Takuji Ohyama

Incorporation of 15N into various nitrogenous compounds in intact soybean nodules after exposure to 15N2 gas

Abstract The intact nodules attached to the upper part of soybean roots were exposed to 15N2 and the incorporation of 15N into various soluble nitrogen constituents was investigated. Results indicated that ammonia, a primary product of N2 fixation, was located in more than two compartments. Ammonia reduced from N2 gas seemed to be incorporated firstly into glutamine especially amido-group nitrogen. Newly fixed nitrogen was secondly incorporated into glutamic acid and alanine in this sequence. These results suggested that fixed ammonia was assimilated by glutamine synthetase/glutamate synthase pathway. Turn-over rate of allantoin plus allantoic acid and serine was relatively high, although apparently these compounds were not primary products of newly fixed ammonia. 15N content of allantoin was always higher than that of allantoic acid. 15N incorporation to aspartic acid and asparagine was relatively slow, especially in early period. In bacteroid fraction there is much amount of ammonia comparing with other...

Assimilation and transport of nitrogenous compounds originated from 15N2 fixation and 15NO2 absorption

Abstract Nodulated leguminous plants utilize both combined nitrogen absorbed by roots and gaseous nitrogen fixed by root nodules. In order to elucidate the physiological role of N2 fixation compared with NO2 absorption, the assimilation and transport of nitrogenous compounds from these nitrogen sources were studied. One part of soybean plants was administered with labeled nitrogen gas and unlabeled nitrate, and another part with unlabeled nitrogen gas and labeled nitrate. After feeding of labeled compounds to roots, ammonia, nitrate, amino acids, amides and allantoin in nodules, roots and stems were separated, and 15N content of them were determined optically using JASCO NIA-l 15N-analyzer. In nodules supplied with 15N2, high 15N contents were found in glutamate, alanine, serine, γ-amino-butyrate, but in roots supplied with 15NO2, asparagine showed the highest 15N content after 8 hr 15N feeding. In stems, aIIantoin showed the highest 15N content in the case of 15N2 treatment, and the ratio (15N from 15N2/...

Nitrogen assimilation in soybean nodules. 2. 15N2 assimilation in bacteroid and cytosol fractions of soybean nodules.

15N assimilation was studied in bacteroid and cytosol fractions of soybean nodules. In the first experiment, after exposing the intact nodules to 15N2 for 5 min and 10 min, most of the fixed 15N was detected in cytosol fraction. In cytosol fraction, 15N content of glutamine was the highest and followed by glutamic acid, alanine, and allantoin in this sequence, whereas, in bacteroid fraction, glutamic acid showed the highest 15N content and alanine and glutamine followed. In the second experiment, 15N assimilation of various 15N-labeled compounds in the separated bacteroid and cytosol fractions was investigated. In the separated bacteroid fraction which was fed with 15NH4, 15N was incorporated very rapidly into glutamic acid, alanine, and aspartic acid, but very slowly into glutamine. From these results, it was suggested that most of the fixed ammonia was exported to cytosol and assimilated via glutamine synthetase to glutamine, then via glutamate synthase to glutamic acid, and from these compound...

NITROGEN ASSIMILATION IN SOYBEAN NODULES : V. Possible Pathway of Allantoin Synthesis in Soybean Nodules

The ureides, allantoin and allantoic acid, have been found to exist in a wide variety of plant species usually in very small amounts, but in some species they play an important role in the storage and transport of nitrogen (19). In soybean plants, a very high content of allantoin has been demonstrated in the stems or shoots, only when the nodulation was well progressed (3, 8, 9, 11, 12, 18). This interesting phenomenon has been discussed in relation to various aspects of soybean physiology, especially the sites and pathways of allantoin synthesis (4, 5, 7, 10, 13–15, 18) and its roles in the vegetative and reproductive growth of soybean plants (6). Using 15N as a tracer, the present authors reported direct evidence that allantoin was vigorously synthesized in nodules, and that most of the allantoin in the shoots of well nodulated soybean plants was supplied from the nodules (14, 15, 18).

A simple method for the preparation, purification, and storage of 15N2 gas for biological nitrogen fixation studies

It is very important to use 15N labeled nitrogen gas (15N2) in studies on biological nitrogen fixation. For example, 15N2 is necessary for direct measurement of the amount of fixed nitrogen, and is useful for studies on the assimilation and transport of fixed nitrogen. However, 15N2 is sometimes troublesome to deal with for the following reasons. Decline in the 15N content of 15N2 gas may occur as a result of contamination with atmospheric N2 during the storage period or application to nitrogen fixing organisms. Also, the 15N2 gas provided commercially in a glass bottle or gas cylinder is technically and economically not convenient for experiments employing small amounts of 15N2. Moreover, purification of 15N2 gas is necessary for biological research, since contamination with the oxidized forms of nitrogen represses biological nitrogen fixation to a certain extent. A simple method for the preparation, purification and storage of 15N2 gas for biological nitrogen fixation studies, was therefore devised.

Soybean Seed Production and Nitrogen Nutrition

The world population is consistently increasing, and it is over 7 billion in 2012, while the land area for agricultural use is limited. Therefore, the increase in crop production per area is very important. Soybean (Glycine max (L.) Merr.) originates from East Asia, and soybean seed is one of the most important protein sources for human and livestock all over the world. Annual production of soybean (262 M (million) t in 2010) is the fourth of the major grain crops, after maize (844 M t), paddy rice (672 M t) and wheat (650 M t) [1]. In the whole world, over 85% of soybean is used for oil and the residue is used for animal feed. Annual soybean seed production has been steadily increasing for recent decades (91 M t in 1980, 109 M t in 1990, 161 M t in 2000, 262 M t in 2010) [1]. The cultivation area of soybean is 102 M ha in 2010. Major soybean production countries (annual production in 2010) are USA (90.6 M t), Brazil (68.5 M t), Argentina (52.7 M t), China (15.1 M t), and India (9.8 M t) in this sequence. Soybean production in Japan in 2010 was only 223,000t and it accounted for 5% of the total consumption in Japan. The world average seed yield is 2.56 t ha-1 in 2010, and is higher in the USA (2.92 t ha-1), Brazil (2.94 t ha-1), and Argentina (2.90 t ha-1) compared with China (1.77 t ha-1), Japan (1.62 t ha-1) and India (1.07 t ha-1) and other countries.

Nitrogen Fixation in Sugarcane

Nitrogen (N) is a major essential element for all organisms, and generally the amount of available N (mainly inorganic nitrogen such as nitrate or ammonia) in soil is limiting factor for natural and agricultural plant production [40]. Biological nitrogen fixation (BNF) is a process by which atmospheric dinitrogen (N2) is reduced into 2 molecules of ammonia (NH3) by the enzyme nitrogenase with 8H+, 8eand 16 Mg ATP. BNF have important role in N cycle in both global ecosystem and agro-ecosystem. Based on the data compiled by Bezdicek and Kennedy in 1988 [11], about 175 million metric tons of nitrogen per year is estimated to be fixed in global ecosystems, in which 90 million metric tones in agricultural land, 50 million metric tones in forest and non-agricultural land, and 35 million metric tones in sea. At that time, nonbiological nitrogen fixation was estimated about 50 million metric tones per year by industrial nitrogen fixation mainly for the synthesis of ammonia fertilizer, and about 20 million metric tones by combustion, and about 10 million metric tones by lightening. In 2009, the production of N fertilizers increased to 106 million metric tones (FAOSTAT), but the amount of BNF still exceeds over non-biological nitrogen fixation.

Transcriptome and Metabolome Analyses Reveal That Nitrate Strongly Promotes Nitrogen and Carbon Metabolism in Soybean Roots, but Tends to Repress It in Nodules

Leguminous plants form root nodules with rhizobia that fix atmospheric dinitrogen (N2) for the nitrogen (N) nutrient. Combined nitrogen sources, particular nitrate, severely repress nodule growth and nitrogen fixation activity in soybeans (Glycine max [L.] Merr.). A microarray-based transcriptome analysis and the metabolome analysis were carried out for the roots and nodules of hydroponically grown soybean plants treated with 5 mM of nitrate for 24 h and compared with control without nitrate. Gene expression ratios of nitrate vs. the control were highly enhanced for those probesets related to nitrate transport and assimilation and carbon metabolism in the roots, but much less so in the nodules, except for the nitrate transport and asparagine synthetase. From the metabolome analysis, the concentration ratios of metabolites for the nitrate treatment vs. the control indicated that most of the amino acids, phosphorous-compounds and organic acids in roots were increased about twofold in the roots, whereas in the nodules most of the concentrations of the amino acids, P-compounds and organic acids were decreased while asparagine increased exceptionally. These results may support the hypothesis that nitrate primarily promotes nitrogen and carbon metabolism in the roots, but mainly represses this metabolism in the nodules.

Role of Nitrogen on Growth and Seed Yield of Soybean and a New Fertilization Technique to Promote Nitrogen Fixation and Seed Yield

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 N2 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.