**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

### **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. [23].*

### **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. [23].*

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.

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

*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 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. [23].*

### **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. [23].*

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.

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 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

### **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. [23].*

*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*

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.
