**5. The N composition transported in xylem sap of soybean plants cultivated with nitrate, ammonium, urea**

Soybean seeds were inoculated with *Bradyrhizobium diazoefficiens* (strain USDA110), and the seedlings were cultivated in an N-free culture solution until 20 DAP were treated with 5 mM-N of nitrate, ammonium, urea for 3 days until 23 DAP [24]. Control plants were cultivated continuously with N-free solution from 20 to 23 DAP. Another group of soybean plants was not inoculated, and seedlings were cultivated with 5 mM NO3 − from 5 to 23 DAP. The xylem sap was collected for 1 h from the cut basal part of the stem on 23 DAP. **Figure 15** shows the concentration of N compounds in xylem sap of soybean plants treated with 3 days of nitrate, ammonium, or urea. Non-nodulated plants were cultivated with NO3 − due to the lack of nodules.

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

### **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. [24].*

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 nonnodulated 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].

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 nonnodulated 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

#### **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. [24].*

*Effects of Application of Various forms of Nitrogen on the Growth of Soybean Nodules and Roots… DOI: http://dx.doi.org/10.5772/intechopen.105348*

### **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. [24].*

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 [28–31]. *De novo* synthesis of purine occurs in the infected cells, and urate is transported to the adjacent uninfected cells and hydrolyzed to allantoin. Some allantoin is further decomposed to allantoate in nodules, then allantoate and allantoin are transported through the xylem. There are two purine degradation pathways in microorganisms, allanotate amidinohydrolase (Pathway A in **Figure 17**) and allantoate amidohydrolase (Pathway B in **Figure 17**). In pathway A, allantoate is hydrolyzed and produces two molecules of urea and one molecule of glyoxylate. On the other hand, in pathway B-left no urea is released but 4 ammonium are sequentially released with one molecule of glyoxylate. Studies of ureide degradation in soybean have been done in the leaves, the sink organ of ureides. Shelp and Ireland [32] reported that allantoate degradation is via pathway A, however, Winkler et al. [33, 34] reported that both the leaf extracts and intact leaves of soybean directly liberate ammonium without releasing urea (Pathway B-left). Werner et al. suggested that allantoate degradation intermediates, ureidoglycine and uredoglycolate are non-enzymatically decayed and release urea and glyoxylate (Pathway B-right). In our results relatively high concentrations of urea were presented in xylem sap and all the parts of soybean, and the concentration was correlated with ureide concentration, suggesting that some part of urea may be derived from ureide degradation, especially in the roots [24].
