Assimilation of Nitrate into Asparagine for Transport in Soybeans

: 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).


Introduction
Nitrate is one of the main forms of nitrogen (N) taken up by plants.A portion of nitrate absorbed by the roots is translocated to the shoots, while another portion is reduced to NO 2 -by nitrate reductase (NR) in the roots.NO 2 − is reduced to NH 3 under the action of nitrite reductase, and the formed NH 3 enters the N assimilation pathway (glutamine synthetase (GS)/glutamate synthetase (GOGAT)) [1][2][3][4][5].In studies of Avena saiiva L., Hordeum vulgare L., Secale cereale L., Triticum aestivum L. and Triticosecale Wittm, when nitrate was exogenously supplied at 0.1-1.0mol•m −3 , it was mainly assimilated in the roots; when the supplied concentration was increased to 20 mol•m −3 , the nitrate was transported to the leaves for assimilation [6].However, in Triticum aestivum L., the supply of nitrate to the roots was found to be associated with the highest levels of asparagine (Asn) [7].In maize, most of the nitrate supplied was assimilated in the leaves, and a small amount was assimilated into glutamine (Gln) in the roots and transported upwards [8].In cucumber, a significant decrease in root Gln content was observed after being supplied with 140 mM nitrate [9], while the Gln accumulation in the cowpea (Vigna unguiculata L.) roots exhibited an increase as the concentration of supplied nitrate increased [10].The application of nitrate to lentil (Lens culinaris Medic.), faba bean (Vicia faba L.), pea (Pisum sativum L.) and Canavalia ensiformes significantly increased the Asn content in the roots [11,12].Ohyama and Kumazawa [13] found that the level of 15 N in Asn present in the roots was considerably greater compared to the other amino acids after the soybean roots were exposed to 15 NO 3 − for a duration of eight hours.According to other researchers, it was discovered that when nitrate was supplied to soybean plants, there was an increase in the amount of Asn in the roots, while the concentration of Gln remained relatively stable [14,15].Ueda et al. [16], using non-nodulated soybeans, also reached similar conclusions.
The xylem and phloem are the main channels for N transport in plants [5,17].In Medicago truncatula, the phloem transports N to underground organs in the form of Asn [18].Important alterations in root N metabolism can be indicated by the transfer of N-containing compounds from the xylem to the shoot in bean (Phaseolus vulgaris L.), cowpea, pea, and lupin (Lupinus albus L.) [19].It was also found that 14 C-labeled Asn was directly transported from the xylem to the phloem of the white lupin when the shoot was cut into a nutrient solution containing 14 C-labeled Asn [20].In soybean roots, nitrate can induce the expression of the asparagine synthase gene SAS and increase the ratio of Asn/Asp transported to the shoots through the xylem [21].The addition of 5 mM nitrate to soybeans led to a notable increase in Asn levels and a decline in Gln levels in the xylem, although these variances were not statistically significant when compared to the control [22].However, Ohyama et al. [23] discovered that providing soybeans with nitrate led to a notable increase in the concentrations of both Asn and Gln in the xylem.
The above research results are the conclusions concerning the direct supply of nitrate to plant roots.Li et al. [24] used a dual-root soybean system to supply nitrate to one side of the root system; the nitrate could be transported to the other side of the root, which systemically regulated the N distribution in the soybean plants.Nevertheless, the research findings on the indirect provision of nitrate to the soybean roots and the process of nitrate assimilation and transportation within the roots remain uncertain.In this study, dual-root soybeans were used, and the nitrate concentration was varied on one side of the roots.The activities of NR and GS and the concentrations of Asn and Gln in the soybean roots were determined in order to further clarify that Asn is the main transport substance for nitrate assimilation by soybean roots.

Materials and Methods
Experiments were conducted at the experimental base of the Northeast Agricultural University of China using sand culture conditions.The soybean variety tested was DongDa1 (G.max L. cv.DongDa1).The seeds were obtained from the College of Agriculture, Northeast Agricultural University, Harbin, China.Dual-root soybean systems and N-free nutrient solutions were prepared according to Li et al. [24].There were two dual-root soybean seedlings per pot.KNO 3 was used as the N source.To ensure that there was an equal K + concentration between the treatments, KNO 3 was added to one side (N+), while KNO 3 was substituted with K 2 SO 4 on the other side (N−) and in the control treatment.At the cotyledon (VC) stage, the frozen soybean nodules grown the previous year were ground and added to the nutrient solution, and the rhizobia were inoculated at about 5 g/L for 5 days.The plants received daily applications of distilled water until reaching the VC stage.From the VC stage to the 4th leaf stage (V4 stage), they were treated with the nutrient solution once a day.From the V4 stage to the end of the experiment, the plants were irrigated with the nutrient solution in the morning and evening.Each side was supplied with 250 mL of distilled water or nutrient solution, staged according to the description of Fehr et al. [25].

Experimental Treatments
Between the VC and V4 periods, the soybean systems with dual-root grafting were supplied with a nutrient solution containing KNO 3 with a N concentration of 25 mg•L −1 on both sides.During the V4 stage, both sides received a nutrient solution devoid of N for a duration of 10 days.The experiment on the short-term change in nitrate concentration in the nitrate supply-withdrawal-resupply was conducted 42 days after grafting.The experiment consisted of three phases, with each treatment phase lasting for 3 days.Four treatments were designed as follows, N LLL , N HHH , N HLL , and N HLH .During all three experimental phases, the N LLL treatment (control) involved the irrigation of a nutrient solution without N on either of the sides.During the three experimental phases, the N+ lateral roots were supplied with a KNO 3 nutrient solution containing 100 mg•L −1 of N in the N HHH treatment (high N), whereas the N− lateral roots were irrigated with a nutrient solution lacking N. At the end of experimental phases I, II, and III, samples were taken from the N LLL and N HHH treatments.During phase I of the N HLL treatment, the N+ lateral roots were irrigated with a KNO 3 nutrient solution containing 100 mg•L −1 of N. In phases II and III, the N+ lateral roots were irrigated with a N-free nutrient solution.Throughout all three experimental phases, the N− lateral roots were consistently irrigated with an N-free nutrient solution.The N HLL treatment samples were collected at the end of experimental phases II and III.In the N HLH treatment, a nutrient solution containing KNO 3 with a N concentration of 100 mg•L −1 was applied in phases I and III.In phase II, a N-free nutrient solution was applied specifically to the N+ lateral roots.The N− side received the N-free nutrient solution during all three experimental phases.At the end of experimental phase III, the samples were collected from the N HLH treatment.Each treatment was replicated three times.

Sampling Methods
After each phase, the soybeans were cut off along the grafting location, and the N+ and N− lateral roots and nodules were collected.In addition, the basal parts of the roots on both sides were cut at approximately 2 cm from the grafting site and divided into peeled skin (phloem + ) and the woody part (xylem + ).Phloem + included the epidermis, cortex, and the phloem, with the phloem serving as the primary transportation pathway; xylem + included the xylem and pith, with the xylem as the primary transport route.The phloem + and xylem + sampling method was described in Li et al. [25].The samples were rinsed with pure water, and the excess liquid was drained using filter paper.After freezing in liquid N 2 , the samples were stored in a refrigerator at −80 • C to determine the concentrations of Asn and Gln in each organ.In addition, the root tips on both sides of each treatment were collected to immediately determine the NR activity and GS activities.

Analytical Methods
The root NR activity was determined using an in vivo assay method, following the approach described by Streeter and Bosler [26].The determination of root GS activity was based on the method described by McCormark et al. [27].The concentrations of Gln and Asn were determined using the method described by Minamisawa et al. [28].The specimens were pulverized using liquid N 2 , treated with 80% ethanol, and then subjected to centrifugation at a speed of 8000× g for a duration of 10 min.The liquid above the sediment was examined using high-performance liquid chromatography (Agilent1100 USA), with the mobile phase comprising 0.1 mol•L −1 sodium acetate buffer solution and acetonitrile at a ratio of 93:7.The mobile phase also included 80% acetonitrile aqueous solution.The analysis was conducted at a column temperature of 40 • C, with a flow rate of 1.0 mL/min.The UV detector used had a wavelength of 254 nm.

Data Analysis
IBM SPSS 21.0 (IBM Corp., Armonk, NY, USA) was utilized to conduct all statistical analyses.Prior to conducting one-way analysis of variance, normality testing was performed on all of the data, followed by the application of Duncan's multiple range test at a significance level of p < 0.05.

Nitrate Reductase Activity in Soybean Roots
The impact of temporary alterations in the nitrate levels on NR function in the roots of the dual-root soybeans is illustrated in Table 1.It can be seen that the NR activity of the roots on both sides did not change significantly as a consequence of the addition and withdrawal of nitrate on the N+ side during the three experimental phases.The values between the treatments were similar and did not reach the significant difference level.

Changes in Glutamine Synthetase Activity in Soybean Roots
Table 2 shows the root GS activity.The GS activity of the N+ and N− lateral roots treated with N HHH was notably higher than that of the N LLL treatment in phases I, II, and III.The findings indicated that providing nitrate to the N+ side at a N concentration of 100 mg•L −1 resulted in a notable enhancement in GS activity in the roots on both sides.Despite not being directly exposed to nitrate, the GS activity of the N− lateral roots also rose when N+ lateral nitrate was added.The N+ lateral roots in the three treatments showed the following GS activities at the end of phase II: N HHH > N HLL > N LLL .The N HHH and N HLL treatment differences were not significant; however, they were significantly different from the N LLL treatment.During phase III, the GS activity of the N+ lateral roots showed the same trend in changes.The findings showed that the GS function of the N+ side rose with the supplementation of nitrate but did not exhibit a notable decline following nitrate removal.The order of N− lateral root GS activities in the three treatments at the end of phase II was as follows: N HHH > N LLL > N HLL .The N HHH treatment exhibited a notable disparity compared to the N LLL and N HLL treatments, while there was no significant distinction between the N HLL and N LLL treatments.During phase III, all three treatments showed similar patterns of change, with no notable distinction between the N HHH and N LLL treatments.The results showed that the N− lateral roots' GS activity was enhanced by the presence of nitrate on the N+ side and declined when nitrate was removed.During phases II and III, the roots' GS activity on the N+ side exhibited a considerable increase compared to the N− side in the N HLL treatment.The findings suggested that supplying nitrate to the N+ side and then subsequently removing it resulted in a reduction in the root GS activity on both sides.Notably, the roots on the N− side, which were not directly exposed to nitrate, exhibited a more pronounced decline in their GS activity.
After phase III, the N+ lateral roots treated with N HLH exhibited a considerably greater GS activity compared to the other three treatments.In particular, the GS activity was approximately 39% higher than that of the N HHH treatment.This suggests that the GS activity of the N+ lateral roots significantly increased following the withdrawal and subsequent resupply of nitrate.Moreover, this method of N supply appeared to stimulate a greater change in GS activity within the roots compared to the continuous supply of nitrate.The GS function of the N− lateral roots in the N HLH treatment was significantly reduced compared to that in the N HHH treatment, but it was higher than those in the N HLL and N LLL treatments, although the distinction was not statistically significant.The GS function of the N+ side during the N HLH treatment exhibited a considerably greater level compared to that of the N− side.The findings indicated that the GS activity of the N-lateral roots increased with the N+ lateral nitrate resupply, but this increase was not significant.

Glutamine and Asparagine Concentration in the Roots
Table 3 shows the root Gln concentration.During the three experimental phases, there were no notable disparities observed in the concentrations of Gln in the N+ lateral roots when compared to the N HHH and N LLL treatments.After phase I, there was no notable disparity in the Gln levels in the N− lateral roots between the N HHH and N LLL treatments.After phases II and III, the level of Gln in the N− lateral roots of the N HHH treatment was notably reduced compared to the N LLL treatment.The above results showed that nitrate supply to the N+ lateral roots slightly increased the Gln concentration in this lateral root, but significantly decreased the concentration in the N− lateral roots when the nitrate supply time was increased on the N+ side.After phase II, the concentration of Gln in the N+ lateral roots under the N HLL treatment exhibited a notable increase compared to the N LLL and N HHH treatments; while in phase III, the concentration of Gln in the N+ lateral roots did not show any significant variation among the three treatments.After phase II, the concentration of Gln in the N− lateral roots was the highest under the N HLL treatment, showing no significant difference compared to the N LLL treatment; however, it was notably higher than that under the N HHH treatment.During phase III, the N HLL treatment was not significantly different from that of the N HHH treatment, but it was significantly lower than that of the N LLL treatment.The above results showed that the change in Gln concentration in the roots was delayed by the modification in nitrate provision on the N+ side.After 3 days of nitrate withdrawal on the N+ side, the Gln concentration in both sides of the roots showed an increase.However, with an increase in the duration of nitrate withdrawal to 6 days, the Gln concentration in the roots on both sides decreased, with a more pronounced decrease observed on the N− side.
After phase III, the Gln concentration in the N+ lateral roots under the N HLH treatment was not significantly different from those of the other three treatments.The concentration of Gln in the N− lateral roots under the N HLH treatment was similar to that of the N LLL treatment and significantly higher than those of the N HHH and N HLL treatments.The above results failed to show whether the Gln concentration in the roots on both sides increased or decreased significantly with the added or withdrawn nitrate on the N+ side.
Table 4 shows the root Asn concentrations.During the three experimental phases, the Asn concentration in the N+ lateral roots under the N HHH treatment was significantly higher compared to that of the N LLL treatment.During phases I and II, there was no significant difference in the Asn concentration in the N− lateral roots between the N HHH and N LLL treatments.During phase III, the Asn concentration in the N− lateral roots under the N HHH treatment was significantly higher comparted to the N LLL treatment.In the three experimental phases, the concentration of Asn in the N+ lateral roots of the N HHH treatment was significantly higher than in the N− lateral roots.The above findings indicated that the supply of nitrate on the N+ side led to a notable increase in the Asn levels within the N+ lateral roots.A significant increase in the Asn concentration in the N− lateral roots occurred when the supply duration of nitrate on the N+ side was extended to 9 days.After phase II, the order of Asn concentration in the N+ lateral roots under the three treatments was N HHH > N LLL > N HLL ; the N HHH treatment reached a significant level of difference compared to the N LLL and N HLL treatments, while there was no significant difference between the N LLL and N HLL treatments.During phase III, the Asn concentration in the N+ lateral roots under the three treatments showed the same change trend as phase II, indicating that the Asn concentration in the N+ lateral roots decreased significantly after nitrate withdrawal on the N+ side.After phase II, there was no significant difference in the Asn concentrations in the N− lateral roots between the N LLL , N HHH , and N HLL treatments.After phase III, the Asn concentrations of the N− lateral roots under the three treatments were N HHH > N LLL > N HLL , and the differences between them were significant.These differences indicated that the concentration of Asn in the N− lateral roots decreased with the increase in nitrate withdrawal days on the N+ side.
After phase III, the Asn concentration in the N+ lateral roots under the N HLH treatment was significantly lower compared to the N HHH treatment and significantly higher compared to the N HLL and N LLL treatments.The N− lateral roots in the N HLH and N HHH treatments showed no notable difference in Asn concentrations, but they were considerably greater compared to those in the N LLL and N HLL treatments.After the supply, withdrawal, and subsequent resupply of nitrate, it was observed that the Asn concentration on both sides of the roots increased again.
The results above showed that the Asn concentration in the roots on both sides rose when nitrate was added to the N+ side but decreased when nitrate was removed.The delay in the N− side change may have been caused by the absence of direct contact with nitrate.
Table 5 shows the Asn concentrations of the phloem + and xylem + on both sides.During the three experimental phases, the Asn concentrations of the phloem + on both sides under the N HHH treatment were significantly greater compared to those under the N LLL treatment; meanwhile, the Asn concentrations of the phloem + on the N+ side were significantly greater compared to those of the N− side under the N HHH treatment.These findings suggest that the provision of nitrate on the N+ side greatly enhanced the Asn concentration in the phloem + on both sides, with the N+ side showing a significantly greater increase compared to the N− side.After phase II, the Asn concentration in the phloem + on both sides varied among the following three treatments, with the order being N HHH > N HLL > N LLL .During phase III, the Asn concentration in the phloem + on both sides under the three treatments had the same trend in changes, with the exception of insignificant differences between the N HLL and N LLL treatments.These results indicated that the Asn concentration in the phloem + on both sides decreased significantly after nitrate withdrawal on the N+ side.
After phase III, the Asn concentration in the N+ lateral phloem + under the N HLH treatment was notably reduced compared to that under the N HHH treatment, but it was considerably higher compared to that under the N LLL and N HLL treatments.The N− side did not show any difference between the N HLH and N HHH treatment; however, the N HLH treatment exhibited a significantly higher concentration compared to the N LLL and N HLL treatments.These results indicated that the Asn concentration in the phloem + on both sides showed a change trend of increase-decrease-increase after the nitrate supply-withdrawalresupply on the N+ side.
The variation in Asn concentration in the xylem + on both sides was similar to that in the roots.That is, when the nitrate was supplied, removed, and then provided again on the N+ side, the Asn concentration in the xylem + on both sides showed an increase-decreaseincrease trend.

Assimilation of Nitrate in Soybean Roots
The root system is the main nitrate-absorbing organ [29].Nitrate supply to soybean plants can promote N metabolism in the roots [30][31][32].A total of 5 mM of nitrate has been shown to significantly enhance nitrate transporters and N assimilation enzymes in the roots [33].NR is an essential enzyme in the process of nitrate assimilation and has a crucial function in the assimilation of nitrate [34].In the three experimental phases, the NR activities of the soybean roots on both sides were unaffected by the change in N+ lateral nitrate concentration.This may be due to the fact that the soybean plants were cultured using a nitrate nutrient solution of 25 mg•L −1 during the pretreatment period.Despite being provided with a N-free nutrient solution for 10 days prior to the experiment, NR is an inducible enzyme that could have induced root NR activity.NR was induced by nitrate in the early stages, so, changing the nitrate concentration during the treatment period had no obvious effect on the root NR activity of both sides, which is consistent with the findings of Hunter [35].
Nitrate is converted into ammonia by NR, and then enters the GS-GOGAT pathway for further N assimilation, where GS activity affects nitrate assimilation and directly influences the Gln and Asn levels in plants [36,37].In this study, the N+ lateral root GS activity increased with the supplementation of nitrate on this side but did not decrease significantly with the withdrawal of nitrate.The GS activity of the N− lateral root increased when nitrate was added to the N+ side and decreased significantly when nitrate was removed.Combined with the findings of a previous study under the same experimental conditions, it was found that the nitrate concentration in the N+ lateral roots was still high after nitrate withdrawal; 2.73% of the nitrate on the N− side was transported from the N+ side (for data, see Li et al., 2021).Therefore, the GS activity in the soybean root exhibited a direct association with the nitrate level.Nitrate concentration may regulate GS activity in roots, and changes in GS activity may further affect the concentration of Gln and Asn in roots on both sides.
Therefore, we measured the root concentrations of Gln and Asn on both sides.We found that direct contact with nitrate on the N+ lateral roots led to an increase in Asn concentration when nitrate was added and a decrease when nitrate was removed.This suggests that the Asn concentration in the roots is influenced by direct exposure to nitrate.This is the same as the results of Amarante et al. [38].Although N− lateral roots did not make contact with nitrate directly, the concentration of Asn increased when nitrate was added to the N+ side and decreased when the nitrate was withdrawn.However, there was a slight delay in the reaction when nitrate was decreased, which matched the variation in the GS activity in the N− lateral roots.This suggests that nitrate transported to the N− lateral roots was assimilated through the action of GS and accumulated as Asn.Alterations in glutamine levels in the roots on either side were not significantly influenced by variations in the concentrations of nitrate supply from lateral N+ roots.It is possible that Asn is the main N transport compound for nitrate assimilation in soybean roots [13].Although Gln is commonly regarded as a direct product of ammonia assimilation after N 2 or nitrate reduction [39], Gln is easily converted into Asn and transported in soybean plants [15,40].

Transport of Asparagine in Soybean Roots
While soybean nodules transport their N fixation products to the shoot as ureides, a large amount of N is exported to the xylem and transported in the form of amides (Gln and Asn) [17].The concentration of Asn in the xylem sap increased with the accumulation of Asn in the soybean shoot especially after nitrate application [38, [41][42][43].Puiatti and Sodek [44] also reached a similar conclusion that the ratio of asparagine/glutamine in soybean xylem sap increased after nitrate application.In peanut (Arachis hypogaea L.), the nitrate supply decreased the Asn concentration in the xylem sap [45].In this study, the Asn concentrations in the xylem + on both sides increased when nitrate was added to the N+ side but decreased when nitrate was removed.This suggests that the concentration of the nitrate supply and time affects the transportation and distribution of Asn in soybean plants.Nitrate was assimilated in the roots, and some of the assimilates were transported through the xylem + as Asn; however, the N− lateral roots did not make contact with the nitrate directly, and the Asn concentration in the xylem + on this side remained controlled by variations in the nitrate concentration on the N+ side.
Asn serves as the primary amino acid component of the xylem and phloem sap in lupin [46], and it acts as the principal N compound in the xylem sap of soybean plants [47,48].The phloem is responsible for delivering a specific amount of N supply from the shoot to the roots of every plant [49].The amino acid transport flux in the soybean phloem regulated the absorption of nitrate by the roots [50].In Medicago truncatula, Asn can be transported from the shoot to the root through the phloem [18].The presence of nitrates led to higher levels of Asn in the xylem and phloem sap of lupin plants [46].Additionally, the phloem of Ricinus communis L. exhibited an increase in Asn levels as a result of the introduction of nitrate [51].In this study, the Asn concentration in the phloem + and xylem + on both sides increased when nitrate was added to the N+ side but decreased when nitrate was removed.The transportation of nitrate in the plant occurs through the phloem + and xylem + , primarily in the form of Asn.Asn assimilated in the roots is transported to the shoot, and nitrate is assimilated into Asn in the shoot and then transported to the root.At the same time, it cannot be ruled out that Asn is assimilated into the root system and then transported in the whole soybean plant.In white lupin, it is possible for Asn to be directly transported from the xylem to the phloem [20].Therefore, it is also possible that Asn is transported between the xylem and phloem in soybean plants.The nitrate content in the basal root peeled skin and woody part was involved in N regulation, and they had a certain regulatory effect on the Asn transport flux and concentration in the N− lateral roots.Thus, the mechanism needs further study.

Conclusions
After being provided to one side of the dual-root soybean root system, nitrate can be transported in two directions within the plant as Asn.The main channels for nitrate assimilation are the phloem + and xylem + .
± 0.11 a Note: N+, nitrate supply side; N−: nitrate-free side.N LLL , no N in three phases; N HHH , supplied 100 mg•L −1 N in three phase; N HLL , supplied 100 mg•L −1 N in phase I and no N in phases II and III; N HLH : supplied 100 mg•L −1 N in phases I and III, and no N in phase II.The values represent the means ± the standard error, with a sample size of three.Significant differences between the treatments at the 5% level (according to Duncan's test) are indicated by the presence of distinct lowercase letters in the longitudinal comparison.
± 4.72 b 117.3 ± 10.79 a Note: N+, nitrate supply side; N−, nitrate-free side.N LLL , no N in three phases; N HHH , supplied 100 mg•L −1 N in three phase; N HLL , supplied 100 mg•L −1 N in phase I and no N in phases II and III; N HLH : supplied 100 mg•L −1 N in phases I and III, and no N in phase II.The values represent the means ± the standard error, with a sample size of three.Significant differences between the treatments at the 5% level (according to Duncan's test) are indicated by the presence of distinct lowercase letters in the longitudinal comparison.* represents the N+ side and N− side comparisons under the same treatment, reaching a 5% significance level in the horizontal comparison.
08 aNote: N+, nitrate supply side; N−, nitrate-free side.N LLL , no N in three phases; N HHH , supplied 100 mg•L −1 N in three phase; N HLL , supplied 100 mg•L −1 N in phase I and no N in phases II and III; N HLH : supplied 100 mg•L −1 N in phases I and III, and no N in phase II.The values represent the means ± the standard error, with a sample size of three.Significant differences between the treatments at the 5% level (according to Duncan's test) are indicated by the presence of distinct lowercase letters in the longitudinal comparison.* represents the N+ side and N− side comparisons under the same treatment, reaching a 5% significance level in the horizontal comparison.

Table 2 .
GS activity in the soybean roots (U/g FW).N in phases I and III, and no N in phase II.The values represent the means ± the standard error, with a sample size of three.Significant differences between the treatments at the 5% level (according to Duncan's test) are indicated by the presence of distinct lowercase letters in the longitudinal comparison.* represents the N+ side and N− side comparisons under the same treatment, reaching a 5% significance level in the horizontal comparison.
Note: N+, nitrate supply side; N−: nitrate-free side.N LLL , no N in three phases; N HHH , supplied 100 mg•L −1 N in three phase; N HLL , supplied 100 mg•L −1 N in phase I and no N in phases II and III; N HLH : supplied 100 mg•L −1

Table 3 .
Glutamine concentration in the roots (µg•g −1 FW).HLL , supplied 100 mg•L −1 N in phase I and no N in phases II and III; N HLH : supplied 100 mg•L −1 N in phases I and III, and no N in phase II.The values represent the means ± the standard error, with a sample size of three.Significant differences between the treatments at the 5% level (according to Duncan's test) are indicated by the presence of distinct lowercase letters in the longitudinal comparison.
Note: N+, nitrate supply side; N−, nitrate-free side.N LLL , no N in three phases; N HHH , supplied 100 mg•L −1 N in three phase; N