 4 addition was not markedly different in soybean plants. However, compared with the R 1 stage, the R 5 stage was associated with a higher proportion of nodule-fixed N in various organs under different conditions for Treatments I, II, and III, indicating a larger contribution of nodule-fixed N to soybean plants at the R 5 stage than at the R 1 stage. In both Treatments II and III, the same N concentration was added to both sides of the root system, and the only difference was related to the 15 N abundance. To distinguish among the three N sources in Treatment II, we calculated the proportion of absorbed 14 N-labeled fertilizer N in various parts of the soybean plants by summing up the proportion of absorbed 14 N-labeled fertilizer N and the proportion of N from nodule fixation in each part of the soybeans from Treatment II and subtracting the proportion of N from nodule fixation in the same parts of the soybean plants from Treatment III (Table 4). In the dual root system, Root non represents non-nodulated roots, Root n represents nodulated roots, and Nodule n represents nodules on the same side as the nodulated roots. 15 Ndff% represents the proportion of 15 N-labeled fertilizer N, 14 Ndff% represents the proportion of unlabeled fertilizer N, and Ndfa% represents the proportion of nodule-fixed N. The values are the means ± standard error (n = 3). Different lowercase letters indicate significant differences between treatments at the 5% level. Three N sources are compared horizontally.
In Treatment II, there were three N sources for soybean plants, i.e., absorbed fertilizer N from Root non , absorbed fertilizer N from Root n , and nodule-fixed N from Root n . Under the NO − 3 and NH + 4 N sources in the R 1 stage, Root non preferentially absorbed the self-assimilated N. At the R 1 stage, 51.3% and 60.7%, respectively, were contributed by absorbed Root n fertilizer N, and 36.3% and 28.6%, respectively, were contributed by nodule-fixed Root n N. At the R 5 stage, the corresponding proportions were 33.8% and 39.4%, 54.0% and 50.4% for absorbed fertilizer N from Root non , absorbed fertilizer N from Root n , and nodule-fixed N from Root n , respectively. These results show that when N was added to both sides of the root system, Root n also preferentially absorbed the N that was assimilated by this root system; the proportion of nodule-fixed N in Root n increased with the increasing N fixation capacity of the nodules. For the N in Nodule n at the R 1 and R 5 stages, nodule-fixed N was preferentially absorbed by Nodule n . Moreover, the proportion of N in the root nodules contributed by the absorbed fertilizer N of the two roots of the dual root system was significantly different; that is, the N from Root non was less than that from Root n . These results show that when N was added to both sides of the root system, the Shoot primarily absorbed fertilizer N at the R 1 stage, whereas nodule-fixed N was primarily absorbed during the R 5 stage. The supplies of fertilizer N from the two roots of the dual root system to the Shoot were almost identical, suggesting the same supply of absorbed fertilizer N to Shoot by each side in the dual root system. A comparison of Treatments I and II revealed no major change in the proportions of N supplied to the Shoot by each root of the dual root system, irrespective of whether N was added to one or both sides. However, after the addition of N to both sides of the root system, the proportion of nodule-fixed N decreased, whereas the proportions of absorbed NO − 3 and NH + 4 supplied to the Shoot by Root non and Root n were similar. Table 5 shows the N accumulation of organs in the dual root system of single-nodule soybeans. In Treatments I, II, and III under the NO − 3 N source for soybean plants, the N accumulation of Root non Agronomy 2020, 10, 397 9 of 17 at the R 1 stage was significantly higher in Treatment III than in Treatments I and II. During the R 5 stage, there was no significant difference among Treatments I, II, or III. At the R 1 stage, the N accumulation of Root n was significantly lower in Treatment I than in Treatments II and III. During the R 5 stage, there was no significant difference among Treatments I, II, and III. The N accumulation of Nodule n at the R 1 and R 5 stages was significantly higher in Treatment I than in Treatment II or III. In Treatment I, the N accumulation in the Shoot at R 1 was significantly lower than in Treatment II or III, and that at R 5 it was significantly lower than in Treatment II. The total N accumulation of Treatment I at the R 1 stage was significantly lower than in Treatment II or III, and the difference at the R 5 stage was not significant. In Treatments I, II, and III under the NH + 4 N source for soybean plants, the N accumulation of Root non in Treatment I at the R 1 and R 5 stages was significantly lower than in Treatments II and III. At the R 1 stage, the N accumulation of Root n in Treatment I was significantly lower than in Treatments II and III, whereas the difference was not significant at the R 5 stage. The N accumulation of Nodule n in Treatment I at the R 1 and R 5 stages was significantly higher than in Treatments II and III. At the R 1 stage, the N accumulation in the Shoot of Treatment I was significantly lower than in Treatments II and III. The R 1 and R 5 stages showed the opposite pattern. In Treatment I, the N accumulation of the Shoot in the R 1 stage was significantly lower than that in Treatments II and III, and the difference at the R 5 stage was not significant. The values are the means ± standard error (n = 3). Different lowercase letters indicate significant differences between treatments at the 5% level. Treatments I, II, and III are compared horizontally.

N Accumulation in Dual Root Soybeans
A comparison of Treatments I, II, and III showed that the N accumulation of Root non and Root n at the R 1 stage in Treatment I was lower than in Treatments II and III, whereas the N accumulation at the R 5 stage was not significantly different among the treatments. This finding indicates that local N application in the early stage of soybean growth affects the accumulation of N in the root, but local N application in the late stage of soybean growth has a weak effect on the accumulation of N in the root, possibly because the ratio of N fixation in the root nodules was increasing during the late stage of soybean growth. N accumulation of Nodule n in Treatment I was significantly higher than in Treatments II and III. This result indicates that the local application of N can inhibit the growth of root nodules on the N-treated side and promotes the growth of root nodules on the N-treated side, thus improving the N-fixing ability of root nodules and leading to an increase in N accumulation. There was no significant difference in N accumulation in each part of the treatment with a NO − 3 vs. NH + 4 N source, indicating that under an N concentration of 50 mg·L −1 , there was no difference in the nutritional effect of NO − 3 vs. NH + 4 on each part.

Accumulation of Fertilizer N and N-Fixing Root Nodules in Dual Root Soybeans
As seen from Table 6, when N was applied on one side, the majority of Root non N came from the fertilizer N absorbed by Root non , whereas a small part came from the root nodules of Root n. When N was not applied to Root n , most of the N came from root nodule N fixation, and a small part came from the fertilizer N absorbed by Root non . Under unilateral N application to soybean roots, most of the N in Nodule n came from the N-fixing root nodules of Root n, and a small part came from fertilizer N absorbed from Root non , which also indicates that not all N needed for root nodule growth came from internal N fixation and some N needed to be absorbed from the roots. In comparing the two N sources in the R 1 and R 5 stages, N fixation from the root nodules was significantly higher than N from fertilizer N absorbed by Root non . The total N accumulation from N-fixing root nodules was much higher than that from fertilizer. In the dual root system, Root non represents non-nodulated roots, Root n represents nodulated roots, and Nodule n represents nodules on the same side as the nodulated roots. 15  Under the condition of bilateral N application, most of the N in Root non came from the fertilizer N absorbed by Root non , and a small part came from the N absorbed and fixed by Root n . However, most of the N in Root n came from the N absorbed and fixed by Root n , whereas a small part came from the fertilizer N absorbed by Root non and transferred to Root n . Most of the N in Nodule n came from N absorbed and fixed by Root n , and a small part came from fertilizer N absorbed by Root non and transferred to Root n nodules. At the same time, it can be observed that not all the N required for root nodule growth comes from self-fixing N, and it is also necessary to absorb N via the roots. In comparing the three N sources in the Shoot at the R 1 and R 5 stages, there was no significant difference between N accumulation in Root non and Root n , but N accumulation was significantly lower than in the N-fixing root nodules of Root n . This suggests that the supply of absorbed fertilizer N to Shoot was the same from the two parts of the dual root systems. As a whole, the total accumulation of N absorbed and fixed by Root n was much higher than the fertilizer N absorbed by Root non , mainly because Root non had no root nodules for N fixation.

N Translocation from Shoot to Roots and Nodules of Soybeans
The fertilizer N absorbed by the two roots of the dual root system and the N fixed by the root nodules were transported to the Shoot, and after assimilation, they were transported to the roots and nodules in certain forms. Due to the different 15 N abundance in various parts of the soybean plants in the treatments (Table 2), we regarded the Root n and the Shoot as one system, in which the Shoot served as a source of 15 N for the Root n . From the R 1 to the R 5 stage, the amount of 15 N increasing in Root n can be obtained from the 15 N accumulation of the R 5 stage minus the 15 N accumulation of the R 1 stage, and this should be equal to the amount of 15 N translocated from the Shoot plus the amount of naturally occurring 15 N that was self-absorbed by the roots of Root n (including the supply by nodules) from the R 1 to the R 5 stage. If we let the amount of N translocated from the Shoot to Root n be x, then the 15 N abundance of the N translocated downwards from the Shoot can be calculated using the mean 15 N abundance in the Shoot at the R 1 and R 5 stages, where x × (f shootR1 +f shootR5 )/2 represents the amount of 15 N translocated from the Shoot to Root n during R 1 -R 5 , N R5 -N R1 represents the N accumulation in Root n during R 1 -R 5 , N R1 -N R5 -x represents the N accumulation from Root n absorbed during R 1 -R 5 (including the N supplied by nodules), (N R5 -N R1 -x) × f nature represents the amount of 15 N in Root n that is self-absorbed by the roots and supplied by the nodules during R 1 -R 5 , N R5 × f R5 represents the total 15 N in Root n at the R 5 stage, and N R1 × f R1 represents the total 15 N in Root n at the R 1 stage. This method can also be used to calculate the amount of N translocated from the Shoot to Nodule n . For Treatments I and II, the calculation is as follows: x where x is the amount of N translocated from the Shoot to Root n or Nodule n , N R1 is the N accumulation in Root n or Nodule n at the R 1 stage, N R5 is the N accumulation in Root n or Nodule n at the R 5 stage, f nature is the natural 15 N abundance, f R1 is the 15 N abundance in Root n or Nodule n at the R 1 stage, f R5 is the 15 N abundance in Root n or Nodule n at the R 5 stage, f shootR1 is the 15 N abundance in the Shoot at the R 1 stage, and f shootR5 is the 15 N abundance in the Shoot at the R 5 stage. Similarly, when calculating the amount of N translocated from the Shoot to Root non , we regarded the Root non and the Shoot as one system. Because 15 N-labeled fertilizer N was added to Root non , the calculation is as follows: where x is the amount of N translocated from the Shoot to Root non , N R1 is the N accumulation in Root non at the R 1 stage, N R5 is the N accumulation in Root non at the R 5 stage, f fertilizer is the 15 N abundance in the fertilizer, f R1 is the 15 N abundance in Root non at the R 1 stage, f R5 is the 15 N abundance in Root non at the R 5 stage, f shootR1 is the 15 N abundance in the Shoot at the R 1 stage, and f shootR5 is the 15 N abundance in the Shoot at the R 5 stage.
The amount of N translocated from the Shoot to Root non , Root n , and Nodule n can be calculated using Equations (4) and (5), as shown in Table 7. In the dual root system, Root n represents nodulated roots, Root non represents non-nodulated roots, and Nodule n represents nodules on the same side as the nodulated roots. ANT (mg) represents the accumulation of N translocated from the Shoot to roots and nodules during R 1 -R 5 , AIN (mg) represents the accumulation of increased N in various parts during R 1 -R 5 , and ANT/AIN (%) represents the proportion of total N from Shoot (NTFS) under AIN in various plant parts. Table 7 shows that for Treatment I, in the NO − 3 and NH + 4 N sources between the R 1 and R 5 stages, the accumulation of increased N in Root non was 28.8 and 16.6 mg, respectively, and the accumulation of N translocated from the Shoot to Root non was 16.1 and 9.5 mg, respectively. The N translocated from the Shoot to Root non accounted for 55.9% and 57.0%, respectively, of the increased accumulation of N in Root non . In Root n , the increased accumulation of N was 46.8 and 41.9 mg, respectively, and the accumulation of N translocated from the Shoot was 13.9 and 14.6 mg, accounting for 29.6% and 34.9%, respectively, of the increased accumulation of N in Root n . In Nodule n , the increased accumulation of N was 73.4 and 68.9 mg, respectively, and the accumulation of N translocated from the Shoot was 6.9 and 8.3 mg, contributing to 9.4% and 12.2%, respectively, of the increased accumulation of N in Nodule n . In Treatment II with NO − 3 and NH + 4 N sources between the R 1 and R 5 stages, the increased accumulation of N in Root non was 18.4 and 26.6 mg, respectively, and the accumulation of N translocated from the Shoot to Root non was 13.0 and 13.8 mg, making up 70.5% and 51.8%, respectively, of the increased accumulation of N in Root non . In Root n , the increased accumulation of N was 45.7 and 43.6 mg, respectively, and the accumulation of N translocated from the Shoot was 22.9 and 17.6 mg, accounting for 50.0% and 40.3%, respectively, of the increased accumulation of N in Root n . In Nodule n , the increased accumulation of N was 53.7 and 47.5 mg, respectively, and the accumulation of N translocated from the Shoot was 8.9 and 7.3 mg, contributing to only 16.6% and 15.3%, respectively, of the increased accumulation of N in Nodule n .

Sources of N in the Roots and Nodules of Soybeans
After adding 15 NO − 3 to soybean (Glycine max L. Merr.) seedlings, Crafts-Brandner and Harper [35] detected 15 N in the reduced N from xylem sap and found that the 15 N abundance tended to increase with time, leading to a conclusion that soybean roots can reduce 15 NO − 3 . Sprent and Thomas [36] indicated that Phaseolus vulgaris and Glycine max directly absorb and transport fertilizer NO − 3 into the shoots for assimilation, whereas Pisum sativum and Vicia faba transport NO − 3 into the shoots after assimilation in the roots. Following the application of different concentrations of NO − 3 to six leguminous crops, Andrews [37] found that the assimilation of absorbed NO − 3 occurred primarily in the roots of Cajanus cajan, Lupinus albus, Trifolium repens, and Pisum sativum, whereas Glycine max, and Phaseolus vulgaris primarily assimilated the absorbed NO − 3 in the shoots. Kiyomiya et al. [38] treated the roots of rice with 13 NH + 4 and then observed the 13 N at the bottoms of the shoots within 2 min. However, the rapid upward transport of 13 N was inhibited after the addition of glutamine synthetase inhibitor, indicating that most of the NH + 4 was assimilated in the roots. In the present study, under 15 NO − 3 and