**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).

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

### **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. [22].*

*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*

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.

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.

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].

#### **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. [22].*

### **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. [22].*




#### **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. [22].*

*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*
