The application of combined nitrogen, especially nitrate, to soybean plants is known to strongly inhibit nodule formation, growth and nitrogen fixation. In the present study, we measured the effects of supplying 5 mM nitrate on the growth of nodules, primary root, and lateral roots under light at 28 °C or dark at 18 °C conditions. Photographs of the nodulated roots were periodically taken by a digital camera at 1-h intervals, and the size of the nodules was measured with newly developed computer software. Nodule growth was depressed approximately 7 h after the addition of nitrate under light conditions. The nodule growth rate under dark conditions was almost half that under light conditions, and nodule growth was further suppressed by the addition of 5 mM nitrate. Similar results were observed for the extending growth rate of the primary root as those for nodule growth supplied with 5 mM nitrate under light/dark conditions. In contrast, the growth of lateral roots was promoted by the addition of 5 mM nitrate. The 2D-PAGE profiles of nodule protein showed similar patterns between the 0 and 5 mM nitrate treatments, which suggested that metabolic integrity may be maintained with the 5 mM nitrate treatment. Further studies are required to confirm whether light or temperature condition may give the primary effect on the growth of nodules and roots.

No abstract

The ammonium produced by nitrogen fixation in the bacteroid is rapidly excreted to cytosol of infected cell of soybean nodules and then assimilated into glutamine and glutamic acid, by glutamine synthetase/glutamate synthase pathway. Most of the nitrogen is further assimilated into ureides, allantoin, and allantoic acid, via purine synthesis, and they are transported through xylem to the shoots. Nitrate absorbed in the roots is reduced by nitrate reductase and nitrite reductase to ammonia either in the roots or leaves. The ammonia is also assimilated by glutamine synthetase/glutamate synthase pathway, and mainly transported by asparagine, and not ureides. The nitrogen transported into leaves is readily utilized for protein synthesis, and then, some of them are decomposed and retransported to roots, apical shoots, and pods via phloem mainly in the form of asparagine.

It has been reported that supply of nitrate to culture solution rapidly and reversibly inhibits nodule growth and nitrogen fixation activity of soybean. In this study, the effects of ammonium, urea, or glutamine on nodule growth and nitrogen fixation activity are compared with that for nitrate. Soybean plants were cultivated with a nitrogen-free nutrient solution, then 1 mM-N of nitrate, ammonium, glutamine, or urea were supplied from 12 DAP until 17 DAP. Repression of nodule growth and nitrogen fixation activity at 17 DAP were observed by ammonium, urea, and glutamine like nitrate, although the inhibitory effects were milder than nitrate. The removal of nitrogen from the culture solutions after nitrogen treatments resulted in a recovery of the nodule growth. It was found that the glutamine treatment followed by N-free cultivation gave highest nitrogen fixation activity about two times of the control. Tracer experiments with 15 N and 13 C were performed to evaluate the translocation of N and C to the different tissues. Culture solutions containing a 15 N-labeled nitrogen source were supplied from 21 DAP, and the whole shoots were exposed to 13 CO 2 for 60 min on 23 DAP, and plants were harvested on 24 DAP. The percentage distribution of 15 N in nodules was highest for ammonium (1.4%) followed by glutamine (0.78%), urea (0.32%) and nitrate (0.25%). The percentage distribution of 13 C in the nodules was highest for the control (11.5%) followed by urea (5.8%), glutamine (2.6%), ammonium (2.3%), and nitrate (2.3%). The inhibitory effects of nitrogen compounds appeared to be related to a decrease in photoassimilate partitioning in the nodules, rather than 15 N transport into the nodules. The free amino acid concentrations after nitrogen treatments were increased in the nodules and leaves by nitrate, in the roots by ammonium, in the stems by urea, and the roots, stems, and leaves by glutamine treatment. The concentrations of asparagine, aspartate, and glutamine were increased after nitrogen treatments. By the long-term supply of nitrogen for 2-weeks, nitrate significantly increased the lateral roots and leaf growth. The long-term supply of urea and glutamine also promoted the lateral roots and leaf growth, but ammonium suppressed them.

Soybean is an important crop for human food and feed for livestock. World soybean production is increasing especially in North and South America. Soybean seeds contain a high percentage of protein about 35-40%, and they require a large amount of nitrogen compared with other crops. Soybean plants make root nodules with rhizobia, and rhizobia can fix atmospheric N 2 and give the fixed N to the host soybean plants. Also, soybean can absorb nitrogen usually nitrate from soil or fertilizers. The amount of total assimilated nitrogen in shoot is proportional to the soybean seed yield either from nitrogen fixation or from nitrogen absorption, and the nitrogen availability is very important for soybean cultivation. Maintenance of a high and long-term nitrogen fixation activity is very important for a high production of soybean. However, application of chemical nitrogen fertilizers usually depresses nodule formation and nitrogen fixation. Nitrate in direct contact with a nodulated part of roots causes severe inhibition of nodule growth and nitrogen fixation, although a distant part of nodules from nitrate application gives no or little effect. Deep placement of slow-release nitrogen fertilizers, coated urea, or lime nitrogen promoted the growth and seed yield and quality of soybean without depressing nitrogen fixation.