ABSTRACT
The specific mechanism by which nitrogen application affects nodulation and nitrogen fixation in legume crops remains uncertain. To further study the effects of nitrogen application on soybean nodulation and nitrogen accumulation, three consecutive tests were performed during the VC-V4, V4-R1 (10 days), and R1-R2 (10 days) growth periods of soybean. In a dual-root soybean system, seedlings on one side were watered with a nutrient solution containing NH4+ or NO3− as the N source (N+ side), and those on the other side were watered with a nitrogen-free nutrient solution (N- side). During the VC-R2 period, on the N+ side, high nitrogen treatment inhibited nodule growth and nitrogenase activity (EC 1.18.6.1), and the inhibition was significantly increased with increasing high nitrogen supply time (10 days, 20 days). When the high nitrogen treatment time reached 20 days, the specific nitrogenase activity (C2H4 μmol−1 g−1 nodule dry mass h−1) was similar to that in the low nitrogen treatment, indicating that the nitrogen fixation capacity per gram of dry mass nodules was almost the same. Therefore, it is assumed that long-term high nitrogen treatment mainly reduces nitrogen fixation by reducing the nodule number. The effect of nitrogen concentration on the roots on the N+ side was greater than that on the N- side. Taken together, these results indicate that nitrogen application affects a contact-dependent local inhibition of root nodule growth, nitrogenase activity, and nitrogen accumulation. The whole plant systematically regulates specific nitrogenase activity, and high nitrogen inhibition is recoverable.
1. Introduction
Soybeans have a large demand for nitrogen, which is mainly obtained by direct inorganic nitrogen uptake and nodule nitrogen fixation at the roots. High concentrations of nitrogen have an inhibitory effect on soybean nodulation and nitrogen fixation (Carroll, Mcneil, and Gresshoff Citation1985; Carroll and Gresshoff Citation1983; Serraj et al. Citation1992; Streeter and Wong Citation1988,), while low concentrations of nitrogen promote nodulation and nitrogen fixation (Gulden and Vessey Citation1998; Hinson Citation1975). Hinson (Citation1975) applied different concentrations of NH4NO3 to soybean roots on one side of a dual-root system using the root-separation method and found that the nodule number and weight on the nitrogen-application side decreased significantly with the increase in nitrogen concentration, and the nodule weight on the nitrogen-free side was also inhibited. Root separation studies by Tanaka, Fujita, and Terasawa (Citation1985) and by Xia et al. (Citation2017) showed that nitrogen locally inhibits soybean nodules; specifically, root nodules in direct contact with nitrate nitrogen and ammonium nitrogen are inhibited, while no significant inhibition of nodules occurs on the other side. Daimon and Yoshioka (Citation2001) used the root-separation method to apply 14 mM nitrate nitrogen to one side of peanut roots and found that after 30 days of nitrate nitrogen treatment, nitrate nitrogen inhibited the nodule nitrogenase activities on both the N+ side and N- side. In the root-separation test by Tanaka, Fujita, and Terasawa (Citation1985), the nitrogenase activity on N- side was also systematically inhibited. Xia et al. (Citation2017) applied nitrogen on one side of a dual-root soybean system and found that the nodule nitrogenase activities on both sides changed synchronously with the nitrogen application concentration. It can be concluded that the effect of nitrogen on nodule number and weight is a local inhibition based on contact, while the effect on the activity of nodule nitrogenase is a systematic regulation in which the aboveground and underground parts interact.
Skrdleta et al. (Citation1980) cultured peas with perlite and found that after application of 20 mM nitrate nitrogen, the nitrogenase activity was significantly inhibited after only one day of treatment and nodule dry weight was inhibited after 3 days of treatment. Streeter (Citation1982) applied 15 mM nitrate nitrogen to soybeans in sand culture for 7 days and found that the nitrogenase activity was inhibited only one day after nitrogen application. Fujikake et al. (Citation2002) applied 5 mM nitrate nitrogen to soybeans by hydroponic culture and found that the nodule growth and nitrogenase activity were rapidly inhibited, and the inhibited nodule growth and nitrogenase activities were completely restored to normal levels after transfer to a nitrogen-free treatment. Yashima et al. (Citation2003) divided soybean roots into upper and lower roots in a two-layered hydroponic pot system and applied 0 or 5 mM nitrate nitrogen to the upper and lower roots, respectively. They found that the effects on nodule growth and nitrogenase activity, in the presence or absence of nitrate nitrogen, can be rapidly and reversibly regulated. This finding indicates that direct inhibition of soybean nodule growth and nitrogenase activity is reversible as nitrogen concentration changes. Bethlenfalvay, Abushakra, and Phillips (Citation1978) cultured peas with vermiculite and applied different concentrations of ammonium nitrogen. They found that the dry weight of plants slightly increased at the concentration of 0–2 mM ammonium nitrogen but exhibited no obvious change at the concentrations of 4 mM, 8 mM and 16 mM ammonium nitrogen. Vessey and Waterer (Citation2010) showed that under hydroponic conditions, pea plants under the nitrogen-free treatment had lower dry weight and nitrogen accumulation than did those with application of 0.5 mM and 1.0 mM ammonium nitrogen. Harper (Citation1974) also found that under hydroponic conditions, the dry weight of soybeans with the application of 7.5 mM nitrate–nitrogen was significantly higher than that of soybeans with the application of 0.75 mM nitrate–nitrogen and nitrogen-free solutions. Ohyama, Nicholas, and Harper (Citation1993) used the 15N tracer method and found that the inhibition of soybean nodulation and nitrogen fixation by nitrate nitrogen and ammonium nitrogen may be related to the high nitrogen accumulation in roots. Singleton and Van Kessel (Citation1987) divided the closed system into two parts, placing the roots on both sides of the closed system, applying nitrogen to both sides and not applying nitrogen, and adding air and Ar (argon) and O2, respectively (mixed gas: Ar: 80%; O2: 20%). The results showed that the nitrogen side of the root received more of the photosynthetic product carbohydrates than the nitrogen-free side of the root.
In the above-described method of rooting, the root system of the legume crop plant was divided into two parts so that neither subsystem remained intact, which may affect the accuracy of the experimental results. In the present study, soybean seedlings were grafted to obtain a double-rooted soybean plant with complete roots on both sides, and the test results were more accurate. Three consecutive single-sided nitrogen application tests were carried out during the V1-R2 period, in which the N+ side was subjected to high and low concentration changes. The soybean nodule number, dry weight and nitrogenase activity were determined, and the dry weight, nitrogen accumulation and C/N ratio in plants and in roots on both sides were also measured. The effects of nitrogen concentration on nodulation and nitrogen fixation as well as soybean growth were systematically studied, providing a reference for analyzing the effects and mechanisms of nitrogen application on soybean nodulation and nitrogen fixation.
2. Materials and methods
This study was conducted at the Experimental Base of Northeast Agricultural University, Harbin City, Heilongjiang Province, China (126° 45’ E and 45° 42’ N), in 2017 and 2018. The annual precipitation is 500–550 mm, and the accumulated temperature ≥10°C is 2700°C.
2.1. Experimental material preparation
Soybean plants with dual-root systems were prepared according to the seedling grafting method described in Xia et al. (Citation2017). The soybean seeds (Glycine max L. cv. HeiNong40) were seeded into fine-sand medium and cultured in an illuminated growth chamber at 30°C for approximately 3 days. Two seedlings were chosen and cut with one seedling slashed downward and the other upward for 0.5–1 cm. Next, after the two seedlings were cross-inserted into one another’s cuts, they were clipped and fixed with a grafting clip. The root systems of the two seedlings were separately planted into fine-sand medium divided by a partition plate in a plastic pot. After the seedlings were grown for 1 week in a weather enclosure, the grafting clip was removed, and the top part of the seedling with the upward incision was cut along the grafting site, leaving only the grafting and the lower parts. The cultivated soybean dual-root system is shown in . Plastic pots used in this experiment were 0.3 m in diameter and 0.28 m in height, each of which was divided into two equal parts with a polycarbonate plastic partition plate. The top of the plate was 2 cm lower than the edge of the pot, and the gaps between the partition plate and the pot were all sealed with glue. Two drainage holes (1 cm in diameter) were drilled at the bottom of the pot, with one for each side of the partition plate. Each pot was filled with 20 kg of sand. Prior to use, the fine sand was thoroughly washed with tap water and then rinsed twice with distilled water.
Figure 1. Double-root soybean material
2.2. Experimental treatments
A nitrogen-containing nutrient solution was used to treat one root system (the N+ side), while a nitrogen-free nutrient solution was used to treat the other root system (the N− side) of soybean plants with dual root systems. (NH4)2SO4 was used for nitrogen addition in 2017, and NaNO3 was used for nitrogen addition in 2018. The addition of various concentrations of nitrogen (25, 100 mg l−1) was applied to the N+ side. To supply nitrogen at different concentrations of 25 mg l−1 and 100 mg l−1, (NH4)2SO4 at concentrations of 117.8 and 471.4 mg l−1 or NaNO3 at concentrations of 151.7 and 607.1 mg l−1 was added into the nutrient solution, respectively. Other nutrient ingredients in the nutrient solution were prepared according to the description provided by Hoagland and Arnon (Citation1950) and Shoukun, Gong, and Zu (Citation2010), with slight modifications. Their concentrations (mg l−1) were KH2PO4, 136.00; MgSO4, 240.00; CaCl2, 220.00; Na2MoO4· H2O, 0.03; CuSO4 · 5H2O, 0.08; ZnSO4 · 7H2O, 0.22; MnCl2 · 4H2O, 4.90; H3BO3, 2.86; FeSO4 · 7H2O, 5.57; and Na2EDTA, 7.45. One root system in the dual- root system was supplied with nitrogen-added nutrient solution (the N+ side), while the other root system received the solution containing only the ingredients listed in above. The concentration of 25 mg l−1 is more suitable for root nodule growth, and 100 mg l−1 inhibits the growth of nodules; thus, the nodules grew in two different environments to facilitate the study of the reversibility of nodulation. The test was completed in four growth stages: VC (unfolded cotyledons), V4 (fourth trifoliate leaf stage), R1 (initial flowering) and R2 (late flowering stage), stages were designated according to the description of Fehr et al. (Citation1971), and the treatments shown in .
Table 1. Nitrogen application concentrations (mg l−1) in this study
The study was divided into two groups. In the first group, the nitrogen concentration changed from low to high nitrogen and then back to low nitrogen. The second set of nitrogen concentrations changed from high nitrogen to low nitrogen and then returned to high nitrogen. Each group consisted of three trials. The test period was VC-V4, V4-R1 (10 days), R1-R2 (10 days).
Prior to full opening of the opposite true leaves, the seedling was irrigated with 250 ml distilled water once a day for each side of the root system. After opening of the opposite true leaves, the prepared nutrient solution was applied once a day, with 250 ml of nitrogen-containing solution for the N+ side and 250 ml of nitrogen-free solution for the N- side. Rhizobium inoculation was conducted for five consecutive days by adding the ground nodular tissue of the previous year into the nutrient solution when the opposite true leaves completely opened. The soybean root nodules (harvested 1 year before and stored in a freezer) were washed, ground, and then added to the nutrient solution at approximately 5 g l−1 for rhizobium inoculation. The experiments was performed with four replications.
2.3. Sampling and determination
2.3.1. Sampling treatments
Three consecutive experiments were performed during the V4, R1 and R2 periods. The aboveground parts along the grafting site were cut on a sunny day from 8:00–10:00. The two parts of the underground roots were washed with distilled water to remove the sand. After blotting with filter paper, the nitrogenase activity of the nodule was determined. After the measurement, the nodule was removed. The number of nodules was determined, and the samples were dried at 65°C to determine the nodule weight, dry weight, nitrogen content and carbon content of the roots and above ground parts.
2.3.2. Measurement
An acetylene reduction assay, as previously described by Gremaud and Harper (Citation1989) and Chen et al. (Citation2013), was used to measure the nitrogenase activity in root nodules. The washed soybean roots were drained with filter paper to remove excess water, and the roots with nodules were placed together in a 500 ml brown jar and covered with a rubber plug that had been punched. A total of 50 ml of air was extracted with a syringe, and 50 ml of acetylene (99.9%) was added for reaction for 2 h. The ethylene content was determined using a GC7900 gas chromatograph analyzer (Model GC7900, Shanghai Techcomp Scientific Instrument Co., Ltd., China). Specific nitrogenase activity (SNA) was expressed as μmoles of ethylene formed per gram dry weight of nodules per hour. Acetylene reduction activity (ARA) was expressed as μmoles of ethylene formed per plant per hour.
For the determination of nitrogen content in plants, K2SO4 and CuSO4 were used as catalysts and concentrated sulfuric acid (H2SO4) for digestion, and the N content was measured with a B324 automatic Kjeldahl analyzer.
To determine the carbon content in plants, carbon in plant samples was oxidized with a certain concentration of potassium dichromate-sulfuric acid solution, and the remaining potassium dichromate was titrated with FeSO4. The amount of potassium dichromate consumed was used to calculate the carbon content.
N accumulation (mg plant−1) = dry matter accumulation (g plant−1) × N content (g kg−1).
An analysis of variance was performed with IBM SPSS Statistics 21.0 (SPSS Inc., Shanghai, China). The data from four replications taken on each sampling date were analyzed separately. T-test was applied to the data, and differences were considered significant corresponded at the 5% or 1% level and presented as the means ± SE.
3. Results and analysis
3.1. Changes in the number and weight of root nodules of soybean plants
shows the changes in nodule number and nodule weight in the three tests. As shown in , in the three consecutive tests on both the N+ side and N- side, the changes in nodule number and nodule weight were both in accordance with the changes in nitrogen application concentration, and the trend was basically the same in 2017 and 2018.
Table 2. Changes in the number and weight of root nodules of soybean plants inVC-V4, V4-R1, R1-R2 growth periods of soybean
At the end of Test I, the application of 100 mg l−1 nitrogen (NHHH) during the VC-V4 period significantly inhibited nodule number and nodule weight on N+ side, but the difference on the N- side was not significant.
At the end of Test II, the performance was basically the same in both years . The results indicate that the change from low nitrogen to high nitrogen during the V4-R1 period inhibited nodule growth on both sides, whereas the change from high nitrogen to low nitrogen did not significantly promote the generation and growth of nodules.
At the end of Test III, the results showed that the longer the duration of treatment with high or low nitrogen, the more obvious was the inhibition or promotion of nodule growth, and the nodule growth can be restored when the nitrogen concentration changes.
3.2. Changes in SNA and ARA in the soybean root nodules
shows the changes in SNA and ARA in the three tests. In both 2017 and 2018, the trend was basically the same. At the end of Test I, the SNA and ARA on the N+ side under NTTT were significantly higher than those under NHHH. Under NHHH, the SNA and ARA on the N- side were extremely significantly higher than those on the N+ side. These results indicated that the supply of 100 mg l−1 nitrogen during the VC-V4 period could significantly inhibit the SNA and ARA on the N+ side, whereas there was no significant effect on the N- side.
Table 3. Changes in specific nitrogenase activity per gram dry weigh and acetylene reduction activity per hour in the soybean root nodules in VC-V4, V4-R1, R1-R2 growth periods of soybean
At the end of Test II, the two-year test showed the same pattern. The results indicated that during the V4-R1 period, with the change from low nitrogen to high nitrogen, the nitrogenase activity was inhibited on both sides, regardless of SNA or ARA, while the change from high nitrogen to low nitrogen promoted SNA on both sides but did not promote ARA.
At the end of Test III, the results indicated that both SNA and ARA were inhibited in the short-term (10 days) high-nitrogen treatment, and the N+ side had a stronger inhibitory effect. The long-term (20 days) high- nitrogen treatment did not significantly inhibit SNA but inhibited ARA on both sides. When low nitrogen treatment resumed, nodule nitrogenase activity was restored.
3.3. Changes in the nitrogen content and C/N ratio of soybean plants
As shown in , in the three consecutive tests, the change over the course of two years was basically the same: the nitrogen content in shoots increased with the increase in nitrogen concentration, and when the nitrogen application concentration decreased, the nitrogen content in shoots decreased accordingly. As shown in , the nitrogen content in the roots on N+ side had the same trend as the nitrogen content in the shoots. The nitrogen content on the N- side also changed with the change in nitrogen concentration on the N+ side, but the magnitude of change was small. Comparing the nitrogen content in the roots on the N+ side with that on the N- side showed that the nitrogen contents in the aboveground part and in the roots on the N+ side were more readily changed with the change in nitrogen concentration, while the nitrogen content in the roots on the N- side also changed but only slightly.
Table 4. Changes in the nitrogen content and C/N ratio of soybean shoots in VC-V4, V4-R1, R1-R2 growth periods of soybean
Table 5. Changes in the nitrogen content and C/N ratio of soybean roots in VC-V4, V4-R1, R1-R2 growth periods of soybean
As shown in and , the effect of the change in nitrogen concentration on the C/N ratio in the tested soybean plants was exactly opposite to the effect on the nitrogen content. High nitrogen decreased the C/N ratio above ground, and when the nitrogen application concentration was changed back to low nitrogen, the C/N ratio recovered. shows the change in the root C/N ratio. During the VC-V4 period, low nitrogen could promote the C/N ratio in roots on both sides and high nitrogen could decrease the C/N ratio. At the end of Test II, the change from low nitrogen to high nitrogen during the V4-R1 period could significantly inhibit the C/N ratio on the N+ side. When the nitrogen application was changed from high nitrogen to low nitrogen, the C/N ratio on the N+ side significantly increased, whereas the C/N ratio on N- side was not obviously affected. At the end of Test III, the longer the high nitrogen treatment time, the greater the extent to which the C/N ratio in the roots on both sides could be inhibited, and when the nitrogen concentration was changed back to low nitrogen, the promotion effect could be restored.
3.4. Changes in the dry weight and nitrogen accumulation of soybean plants
As shown in , the high nitrogen treatment promoted the dry weight of the shoots ; when it changed from high nitrogen to low nitrogen, the dry weight of the shoots did not decrease significantly. As shown in , during the VC-V4 period, high nitrogen could promote root dry weight. At the end of Test II, the differences among the four treatments were significant on N+ side, and the descending order of root dry weight was NHHH>NHTT>NTHH>NTTT; the differences among the four treatments were not significant on the N- side. These results indicated the root dry weight could be maintained at a high level even in the low-nitrogen environment as long as the high-nitrogen treatment was carried out during the early period. At the end of Test III, the differences among NTTT, NTHH and NTHT were not significant, nor were the differences among NHHH, NHTT and NTHH, indicating that the change in nitrogen concentration during the R1-R2 period could change the root dry weight, but the effect was not as obvious as during the V4-R1 period; in addition, the high-nitrogen treatment in the early period could still maintain a high dry weight level after the decrease in nitrogen concentration.
Table 6. Changes in the dry weight and nitrogen accumulation of soybean shoots in VC-V4, V4-R1, R1-R2 growth periods of soybean
Table 7. Changes in the dry weight and nitrogen accumulation of soybean roots in VC-V4, V4-R1, R1-R2 growth periods of soybean
As shown in , the accumulation of nitrogen in the shoots is consistent with the change in nitrogen content and dry weight of soybean shoots. Additionally, as shown in , Test 1 and Test 2 show that the high nitrogen treatment could significantly promote the increase in nitrogen accumulation on both sides, whereas low nitrogen reduced the accumulation of nitrogen. At the end of Test III, the longer the high nitrogen treatment time, the greater the increase in nitrogen accumulation on both sides of the roots. When the nitrogen was changed back to low nitrogen, the nitrogen accumulation on both sides also decreased.
4. Discussion
4.1. Relationship among soybean nodulation, nitrogen fixation and nitrogen application
Daimon et al. (Citation2015) applied nitrate nitrogen at concentrations of 0.7 mM, 3.5 mM, and 14.0 mM to peanut under sand cultivation conditions and found that the nodule number, fresh weight and nitrogenase activity were relatively high under 0.7 mM, were the highest under 3.5 mM, and were the lowest under 14.0 mM. Gan et al. (Citation2004) cultured soybeans in perlite and found that the nodule number, nodule weight and nitrogen fixation amount were significantly decreased under a high nitrogen concentration (10 mM) and significantly increased under a low nitrogen concentration (1 mM or 3.75 mM). Harper (Citation1974) found that soybeans grown under hydroponic conditions had a higher nodule nitrogenase activity under low-nitrogen than under nitrogen-free conditions. Gulden and Vessey (Citation1997) found similar patterns in peas and soybeans cultured in sand, in which low concentrations of ammonium nitrogen promoted the formation and growth of plant nodules. This study showed that the nodule number and nodule weight under NTTT on the N+ side were significantly higher than those under NHHH in all the three tests. The SNA under NTTT was significantly higher than that under NHHH in Test I, and the difference between these two treatments was not significant with the increase in time until Test II and Test III. The ARA under NTTT was significantly higher than that under NHHH in all the three tests, which was due to the decrease in nodule number and nodule weight caused by high nitrogen. Carroll and Gresshoff (Citation1983) applied the root-separation method in Trifolium repens to supply different concentrations of nitrate nitrogen and found that the nodule number and nodule weight on the N+ side decreased significantly with the increase in nitrogen application level, whereas the nodule number and nodule weight on the N- side were not inhibited. Cho and Harper (Citation1991) used the root-separation method to study soybeans and found that when 5 mM nitrate nitrogen was applied, the nodule number, nodule weight, and nitrogenase activity on the N+ side were all significantly smaller than those on the N- side. Xia et al. (Citation2017) applied different concentrations of nitrogen on the same side of the soybean dual-root systems and found that the nodule number and nodule weight on the N+ side increased first and then decreased with the increase in nitrogen concentration, whereas the nodule number and weight did not change with concentration on the N- side. The nodule nitrogenase activity increased first and then decreased with the increase in nitrogen concentration on both the N+ side and N- side, with the peak occurring at a concentration of 50 mg l−1. Both the SNA and ARA exhibited synchronism with changes in nitrogen concentration on both the N+ side and N- side in that on both sides, the SNA and ARA decreased as the nitrogen concentration increased and decreased as the nitrogen concentration increased. In the present study, on the N- side, nodule number and nodule weight under NTTT were not significantly different from those under NHHH at the end of Test I and Test II but were higher than those under NHHH at the end of Test III. Moreover, the nodule number and weight on the N- side were higher than those on the N+ side after culture in Test II and Test III. SNA on the N- side under NTTT was not significantly different from that under NHHH in Test I and Test II but was significantly higher than that under NTTT in Test III. The ARA on the N- side under NTTT was not significantly different from that under NHHH in all three tests. Moreover, under NTTT, the ARA on the N- side was higher than that on the N+ side at the end of Test II and Test III; under NHHH, the ARA during Test I, Test II, and Test III on the N- side was extremely significantly higher than that on N+ side. For SNA, under NTTT, no significant difference existed between sides in all the three tests. Under NHHH, the SNA on the N- side was significantly higher than that on the N+ side in Test I, but the difference in SNA between sides was not significant after the culture of Test II and Test III. Yashima et al. (Citation2005) used a two-layered pot system to divide soybean roots into upper and lower parts, and they found that long-term supply of low-concentration nitrogen (1 mM) to lower roots could promote nodulation and nitrogen fixation in upper roots. At 40 days after the nitrogen concentration applied to the lower roots was changed from 0 mM to 5 mM, the nodule number, nodule weight and nitrogenase activity in the upper and lower roots were significantly reduced, and at 40 days after the nitrogen concentration applied to the lower roots was changed from 5 mM to 0 mM, the nodule number, nodule weight and nitrogenase activity in the upper and lower roots were all significantly restored.
4.2. Relationship among soybean nodulation, nitrogen fixation, nitrogen content and C/N ratio of plants
This study shows that when supplying nitrogen to one side of roots for dual-root soybeans, the nitrogen content in the shoots and roots increased with the increase in nitrogen concentration and decreased with the decrease in nitrogen concentration, and the longer the nitrogen treatment time was, the more obvious was the effect. At high nitrogen concentrations, the nitrogen content in roots on the N+ side was significantly higher than that on the N- side, which is similar to the findings of Cho and Harper (Citation1991) and Harper (Citation1974). Bacanamwo and Harper (Citation2010) suggested that soybean nodule nitrogenase activity is inhibited by nitrate nitrogen and may be attributed to the concentrations of nitrogen and C in plant tissues. In the study by Oti-Boateng, Wallace, and Silsbury (Citation1994), 4 days after the top of soybeans was removed, the plant nitrogen content increased, whereas the nitrogenase activity did not change; after 6 days, the nitrogen content increased sharply, the C/N ratio decreased significantly, and the nitrogenase activity was inhibited. In this study, the C/N ratio in the aboveground parts and roots of soybean changed with the change in nitrogen concentration; the higher the nitrogen concentration was, the lower the C/N ratio in the aboveground part and in the roots on the N+ side, indicating that a change in the N concentration on the N+ side could significantly change the C/N ratio of aboveground parts and roots on the N+ side in soybean. Under high-nitrogen treatments, roots on the N+ side had a lower C/N ratio than those on the N- side, while under low-nitrogen treatments, roots on both sides had no significant difference. These results suggested that while nitrogen inhibits nitrogenase activity, the C/N ratio in roots also decreases, and this pattern is more obvious on the N+ side. This result corroborates the views of Bacanamwo and Harper (Citation2010) and Oti-Boateng, Wallace, and Silsbury (Citation1994). Whether the effect of changes in nitrogen application concentration on the C/N ratio in soybean plants can be attributed to nitrogen inhibition of nodulation or nitrogen fixation and whether nitrogen inhibition of nodulation and nitrogen fixation is regulated by the C/N ratio in plants requires further study.
4.3. Relationship between nitrogen accumulation and nitrogen application in soybean
Bethlenfalvay, Abushakra, and Phillips (Citation1978) and Harper (Citation1974) considered that nitrogen application rate affected dry weight and nitrogen accumulation in soybeans. Gan et al. (Citation2004) indicated that as nitrogen concentration increased, dry matter accumulation and nitrogen accumulation in soybeans increased accordingly, but high concentrations of nitrogen (10 mM) inhibited soybean growth. Gulden and Vessey (Citation1998) and Silsbury, Catchpoole, and Wallace (Citation1986) showed that nitrogen application had a positive effect on dry weight in both roots and aboveground parts in soybean and white clover, and Yashima et al. (Citation2003) reported similar findings in a soybean study. In the present study, when nitrogen was applied to one side of a dual-root soybean, high nitrogen concentration could significantly promote the growth and nitrogen accumulation in the aboveground parts of the plants and in the roots on both sides. Although nitrogen application could promote the accumulation of dry matter and nitrogen in roots on the N- side, the promotion of root growth and N accumulation was more significant on the N+ side, especially at high nitrogen concentrations, which indicated that soybean roots shows a tendency toward high concentrations of nitrogen. It is worth mentioning that at high nitrogen levels, the nitrogen content and nitrogen accumulation in roots on the N+ side were significantly higher than those on the N- side, indicating that nitrogen has a local effect on nitrogen content and nitrogen accumulation in roots and more readily promotes nitrogen accumulation in the roots with direct contact of nitrogen. The accumulation of nitrogen was more obvious than the accumulation of dry matter, which may be due to the fact that when roots on the N+ side encountered a high nitrogen concentration, nitrogen was stored in a certain form after nitrogen uptake, thereby increasing the nitrogen content. The promotion effect on dry matter accumulation by nitrogen application was not as great as that on nitrogen content and nitrogen accumulation.
5. Conclusions
During the VC-R2 period for the dual-root soybean system, nitrogen was applied to one side and not to the other. The nodule number, nodule weight, and ARA under NHHH on the N+ side were inhibited; the SNA under the high-nitrogen treatment was inhibited during the initial period of nitrogen application, but as the nitrogen supply time was prolonged, the inhibition was weakened. After the high-nitrogen treatment was changed to low nitrogen, the inhibition of high nitrogen could be restored. The N- side exhibited a significant change only in SNA, indicating that the nodule number, nodule weight, and ARA were locally regulated by nitrogen application, while the SNA was systematically regulated by the whole plant, and nitrogen application could promote root growth on the N+ side, demonstrating the nitrogen tendency for the root system.
Acknowledgments
We thank American Journal Experts for its linguistic assistance during the preparation of this manuscript.
Disclosure Statement
No potential conflict of interest was reported by the authors
Additional information
Funding
References
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