**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 (mm<sup>2</sup> ) 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

### **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. [19].*

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.

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 *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*

(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

**Figure 5.**

*2D-Page of nodule soluble proteins with 0 mM (A) or 5 mM (B) nitrate. From Saito et al. [19].*

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].
