Estimation of nitrogen fixation rate of soybean (Glycine max (L.) Merr.) by micro-scale relative ureide analysis using root bleeding xylem sap and apoplast fluid in stem

Soybean plants require a large amount of nitrogen compared with cereal crops, such as rice or wheat, and the N is derived from three sources; N derived from atmospheric N2 (Ndfa) by symbiotic nitrogen fixation, N derived from soil (Ndfs) and N derived from fertilizer (Ndff). To improve the N fertilization or cultivation methods, it is important to evaluate the percentage proportion of the N origins. A relative ureide method is a simple and reliable method for estimating the percentage of nitrogen depending on nitrogen fixation (%Ndfa) in a field grown soybean (Herridge et al. 1990, Takahashi et al. 1992, 1993). Recently papers have been published using a relative ureide method for estimating %Ndfa (Nohara et al. 2005, Nagumo et al. 2010, Takakai et al. 2010, Murata et al. 2012). This method is based on the fact that Ishizuka et al. (Kushizaki et al. 1964, Ishizuka 1970) found that nodulating soybean accumulated a high concentration of ureides (allantoin and allantoic acid) in the stem. Then it was found that the principal transport form of N fixed by the nodules is mainly ureides (Matsumoto et al. 1977, Ohyama and Kumazawa 1978), but major forms of N from roots are nitrate and amides (Ohyama and Kumazawa 1979, Ohyama et al. 2009). MacClure and Israel (1979) analyzed transport of nitrogen in the xylem sap of soybean plants, and proposed the percent ureide-N in xylem sap may be used as an indicator of the relative contribution of N2 fixation to the total input of plant N. Herridge and Peoples (1990) compared the ureide assay and N method, and they proposed the calibration equations. They calculated the relative abundance of ureide-N in root bleeding sap, and vacuum-extracted sap by (100 x ureide-N/(ureide-N + α-amino-N + nitrate-N), and stem hot water extracts by (100 x ureide-N/(ureide-N + nitrate-N). We proposed the eqation as 100 x ureide-N/ (ureide-N + amide-N + nitrate-N), where amide-N is "2 x α -amino-N", because major form of amino acids and amides is asparagine (McClure and Israel, 1979, Herridge 1984, Ohyama et al. 1989). When plants are periodically harvested (eg. R1, R5 and R7 stages), and the nitrogen fixation activity and nitrate absorption rate could be estimated from the relative ureide percent and total N increase in the shoots (Takahashi et al. 1992, 1993, Ohyama 1990, Tewari et al. 2004, 2011, Ohyama and Toan 2006). For relative ureide analysis, the concentrations of