Open access peer-reviewed chapter
Abstract
Soybean plants require a large amount of nitrogen either from nitrogen fixation in nodules or nitrogen absorption from roots. It is known that nitrate, a major inorganic nitrogen compound in upland soils, represses nodule growth and nitrogen fixation. Rapid and reversible inhibition of nodule growth and nitrogen fixation activity was found in the hydroponically cultivated soybeans after changing the nutrient solution with or without nitrate. Isotope tracer analysis revealed that the major cause of this inhibition depended on the changes in the partitioning of photo-assimilate between nodules and roots and was not directly related to the transported N compounds. Transcriptome and metabolome analyses supported that nitrate strongly promotes nitrogen and carbon metabolism in the roots but represses them in the nodules. The application of ammonium, glutamine, or urea also inhibited the nodule growth and nitrogen fixation like nitrate, although the inhibition was lower than that of nitrate. The degree of inhibition was related to the decrease in carbon isotope partitioning into the nodules, rather than the import of nitrogen isotope to nodules. Urea was detected in xylem sap and all parts of soybean, and some urea might be originated from ureide degradation.
Keywords
- soybean
- nitrogen fixation
- nodules
- roots
- nitrate
- ammonium
- urea
- ureides
1. Introduction
1.1 Characteristics of nitrogen assimilation in soybean
Currently, soybean seed production is increasing worldwide, and the annual production in 2019 was 334 million tons [1]. The percentage composition of soybean seeds is proteins (35%), lipids (19%), carbohydrates (28%), minerals (5%), and water (13%) [2]. Soybean plants originate from East Asia, and Asian people including the Japanese eat various kinds of traditional soy foods such as Tofu, Miso, Shoyu, Natto, etc. [3]. Traditional Eastern soy foods are now gradually accepted by Western people for promoting their health, as well as meat substitutes made from soybean. In addition, soybean seeds are very important for feeding livestock.
Because soybean seeds contain a higher amount of protein than cereals and other legume seeds, soybean requires a large amount of nitrogen for high seed yield [4]. One of soybean seeds production requires 70–90 kgN assimilation, therefore, the world average yield of 2.77 t ha−1 in 2019 requires as high as 200–250 kgN ha−1. Soybean plants depend on nitrogen fixation by root nodules (Ndfa), and the nitrogen is absorbed from soil (Ndfs) or fertilizer (Ndff) when applied. The N availability from soil mineralization during the soybean cultivation period varies widely depending on the soil fertility, but it is about 50 kgN ha−1 in Japan. Therefore, nitrogen fixation is the main source, and about 60–75% of N was derived from N derived from Ndfa in Niigata, Japan [5]. The root nodule is a symbiotic organ with soil bacteria, rhizobia. Figure 1A shows the photograph of nodulated roots of hydroponically cultivated soybean. Soybean nodules can be visible from 8 days after planting and grow and start to fix N2 around 15–20 DAP, when rhizobia were inoculated to seeds [6]. The cross-section of a soybean nodule is shown in Figure 1B. Infected rhizobia live in the red central zone of the nodule due to a high concentration of leghemoglobin, which bind to O2 supporting respiration and nitrogen fixation by rhizobia [6].
Figure 1.
Photographs of root nodules of hydroponically cultivated soybean. (A) Root nodules attached to the roots of soybean. (B) Cross-section of a soybean nodule.
Another characteristic of N assimilation in soybean is that soybean needs about 80% of N after the beginning of flowering, which is quite different from the paddy rice which assimilates only 20% of N is assimilated after heading [5]. Therefore, a long-lasting high nitrogen fixation activity after flowering is essential for high soybean seed yield. However, nitrogen fixation activity tends to decrease during the pod filling stage, due to competition of nutrients mainly photo-assimilate between seeds and nodules. Therefore, supplemental application of N fertilizers may be beneficial to support vigorous shoot growth and photosynthetic activity during the pod filling stage. However, a basal application of chemical N fertilizers often inhibits the nodule growth and nitrogen fixation activity, and they will be lost by nitrate leaching and denitrification. So, it is necessary to harmonize the N fertilization and N fixation to obtain a constant and high seed yield of soybean [7, 8].
1.2 Nitrogen inhibition on the growth and nitrogen fixation activity of soybean nodules
The inhibitory effects of combined nitrogen, especially nitrate, on nodule formation and nitrogen fixation of legumes have been studied for over 100 years [9]. According to Streeter’s review, the responses can be divided into three classes, the number of nodules per root, nitrogenase activity per unit mass of the nodules, and the nodule mass per plant [10]. The effects of nitrate concentration on the magnitude of the three responses are different. A relatively high nitrate concentration is required for the inhibition of nodule number per plant, followed by nitrogenase activity per nodule mass. The concentration effect on nodule mass is more sensitive than nodule number and nitrogen fixation activity, while low levels of nitrate stimulate nodule growth through the promotion of shoot growth.
Concerning the effects of nitrate on nodule growth, there are two different situations, the first is a direct effect or local effect in which nodules are in direct contact with the solution containing nitrate, and the second is an indirect or systemic effect in which nodules are not directly contacted with nitrate and nitrate is absorbed from the distant part of the roots [11]. In the direct effect, when soybean plants were hydroponically cultivated, the addition of 5 mM nitrate in culture solution rapidly stopped the individual nodule growth within one day as well as decreased N2 fixation activity measured by C2H2 reduction activity [12, 13].
Figure 2 shows the effect of 5 mM nitrate supply to the culture solution on the nodule growth of soybean plants. Soybean seeds were inoculated with
Figure 2.
Effect of 0 mM (A) or 5 mM (B) nitrate on the growth of nodules of hydroponically cultivated soybean from 11 days to 19 days after planting. (A) Root nodules attached to the roots of soybean cultivated with 0 mM NO3−. (B) Root nodules attached to the roots of soybean cultivated with 5 mM NO3−. (A) 0 mM NO3−, (B) 0 mM NO3−.
Figure 3.
Growth response of soybean nodules to 0 mM or 5 mM nitrate application in the culture solution. (A) Photographs of nodulated roots after nitrate treatments to 0 mM (blue arrows) or 5 mM nitrate (red arrows) from 10 DAP to 24 DAP. (B) Graphs of the changes in nodule diameter after nitrate treatments from 10 DAP to 24 DAP. (a) 0 mM NO3−, (b) 5 mM NO3−, and (c) 0,5,0 mM NO3−, white background: 0 mM NO3−, gray background: 5 mM NO3−. From Fujikake et al. [
As for the indirect or systemic effect of nitrate, Tanaka et al. [15] reported that nitrate supplied to one side of the split root system of soybean did not inhibit the nodule growth and nitrogen fixation activity of the other side of the roots supplied N-free medium. The upper and lower root systems were separated vertically by a two-layered pot system, the concentration and period of nitrate supply from lower roots gave a different effect on the nodule growth in the upper roots [16]. The long-term supply of a high concentration (5 mM) of nitrate from the lower roots inhibited the nodule growth (DW) of the upper roots, but the continuous supply of a low concentration (1 mM) of nitrate in the lower roots promoted the nodule growth in the upper roots through the increased in shoot growth and photosynthetic activity.
Concerning the chemical forms of N, the inhibitory responses were more sensitive to nitrate than to ammonium, and urea was only slightly inhibitory [17, 18]. The physiological meaning of the different responses to nitrogen compounds is not well understood. In this review, we would like to introduce recent advances in nitrate inhibition to nodule growth and nitrogen fixation activity first, and the effect of nitrate, ammonium, urea, and glutamine on the inhibition of nodule growth was compared.
2. Short-term effect of nitrate supply on nodule and root growth of soybean
Saito et al. [19] investigated the short-term responses of nodule growth to the 5 mM nitrate supply at one-hour intervals for 16 h under light and dark conditions (Figure 4A). Inoculated soybean plants were cultivated in a photo chamber under 16 h light at 28°C and 8 h dark at 18°C conditions. The nodule size is indicated by the nodule area (mm2) measured by a time-lapse camera and the nodule area measuring software NODAME [20, 21]. Under light conditions, the nodule growth of 13 DAP plants was constant from the start of light period to the end of the 16 h light. When 5 mM NO3− was applied under light conditions, the increase in the nodule area was the same until the initial 2 h and became significantly repressed after 7 h. The increase in the nodule area with 5 mM NO3− during the 16 h was about 60% of that with 0 mM NO3− under light conditions. This result indicated that nitrate inhibition on nodule growth begins very rapid at a few h after the addition of nitrate to the culture solution. Under dark conditions with 0 mM NO3− the nodule growth was more severely depressed (42% of 0 mM NO3− under light conditions at 16 h) than that treated with 5 mM NO3− under light conditions. The nodule growth was most severely repressed under dark conditions with 5 mM NO3− (25% of 0 mM NO3− under light conditions at 16 h) among treatments. These results support the former hypothesis that the addition of NO3− on the nodulated roots is repressed mainly through the decrease in photo-assimilate supply to the nodules because dark conditions repressed the nodule growth, and dark plus nitrate additively repressed the nodule growth.
Figure 4.
(A) Increase in nodule area with 0 mM or 5 mM NO3− under light or dark conditions, (B) Increase in primary root length with 0 mM or 5 mM NO3− under light or dark conditions, and (C) Increase in lateral root length with 0 mM or 5 mM NO3− under light or dark conditions. From Saito et al. [
Similar responses are observed in the increase in primary root growth with 0 mM or 5 mM NO3− under light or dark conditions (Figure 4B). The primary root growth was the fastest with 0 mM NO3− under light conditions, and that with 5 mM NO3− repressed (73% at 16 h), 0 mM NO3− under dark conditions (40% at 16 h), and 5 mM NO3− under dark conditions (29% at 16 h).
Interestingly, the opposite responses to nitrate were observed for the growth of lateral roots (Figure 4C). Under light conditions, the increase in the lateral roots with 5 mM NO3− was promoted to 121% of the 0 mM under light conditions. The increase in the lateral roots was repressed in 0 mM NO3− under dark conditions (51% at 16 h), but 5 mM NO3− still increased the lateral root growth under dark conditions compared with 0 mM NO3−.
Initial transport of photo-assimilate labeled with 11CO2 exposed to the matured leaf showed that 11C was leached to the roots and nodules within 1 h, and the distribution of photo-assimilate was higher in the root parts in contact with 5 mM NO3− solution compared with 0 mM NO3− solution [13]. Quantitative analysis of photo-assimilate transport was conducted using 14CO2 as a tracer, which was supplied to the whole shoot for 2 h, then the distribution of 14C was investigated among the organs of soybean plants supplied with 0 mM or 5 mM NO3−. The percentage distribution of 14C in roots and nodules was 5.2 and 9.1% of total fixed 14C in the plants with 0 mM NO3−, while those in the roots and nodules changed to 9.1 and 4.3%, respectively [13]. The increase in 14C was mainly in the lateral roots after supplying 5 mM NO3−, while 14C distribution in the primary roots was not changed between 0 mM and 5 mM NO3− [13].
The inhibitory effect of nitrate on nodule growth was shown to be reversible, and when 5 mM NO3− was changed to 0 mM NO3−, the nodule growth recovered in a day, when 5 mM NO3− treatment continued for 14 days [12]. This means the physiological function of nodules may be maintained under 5 mM NO3−, while the nodule growth was almost completely stopped. Figure 5 shows the 2D-PAGE of soybean nodule extract cultivated with 0 mM (A), and 5 mM NO3− (B) treatment from 10 DAP to 34 DAP. The patterns and intensities of the protein spots were similar between the 0 mM and 5 mM NO3− treatments after 24 days of treatment. Therefore, the functions of nodule were maintained under 5 mM NO3− conditions, although nodule growth and nitrogen fixation activity were strongly repressed. This might suggest that nodule function is not disintegrated in the nodule in direct contact with NO3−, whereas carbohydrate deficiency temporarily retard the nodule growth and nitrogen fixation activity. Therefore, after removal of NO3− from the culture solution quickly recovers the nodule growth and nitrogen fixation activity [12, 13].
Figure 5.
2D-Page of nodule soluble proteins with 0 mM (A) or 5 mM (B) nitrate. From Saito et al. [
3. Transcriptome and metabolome analyses of nitrate inhibition
The effect of nitrate supply on the gene expression and metabolite changes in nodules and roots were investigated by transcriptome and metabolome analysis [22]. After supplying 5 mM NO3− for 24 h to the nodulated soybean plants at 19 DAP, mRNA was extracted from the nodules and roots, and the cDNAs were hybridized with soybean oligo DNA microarray [22]. The results were compared with control plants supplied with 0 mM NO3−. Figure 6 shows the numbers of probe sets in the roots and nodules, which were up-regulated (4-fold, 2-fold) or down-regulated (4-fold, 2-fold) following 5 mM NO3− supply for 24 h compared with the control plants with 0 mM NO3−. The number in the 4-fold up-regulated probe sets in roots was 142 and higher than that in the nodules (78). The number of common probe sets 4-fold up-regulated both in roots and nodules was 9. On the other hand, the number of 4-fold down-regulated probe sets in the roots was 92 and lower than that in nodules (116). Similar trends were observed for the 2-fold up-regulation (C), and 2-fold down-regulation (D).
Figure 6.
Numbers of probe sets in the roots and nodules up-regulated or down-regulated by more than 4-fold or 2-fold following the addition of 5 mM nitrate to medium at the level of p < 0.05. From Ishikawa et al. [
The results showed that the 5 mM NO3− supply highly enhanced the gene expression in the roots related to nitrate transporter and metabolisms such as nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), glutamate synthase (GOGAT), and Asparagine synthetase (AS) (Figure 7). The genes for ureide synthesis through purine synthesis and degradation in the roots were slightly increased. The gene expression related to nitrate transport and metabolism in nodules was also slightly promoted after NO3− supply, but not so high as in the roots. Figure 8 shows the ratios of gene expression related to C metabolism, glycolysis, and TCA cycle in roots (left) and nodules (right). The gene expression of the enzymes in glycolysis and TCA cycle in the roots were promoted by NO3−, while those in the nodules were mostly depressed.
Figure 7.
Comparison of the ratios of gene expression related to N metabolism in roots or root nodules with 5 mM or 0 mM nitrate (+N/−N). From Ishikawa et al. [
Figure 8.
Ratios of gene expression related to C metabolism (Glycolysis and TCA cycle in roots and nodules with 5 mM or 0 mM nitrate (+N/−N). From Ishikawa et al. [
Metabolome analysis was conducted using the same plants for the transcriptome analysis as above. The ratios of the concentrations of nitrogen compounds, phosphorus compounds, and organic acids concentrations in roots and nodules after 5 mM NO3− treatment were compared with 0 mM NO3− treatment as shown in Figure 9. Most of the nitrogen compounds in the roots were increased by NO3− treatment, especially allantoic acid (3.1-fold), glutamine (2.4-fold), and asparagine (2.1-fold). On the other hand, asparagine and N-acetylglucosamine in nodules were increased, but some amino acids were decreased such as alanine. For the phosphorous compounds, 5 mM NO3− treatment increased ATP concentration in the roots (3.9-fold) but decreased in nodules (0.7-fold). Similarly, most of the ratios of phosphorous compounds show over 1-fold in the roots, but less than 1-fold in nodules. The same was true in organic acids in roots and nodules.
Figure 9.
Ratios of N and P metabolite concentrations in roots and nodules with 5 mM or 0 mM nitrate (+N/−N). From Ishikawa et al. [
Both transcriptome and metabolome analysis indicated that NO3− treatment caused the promotion of C and N metabolism in the roots, while depressed C metabolism in the nodules. This agrees with the changes in the photo-assimilate supply from nodules to the roots [13].
4. Effects of nitrate, ammonium, urea, and glutamine on nodule growth and nitrogen fixation activity
In addition to the inhibitory effect of nitrate on nodule growth and nitrogenase activity, the repressive effect of ammonium is also reported [14], whereas urea did not reduce nodule dry weight and nitrogen fixation activity in hydroponically grown soybean [17, 18]. The difference in inhibitory effects by the forms of N compounds is not fully understood yet. Yamashita et al. [23] investigated the effect of various forms of nitrogen; nitrate, ammonium, urea, and glutamine on the quick and reversible inhibition of nodule growth and nitrogen fixation activity of soybean plants.
Soybean plants were cultivated in a nitrogen-free nutrient solution in a glass bottle, and nitrate, ammonium, glutamine, or urea (1 mM-N) were supplied from 12 days after planting (DAP) to 17 DAP. The increase in individual nodule growth expressed by nodule volume (mm3) was shown in Figure 10. The inhibitory effects on the nodule growth were severe in nitrate and ammonium treatments, and those by urea and glutamine were smaller than in nitrate and ammonium. On 17 DAP, the increase in nodule dry volume was low in nitrate and ammonium medium in urea and glutamine compared with the control with N-free solution. After N-based solutions were replaced by N-free solutions from 17 DAP to 24 DAP, the nodule growth in all treatments showed the recoveries. These results indicated that the rapid and reversible inhibition is not only by nitrate but also by ammonium, urea, and glutamine. Figure 11A shows the dry weight of nodules per plant on 17 DAP. The application of 1 mM-N of nitrate, ammonium, urea, and glutamine for 5 days depressed the nodule weight to 45, 75, 76, and 65% of the control nodules on 17 DAP. After N-free solution was supplied for 6 days following 5-day N treatment, the nodule dry weight on 24 DAP increased in all N treatments similar to the control plants (Figure 11B). Figure 11C shows the ARA per a single plant, and the activities were depressed in nitrate, ammonium, urea, and glutamine treatment like the decrease in the nodule dry weight (Figure 11A). The specific ARA per g DW of nodules on 17DAP were almost the same (Figure 11D), so the decrease in ARA per plant is due to lower nodule dry weight and not by a decline in the specific ARA. In this experiment, the DW and N concentrations of roots, stems, and leaves increased after 5 days of N application on 24 DAP compared with the control plants supplied with N-free solution.
Figure 10.
Changes in nodule volume from 12 to 24 DAP for treatments with control (N-free), nitrate, ammonium, urea, or glutamine from 12 to 17 DAP, thereafter cultivated with a N-free culture solution (Experiment 2). Shaded background indicates N treatment period, and white background indicates cultivation with N-free medium. Average and standard error are shown (n = 5). From Yamashita et al. [
Figure 11.
Comparison of the dry weight of nodules at 17 DAP (A) and at 24 DAP (B), and acetylene reduction activity per plant (C), and specific acetylene reduction activity per nodule g dry weight (D) on 17 DAP of soybean plants supplied with control (N-free), nitrate, ammonium, urea, or glutamine from 12 to 17 DAP, followed by supplying N-free solution from 17 DAP to 24 DAP. Averages and standard errors are shown (n = 4). Different letters above the column indicate significant differences at <0.05 by Tukey’s test. From Yamashita et al. [
15N-labeled 1 mM-N nitrate, ammonium, urea, or glutamine was supplied for 3 days from 21 to 24 DAP, in which the solutions were renewed every day. The whole shoot was enclosed in a plastic bag on 23 DAP, and 13CO2 was exposed to the plants for 1 h. Then the plants supplied with 15N and 13C were harvested on 24 DAP, 26 h after 13CO2 exposure. The labeling of 15N and 13C in each organ was determined by Mass spectrometry. The amount of 15N in nodules (Figure 12A) was 0.14 mg from nitrate-15N, 0.26 mg from ammonium-15N, 0.14 mg from urea-15N, and 0.29 mg from glutamine-15N. It is interesting to note that the amount of 15N in nodules is not related to the decrease in nodule DW (Figure 11A) and ARA (Figure 11C). Figure 12B shows the amount of 13C in nodules, and it was 85 mg in control, 27 mg in nitrate, 34 mg in ammonium, 54 mg in urea, and 34 mg in glutamine treatment. The decrease in the amount of 13C in nodules was similar to the nodule DW (Figure 11A) and ARA (Figure 11C) among treatments. This result supports the hypothesis that the depression of nodule growth and nitrogen fixation activity is related to the decline in photoassimilates partitioning but not N supply.
Figure 12.
(A) Amounts of N derived from 15N-labeled source in each tissue of soybean plants on 15 DAP supplied for 3 days from 21 to 24 DAP. (B) amounts of C derived from 13C-labeled CO2 in each tissue of soybean plants on 24 DAP supplied for 1 hour at 23 DAP. From Yamashita et al. [
Changes in the free amino acid concentrations in nodules, roots, stems, and leaves were shown in Figure 13. The nitrate treatment significantly increased the concentration of the amino acids, especially Asp, Asn, and Glu in the nodules. The application of ammonia, urea, or glutamine also increased the concentrations of Asp and Asn in nodules compared with control plants although the increases were not higher than nitrate treatment. On the other hand, the amino acid concentrations in the roots show that ammonium treatment remarkably increased the Asn and Asp concentrations, the urea and glutamine treatments also increased the Asn concentration in the roots, but the increase in Asn in the roots treated with nitrate was relatively low. The increase in the Asn and Asp concentrations was the highest in the stems of plants supplied with urea, followed by glutamine, ammonium, and nitrate treatments. The increase in Asn and Asp was observed from nitrate, ammonium, urea, and glutamine treatments.
Figure 13.
Free amino acid concentrations in each tissue of soybean plants on 24 DAP supplied with various N compounds from 21 to 24 DAP. From Yamashita et al. [
Figure 14A shows the effects of nitrogen compounds on total root length on 34 DAP after long-term nitrogen treatment for 2 weeks. The application of nitrate promoted the total root length by over 2-fold compared with the control plants. On the other hand, the application of ammonium inhibited the root length by only a half of the control. The application of urea and glutamine slightly increased the total root length. Similar trends were observed for the root dry weight (Figure 14B). Figure 14C and D show the increase in primary root and lateral roots for the first week of N application from 20 to 27 DAP, and the second week from 27 to 34 DAP. By NO3− application, the growth of the lateral roots was promoted, but the primary root was not. On the other hand, the inhibitory effects of NH4+ were evident both for the primary root and the lateral roots. The promotive effects on the length of the primary root and not on the lateral root length were observed by urea and glutamine applications like nitrate.
Figure 14.
Total root length (A) and the dry weight of roots (B) on 34 DAP after two weeks of the treatments with various forms of N compounds, and the increase in the primary root length (C), and lateral root length (D) for the first week (blue bar) and the second week (red bar). (A) Total root length, (B) Root dry weight, (C) Increase in primary root length, and (D) Increase in lateral root length. From Yamashita et al. [
5. The N composition transported in xylem sap of soybean plants cultivated with nitrate, ammonium, urea
Soybean seeds were inoculated with
Figure 15.
Nitrogen concentrations of N compounds in xylem sap of nodulated soybean plants treated with control (N-free), nitrate, ammonium, urea, and non-nodulated plants with nitrate. From Ono et al. [
The concentration of ureides (sum of allantoin and allantoic acid) was the highest in control plants depending on nitrogen fixation, followed by ammonium, and urea treatments. The concentration of ureides was very low in nodulated plants treated with nitrate. A small amount of ureides was present in non-nodulated plants. This result is in accordance with the effect of nitrogen compounds on nitrogen fixation activity because most of the ureides in xylem sap originated from fixed nitrogen in nodules, although a small amount is produced in the roots. Nitrate was detected only in the xylem sap of soybean treated with nitrate, and the concentration of nitrate accounted for about 50% of total N in xylem sap either in nodulated or non-nodulated plants supplied with NO3−. The asparagine concentration was the lowest in control plants totally depended on nitrogen fixation, and it was higher in ammonium and urea treatments. The concentration of glutamine was also higher in ammonium and urea treatments, but very low in nitrate treatment. It is interesting that urea is always present in xylem sap and it was higher in control, ammonium, and urea treatments.
The concentrations of ureides and urea or arginine and urea in xylem sap were plotted in Figure 16. The concentration of urea was positively correlated with ureides concentration (Figure 16A), but the correlation was not observed between urea and arginine in xylem sap (Figure 16B) which is the alternative precursor of urea production [25]. Appreciable amounts of urea were present in all the organs for all treatments, and the positive correlations between urea and ureides were observed in the nodules, roots, stems, and leaves. This may indicate that some urea originated from ureides in soybean plants, especially in the roots [24].
Figure 16.
Correlations between the concentrations of ureides and urea (A), and arginine and urea (B) in the soybean xylem sap. (A) Ureide vs. Urea (B) Arginine vs. Urea. From Ono et al. [
The ureides, allantoin, and allantoate, are universal metabolites in all organisms including plants, animals, and microorganisms generated by the degradation of futile purines. Soybean plants transport the fixed nitrogen in the nodules mainly in the form of ureides (ca. 80–90% of total N) supplemented with amides and amino acids [26, 27]. On the other hand, nitrate and asparagine are the principal forms of N transport in the xylem sap of the non-nodulated soybean plants [26]. A small percentage of N about 10% was transported in the form of ureides from the non-nodulated roots, which means some ureides can be synthesized in the roots as well as nodules. The concentrations of ureides, nitrate, and amide N transported through xylem sap could be used to evaluate the percentage dependence of N derived from nitrogen fixation [27].
Figure 17 shows a model of ureide synthesis in the nodules and ureide degradation in soybean. The fixed ammonia from N2 in the bacteroid, a symbiotic state of rhizobia, is rapidly excreted to the cytosol of the infected cells, then the ammonium is assimilated by glutamine synthetase (GS)/glutamate synthase (GOGAT) pathway to glutamine [28, 29, 30, 31].
Figure 17.
A model of ureide synthesis in the nodules and ureide degradation in soybean. GS: glutamine synthetase; GOGAT: glutamate synthase; XDH: xanthine dehydrogenase; Xan: xanthine; HIU: hydroxyisourate; OHCU: 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline; UGlyAH: ureidoglycine aminohydrolase. From Ono et al. [
6. Conclusion
Rapid and reversible repression of nodule growth and nitrogen fixation activity of nodulated soybean were observed when nitrate was supplied in the culture solution. This may be caused by the decrease in the photo-assimilate partitioning to the nodules and not by the transport of N compounds from applied N in the solution. Transcriptome and metabolome analysis supported the above hypothesis. Conversely, the C and N metabolism in the roots was promoted by the application of nitrate. A similar rapid and reversible repression of nodule growth and nitrogen fixation activity was also observed when ammonium, urea, or glutamine was supplied as same as nitrate, however, the inhibitory effect was stronger in nitrate compared with ammonium, urea, or glutamine. Urea was detected in xylem sap and all parts of soybean, and some of the urea originated from ureide degradation.
The plant shoots and roots exchange C and N through the xylem and phloem transport systems [35]. The C and N metabolism is regulated by complex mechanisms to optimize plant organ development and growth. So, understanding the CN metabolism can be related to the agricultural crop production, and maintenance of the agroecosystem.
Acknowledgments
This research was partially supported by the Grants-in-Aid for Scientific Research (No. 18380049, No. 26292036) from Japanese Society for the Promotion of Science.
References
- 1.
FAOSTAT. Available from: https://www.fao.org/faostat/en/#data - 2.
Ohyama T. The role of legume-rhizobium symbiosis in sustainable agriculture. In: Sulieman S, Tran LP, editors. Legume Nitrogen Fixation in Soils with Low Phosphorus Availability. Berlin: Springer; 2017. pp. 1-20. DOI: 10.1007/978-3-319-55729-8_1 - 3.
Ohyama T. Nutrition of soybean seeds. In: Ohyama et al., editors. Traditional and Modern Japanese Soy Foods-Manufacturing, Nutrition and Cuisine of a Variety of Soy Foods for Health and Joy of Taste. New York: NOVA Publishers; 2013. pp. 1-10 - 4.
Ohyama T et al. Soybean seed production and nitrogen nutrition. In: Board JE, editor. A Comprehensive Survey of International Soybean Research-Genetics, Physiology, Agronomy and Nitrogen Relationships. London: InTech; 2013. pp. 115-157 - 5.
Ohyama T et al. Role of nitrogen on growth and seed yield of soybean and a new fertilization technique to promote nitrogen fixation and seed yield. In: Kasai M, editor. Soybean, The Basis of Yield, Biomass and Productivity. London: InTech; 2017. pp. 153-185 - 6.
Sato T, Onoma N, Fujikake H, Ohtake N, Sueyoshi K, Ohyama T. Changes in four leghemoglobin components in nodules of hypernodulating soybean ( Glycine max [L] Merr.) mutant and its parent in the early nodule developmental stage. Plant and Soil. 2001;237 :129-135 - 7.
Harper JE. Soil and symbiotic nitrogen requirements for optimum soybean production. Crop Science. 1974; 14 (2):255 - 8.
Rabie RK, Kumazawa K. Effect of nitrate application and shade treatment on the nitrogen fixation and yield of soybean plant. Soil Science & Plant Nutrition. 1979; 25 (4):467 - 9.
Fred EB, Graul EJ. The effect of soluble nitrogenous salts on nodule formation. Journal of American Society of Agronomy. 1916; 8 (5):316-328 - 10.
Streeter J. Inhibition of legume nodule formation and N2 fixation by nitrate. CRC Critical Reviews in Plant Sciences. 1988; 7 (1):1-23 - 11.
Ohyama T et al. Effect of nitrate on nodulation and nitrogen fixation of soybean. In: El-Shemy HA, editor. Soybean, Physiology and Biochemistry. London: InTech; 2011. pp. 333-364 - 12.
Fujikake H, Yashima H, Sato T, Ohtake N, Sueyoshi K, Ohyama T. Rapid and reversible nitrate inhibition of nodule growth and N2 fixation activity in soybean ( Glycine max (L.) Merr.). Soil Science & Plant Nutrition. 2002;48 (2):211-217 - 13.
Fujikake H, Yamazaki A, Ohtake N, et al. Quick and reversible inhibition of soybean root nodule growth by nitrate involves a decrease in sucrose supply to nodules. Journal of Experimental Botany. 2003; 54 (386):1379-1388 - 14.
Imsande J. Inhibition of nodule development in soybean by nitrate or reduced nitrogen. Journal of Experimental Botany. 1986; 37 (3):348-355 - 15.
Tanaka A, Fujita K, Terasawa H. Growth and dinitrogen fixation of soybean root system affected by partial exposure to nitrate. Soil Science & Plant Nutrition. 1985; 31 (4):637-645 - 16.
Yashima H et al. Long-term effect of nitrate application from lower part of roots on nodulation and N2 fixation in upper part of roots of soybean ( Glycine max (L.) Merr.) in two-layered pot experiment. Soil Science & Plant Nutrition. 2005;51 (7):981-990 - 17.
Vigue JT, Harper JE, Hageman RH, Peters DB. Nodulation of soybeans grown hydroponically on urea. Crop Science. 1977; 17 (1):169-172 - 18.
Ohyama T, Nicholas JC, Harper JE. Assimilation of 15N2 and 15NO3− by partially nitrate tolerant nodulation mutants of soybean. Journal of Experimental Botany. 1993; 44 (269):1739-1747 - 19.
Saito A et al. Effect of nitrate on nodule and root growth of soybean (Glycine max (L.) Merr.). International Journal of Molecular Sciences. 2014:4464-4480. DOI: 10.3390/ijms15034464 - 20.
Available from: https://www.google.com/search?client=firefox-b-d&q=NODAME - 21.
Tanabata S, Tanabata T, Saito A, et al. Computational image analysis method for measuring size of nodule growth in soybean. Japanese Society of Soil Science and Plant Nutrition. 2014; 85 (1):43-47. (in Japanese) - 22.
Ishikawa S, Ono Y, Ohtake N, Sueyoshi K, Tanabata S, Ohyama T. Transcriptome and metabolome analyses reveal that nitrate strongly promotes nitrogen and carbon metabolism in soybean roots, but tends to repress it in nodules. Plants. 2018; 7 (32). DOI: 10.3390/plants7020032 - 23.
Yamashita N, Tanabata S, Ohtake N, Sueyoshi K, Sato T, Highchi K, et al. Effects of different chemical forms of nitrogen on the quick and reversible inhibition of soybean nodule growth and nitrogen fixation activity. Frontiers in Plant Science. 2019; 10 (131). DOI: 10.3389/fpls.2019.00131 - 24.
Ono Y, Fukasawa M, Sueyoshi K, Ohtake N, Sato T, Tanabata S, et al. Application of nitrate, ammonium, or urea changes the concentrations of ureides, urea, amino acids and other metabolites in Xylem Sap and in the organs of soybean plants ( Glycine max (L.) Merr.). International Journal of Molecular Sciences. 2021;22 (4593):4573. DOI: 10.3390/ijms22094573 - 25.
Blume C, Ost J, Mühlenbruch M, Peterhänsel C, Laxa M. Low CO2 indices urea cycle intermediate accumulation in Arabidopsis thaliana. PLoS One. 2019; 14 (1):e0210342 - 26.
Takahashi Y, Chinushi T, Nagumo Y, Nakano T, Ohyama T. Relative concentration of ureides-N in root-bleeding sap of nodulated and non-nodulated soybean isoline. Japanese Society of Soil Science and Plant Nutrition. 1991; 62 (4):431-433 - 27.
Takahashi Y, Chinushi T, Ohyama T. Quantitative estimation of N2 fixation and N absorption rate in field grown soybean plants by relative ureide method. Bullut in Faculty of Agriculture Niigata University. 1993; 45 :91-105 - 28.
Ohyama T, Kumazawa K. Incorporation of 15N into various nitrogenous compounds in intact soybean nodules after exposure to 15N2 gas. Soil Science & Plant Nutrition. 1978; 24 (4):525-533 - 29.
Ohyama T, Kumazawa K. Assimilation and transport of nitrogenenous compounds originated from 15N2 fixation and 15NO3 absorption. Soil Science & Plant Nutrition. 1979; 25 (1):9-19 - 30.
Ohyama T, Kumazawa K. Nitrogen assimilation in soybean nodules. I. The role of GS/GOGAT system in the assimilation of ammonia produced by N2 fixation. Soil Science & Plant Nutrition. 1980; 26 (1):109-115 - 31.
Ohyama T, Kumazawa K. Nitrogen assimilation in soybean nodules. II. 15N2 assimilation in bacteroid and cytosol fractions of soybean nodules. Soil Science & Plant Nutrition. 1980; 26 (2):205-213 - 32.
Shelp BJ, Ireland RJ. Ureide metabolism in leaves of nitrogen-fixing soybean plants. Plant Physiology. 1985; 77 (3):779-783. DOI: 10.1104/pp.77.3.779 - 33.
Winkler RG, Polacco JC, Blevins DG, Randall DD. Enzymatic degradation of allantoate in developing soybeans. Plant Physiology. 1985; 79 (3):787-793. DOI: 10.1104/pp.79.3.787 - 34.
Winkler RG, Blevins DG, Polacco JC, Randall DD. Ureide catabolism of soybeans. II. Pathway of catabolism in intact leaf tissue. Plant Physiology. 1987; 83 (3):585-591. DOI: 10.1104/pp.83.3.585 - 35.
Marouane B, Mitsui T, Sueyoshi K, Ohyama T. Recent advances in carbon and nitrogen metabolismin C3 plants. International Journal of Molecular Sciences. 2021; 22 (1):318. DOI: 10.3390/ijms22010318