**3. Characteristics of nitrogen nutrition of soybean**

### **3.1. Characteristics of nitrogen nutrition related to seed yield**

Soybean seed contains an extraordinary high concentration of protein about 35-40% based on the seed weight. Many field researches showed the soybean seed yield is proportional to the total assimilated N in plants. Figure 10 shows the relationship between total amounts of N in soybean shoot at the R7 stage and seed yield in rotated paddy field in Nagakura from 1989-1991 [21]. The seed yield exhibited a linear correlation (r=0.855) with the amount of ni‐ trogen accumulation.

times more than in the case of rice [13]. Soybean plants assimilate the N from three sources, 1) N derived from symbiotic N2 fixation by root nodules (Ndfa), 2) absorbed N from soil mineralized N (Ndfs), and 3) N derived from fertilizer when applied (Ndff) (Figure 8). For the maximum seed yield of soybean, it is necessary to use both N2 fixation and absorbed N from roots [14-15]. When only N2 fixation is available to the plant vigorous vegetative growth does not occur, which results in reduced seed yield. On the other hand, a heavy sup‐ ply of N often depresses nodule development and N2 fixation activity and induces nodule senescence, which also results in reduced seed yield. In addition, a heavy supply of N from fertilizer or from the soil causes luxuriant shoot growth, which result in lodging and poor pod formation. Therefore, for soybean cultivation no nitrogen fertilizer is applied or only a

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

small amount of N fertilizer is applied as a "starter N" to promote the initial growth.

**Figure 8.** Three sources of nitrogen for soybean plants, nitrogen fixed in root nodules, nitrogen absorbed by roots

In Niigata fields, about 60-75% of N assimilation in soybean was estimated to derive from N2 fixation [16,17]. Figure 9 shows the growth of cultivar "Enrei" and the non-nodulated iso‐ genic line "En1282" planted in the same Nagakura field. It is obvious that non-nodulated soybean grew very poor with pale leaf color due to N deficiency by the lack of nitrogen fixa‐ tion. It is said that the legume nitrogen fixation is variable, but it is a valuable process in

Soybean seed contains an extraordinary high concentration of protein about 35-40% based on the seed weight. Many field researches showed the soybean seed yield is proportional to

**3. Characteristics of nitrogen nutrition of soybean**

**3.1. Characteristics of nitrogen nutrition related to seed yield**

from fertilizer and from soil nitrogen.

Relationships

122

world agriculture [18-20].

**Figure 9.** Comparison of the growth of nodulating soybean cv. "Enrei" (left) and the non-nodulated mutant "En1282" (right) cultivated in Nagakura field.

**Figure 10.** Relationship between amount of nitrogen accumulated in soybean shoot at R7 stage and seed yield of soybean cv. "Enrei". In Nagakura field with various fertilizer treatments.[21]

The protein concentration in soybean seeds is about 4 times higher than cereal seeds such as rice grain (Figure 11) [12]. Due to a high concentration of seed protein, 1t of soybean seed production requires about 70-90 kg of N, while 1t of rice grain requires only 20 kg of N. Soy‐

bean plants assimilate about 20 % of total N until initial flowering stage (R1 stage), and 80% of N during the reproductive stage. On the other hand, rice assimilates about 80% of N until flowering. Therefore, the continuous assimilation of nitrogen after initial flowering stage is essential for good growth and high seed yield in soybean cultivation.

**Figure 11.** Comparison of the nitrogen assimilation and distribution pattern of soybean and rice.

To obtain high seed yield of soybean, good nodulation and high and long lasting nitrogen fixation activity are very important (Figure 12). Nodule formation and nodule growth are influenced by various soil conditions (water content, pH, nutrition) and climatic conditions (solar radiation, temperature, rain fall etc). Soybean can fix atmospheric N2 by their root nodules associated with soil bacteria, bradyrhizobia. In addition, soybean can absorb inor‐ ganic nitrogen, such as nitrate and ammonia from soil or fertilizer. Usually a high yield of soybean was obtained in a field with high soil fertility. By supplying a constant but low con‐ centration of nitrogen either from soil or organic manure, soybean growth will occur with‐ out depressing nodulation and nitrogen fixation activity. However, it is well known that a high concentration of mineral N depresses nodule formation and nitrogen fixation activity. Especially, nitrate, the most abundant inorganic nitrogen in upland fields, severely inhibits nodulation and nitrogen fixation of soybean, when nodulated roots are in direct contact with the soil solution containing nitrate [22-24].

**Figure 12.** Comparison of the time course of nitrogen assimilation in soybean plants with a low yield (left) and a high yield (right).

**Figure 13.** Chemical formula of ureides (allantoin and allantoic acid), nitrate and asparagine.

#### **3.2. Nitrogen assimilation in nodules**

bean plants assimilate about 20 % of total N until initial flowering stage (R1 stage), and 80% of N during the reproductive stage. On the other hand, rice assimilates about 80% of N until flowering. Therefore, the continuous assimilation of nitrogen after initial flowering stage is

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

essential for good growth and high seed yield in soybean cultivation.

Relationships

124

**Figure 11.** Comparison of the nitrogen assimilation and distribution pattern of soybean and rice.

with the soil solution containing nitrate [22-24].

To obtain high seed yield of soybean, good nodulation and high and long lasting nitrogen fixation activity are very important (Figure 12). Nodule formation and nodule growth are influenced by various soil conditions (water content, pH, nutrition) and climatic conditions (solar radiation, temperature, rain fall etc). Soybean can fix atmospheric N2 by their root nodules associated with soil bacteria, bradyrhizobia. In addition, soybean can absorb inor‐ ganic nitrogen, such as nitrate and ammonia from soil or fertilizer. Usually a high yield of soybean was obtained in a field with high soil fertility. By supplying a constant but low con‐ centration of nitrogen either from soil or organic manure, soybean growth will occur with‐ out depressing nodulation and nitrogen fixation activity. However, it is well known that a high concentration of mineral N depresses nodule formation and nitrogen fixation activity. Especially, nitrate, the most abundant inorganic nitrogen in upland fields, severely inhibits nodulation and nitrogen fixation of soybean, when nodulated roots are in direct contact

Ammonia is known to be the initial product of biological nitrogen fixation by the enzyme nitrogenase [25]. After discovering an enzyme glutamate synthase (GOGAT) in *Aerobacter aerogenes* [26], it was confirmed that ammonia can be assimilated via glutamine synthetase (GS) and glutamate synthase (GOGAT) pathway in soybean nodules by15N tracer experi‐

ments [27,28]. The 15N assimilation was investigated in the cytosol (plant cytoplasm) and bacteroid fractions of soybean nodules [29]. The result suggested that most of the fixed N is immediately exported from the bacteroid to the plant cytosol and assimilated via GS/ GOGAT pathway into various amino acids via transamination from glutamate. Ureides, al‐ lantoin and allantoic acid are synthesized from amino acids and amides in the cytosol (Fig‐ ure 13,14). Kushizaki et al. discovered that nodulated soybean plants contain a large amount of ureides in their stems, while non-nodulating isolines contain much less [30]. Reviews on ureide biosynthesis in legume nodules were published [31,32].

**Figure 14.** A model of the flow of fixed N2 in infected cell and uninfected cell of soybean nodule.

### **3.3. Nitrogen absorption and assimilation in soybean roots**

The outlines of absorption and metabolism of ammonium and nitrate in plant cells are shown in Figure 15. Ammonium (NH4 + ) and nitrate (NO3 - ) are major sources of inorganic ni‐ trogen in soil. Ammonium is the most reduced form of nitrogen and nitrate is the most oxi‐ dized form. The NH4 + ion is absorbed through the membrane bound protein, ammonium transporter. The NO3 ion is absorbed through the nitrate transporter with 2H+ co-transport. There are two types of nitrate transporter, a high affinity nitrate transporter system (HATS) and a low affinity nitrate transporter system (LATS) [33]. The kinetics of the absorption rate versus nitrate concentration indicated the presence of only one HATS, having a Km value of 19 µ mole in soybean roots.

**Figure 15.** A model of absorption and metabolism of ammonium and nitrate in plant cell.

ments [27,28]. The 15N assimilation was investigated in the cytosol (plant cytoplasm) and bacteroid fractions of soybean nodules [29]. The result suggested that most of the fixed N is immediately exported from the bacteroid to the plant cytosol and assimilated via GS/ GOGAT pathway into various amino acids via transamination from glutamate. Ureides, al‐ lantoin and allantoic acid are synthesized from amino acids and amides in the cytosol (Fig‐ ure 13,14). Kushizaki et al. discovered that nodulated soybean plants contain a large amount of ureides in their stems, while non-nodulating isolines contain much less [30]. Reviews on

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

ureide biosynthesis in legume nodules were published [31,32].

**Figure 14.** A model of the flow of fixed N2 in infected cell and uninfected cell of soybean nodule.

+

The outlines of absorption and metabolism of ammonium and nitrate in plant cells are

trogen in soil. Ammonium is the most reduced form of nitrogen and nitrate is the most oxi‐

There are two types of nitrate transporter, a high affinity nitrate transporter system (HATS) and a low affinity nitrate transporter system (LATS) [33]. The kinetics of the absorption rate versus nitrate concentration indicated the presence of only one HATS, having a Km value of

) and nitrate (NO3


ion is absorbed through the membrane bound protein, ammonium

ion is absorbed through the nitrate transporter with 2H+ co-transport.

) are major sources of inorganic ni‐

**3.3. Nitrogen absorption and assimilation in soybean roots**

shown in Figure 15. Ammonium (NH4

+


dized form. The NH4

Relationships

126

transporter. The NO3

19 µ mole in soybean roots.

The diurnal rhythm in NO3 absorption by intact soybean plants was investigated by sam‐ pling the culture solution every 15 min [34]. The NO3 - absorption rate was different between day (1.10 mgN L-1 h-1) and night period (0.77 mgN L-1 h-1), and the nitrate absorption rate at night was about 60-75% of that in the daytime. The temporary interruption of NO3 - absorp‐ tion was observed twice a day at dawn and dusk. The changes in NO3 - absorption rate were not controlled by the shoots, because the rhythm continued under the extended dark period or by cutting the shoots [34]. When the roots were put in the water bath at a constant tem‐ perature of 30o C, the rhythm of NO3 absorption disappeared. This suggests that the nitrate absorption rate of soybean roots may be controlled by monitoring temperature changes by the root.

Some parts of the NO3 - absorbed in the root cell is reduced to nitrite (NO2 - ) by nitrate reduc‐ tase (NR) in the cytosol, and NO2 is reduced to ammonia by nitrite reductase (NiR) in the plastids. Then amino acids are formed followed by the assimilation via GS/GOGAT path‐ way. When a high concentration of NO3 is supplied, a part of NO3 - is temporarily stored in the vacuoles of cortex cells in roots. Some parts of NO3 are transported cell to cell via the symplast pathway and effluxed in the stele and transported via the xylem with the transpi‐ ration stream in the form of NO3 - . Plant NR requires NADH or NAD(P)H which means both NADH and NADPH act as electron donors. In soybean, there are two types of NAD(P)H-NR and one type of NADH-NR [15].

After adding 15NO3 - in the solution, the 15N concentration of asparagine increased markedly, indicating that asparagine is a major assimilatory compound of NO3 - in soybean roots. Ni‐

trogen assimilation and transport of plants supplied with 15NO3 was investigated by analyz‐ ing xylem sap collected from decapitated soybean plants [35]. Some part of the NO3 absorbed in the roots was immediately exported to the shoots, whereas another parts of the NO3 was temporarily stored in the vacuoles of root cells and then gradually released to the xylem. On the other hand, some other parts of the NO3 was reduced and assimilated in the roots and synthesized into asparagine.

**Figure 16.** Comparison of the fate of fixed N2 in nodules and absorbed nitrate in roots.

#### **3.4. Comparison of the fate of fixed N2 and absorbed NO3 in soybean plant**

The labeling patterns of ureides and amino acids were compared from the data of 15N2 and 15NO3 feedings [36]. The result proved that the ureides in stems are mainly derived from fixed N2, and only a small amount of ureides is synthesized in the root. Figure 16 shows the metabolic pathways and transport of N derived from N2 fixation and NO3 absorption in soybean plants.

#### **3.5. Effect of combined nitrogen on nodule growth and nitrogen fixation activity**

It is well known that a heavy supply of nitrogen fertilizer causes the inhibition of nodulation and nitrogen fixation. The inhibitory effect of combined nitrogen depends on the forms, con‐ centration, application site, and soybean growth stage. The inhibitory effect of nitrate is stronger than urea or ammonium. Direct and indirect effects of nitrate have been known. Di‐ rect or local effect of nitrate is the effect of nitrate in direct contact with the nodulated part of roots. When nitrate was supplied to the hydroponically grown soybean roots, the nitrate in‐ hibition on nodule growth and nitrogen fixation was shown to be rapid and reversible [22-24]. On the other hand, indirect or systemic effect of nitrate, which means the effect of nitrate absorbed from distant part of the roots, depended on the site, duration and concen‐ trations of nitrate supply [37,38]. The indirect effect of nitrate was investigated by a two-lay‐ ered pot system separating the upper roots and lower roots. A continuous high concentration of nitrate (5mM) supply in the lower roots depressed the nodulation and ni‐ trogen fixation of the upper roots. However, a continuous supply of a low concentration of nitrate (1 mM) resulted in the promotion of nodulation and nitrogen fixation activity in the upper roots [38].

#### **3.6. Nitrogen metabolism in soybean leaves**

trogen assimilation and transport of plants supplied with 15NO3

**Figure 16.** Comparison of the fate of fixed N2 in nodules and absorbed nitrate in roots.

metabolic pathways and transport of N derived from N2 fixation and NO3

**3.5. Effect of combined nitrogen on nodule growth and nitrogen fixation activity**

**3.4. Comparison of the fate of fixed N2 and absorbed NO3**

xylem. On the other hand, some other parts of the NO3

roots and synthesized into asparagine.

NO3 -

Relationships

128

15NO3 -

soybean plants.

ing xylem sap collected from decapitated soybean plants [35]. Some part of the NO3

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

absorbed in the roots was immediately exported to the shoots, whereas another parts of the

was temporarily stored in the vacuoles of root cells and then gradually released to the


**-**

The labeling patterns of ureides and amino acids were compared from the data of 15N2 and

It is well known that a heavy supply of nitrogen fertilizer causes the inhibition of nodulation and nitrogen fixation. The inhibitory effect of combined nitrogen depends on the forms, con‐ centration, application site, and soybean growth stage. The inhibitory effect of nitrate is stronger than urea or ammonium. Direct and indirect effects of nitrate have been known. Di‐ rect or local effect of nitrate is the effect of nitrate in direct contact with the nodulated part of roots. When nitrate was supplied to the hydroponically grown soybean roots, the nitrate in‐

 feedings [36]. The result proved that the ureides in stems are mainly derived from fixed N2, and only a small amount of ureides is synthesized in the root. Figure 16 shows the

 **in soybean plant**


absorption in


was investigated by analyz‐

was reduced and assimilated in the


Plant leaves are the important organ for nitrogen metabolism, in addition to photosynthetic activity. The N absorbed from roots or fixed in root nodules is transported via the xylem in stems and petioles, and the N in leaves is translocated to the sink organs such as pods and seeds via the phloem. The flow of N in leaves was investigated by petiole girdling and 15N2 or 15NO3 treatment [39]. By petiole girdling treatment, the accumulation of amino acids (x 2.5) especially asparagine (x 8.8) in leaf blades was observed, indicating that these com‐ pounds are the transport forms from leaves to sink organ via the phloem. However, nitrate and ureides are not accumulated in girdled leaves compared with intact leaves, suggesting that nitrate and ureides are not transported from leaves to sinks via the phloem (Figure 17). There are two different ureide degradation pathways in soybean leaves, either by allantoate amidinohyfrolase or by allantoate amidohydrolase [40].

**Figure 17.** A model of N flow in a soybean leaf.

A comparative study on the nitrogen metabolism and transport was done at the pod fill‐ ing stage by 15N2 or 15NO3 treatment [41]. Based on the results obtained, we proposed a model of N flow derived from N2 and NO3 in soybean plants as shown in Figure 18 [42]. The N derived from N2 fixed by the root nodules is rapidly assimilated into ureides (allan‐ toin and allantoate), and some ureides are directly transported to pods and used for seed development. Ureides are also used for leaf protein synthesis, but the contribution is rela‐ tively lower than N derived from NO3 absorbed from the roots. On the other hand, some part of NO3 absorbed from the roots is immediately reduced in the roots, and transported in the form of amino acids, especially asparagine. Another part of NO3 is transported to the leaf blades via transpiration, and assimilated into leaf protein. The remobilization of storage protein in leaves and roots may be a major source for seed N source in the case of NO3 nutrition.

**Figure 18.** A model of N flow in soybean plant originated from N2 (left) and NO3 - (Right). AA: amino acids, P: protein

#### **3.6. Assimilation of nitrogen in pods and seeds**

Figure 19 shows the outline of the N flow in soybean pod and seed (cotyledon). Ureides are transported from the root nodules via the xylem and are accumulated in the pod. Al‐ lantoin and allantoic acids are metabolized into amino acids in the pod or seed coat and excreted to the inside of seed coat. Asparagine from roots via the xylem or from leaves via phloem is also metabolized to amino acids and then transported into the apoplast space between the seed coat and cotyledon. The cotyledon cells absorb amino acids from the apoplast and they synthesize storage proteins and accumulate them into protein bodies. Rainbird et al. reported that glutamine is the principal N supply to the cotyledon, contri‐ buting 55% of the embryo nitrogen requirement, and 20% comes from asparagine, with negligible amounts from ureides, allantoin and allantoic acid. Haga and Sodek also report‐ ed that glutamine was the most efficient source in terms of protein accumulation in the cultured soybean cotyledons, while asparagine was less efficient and allantoin was a poor source of nitrogen. Ohtake et al. reported that the rapid N transport to pods and seeds in N-deficient soybean plants were faster compared with N-sufficient plants.

**Figure 19.** The model of nitrogen assimilation in soybean pod and seed.

A comparative study on the nitrogen metabolism and transport was done at the pod fill‐

The N derived from N2 fixed by the root nodules is rapidly assimilated into ureides (allan‐ toin and allantoate), and some ureides are directly transported to pods and used for seed development. Ureides are also used for leaf protein synthesis, but the contribution is rela‐

the leaf blades via transpiration, and assimilated into leaf protein. The remobilization of storage protein in leaves and roots may be a major source for seed N source in the case of

Figure 19 shows the outline of the N flow in soybean pod and seed (cotyledon). Ureides are transported from the root nodules via the xylem and are accumulated in the pod. Al‐ lantoin and allantoic acids are metabolized into amino acids in the pod or seed coat and excreted to the inside of seed coat. Asparagine from roots via the xylem or from leaves via phloem is also metabolized to amino acids and then transported into the apoplast space between the seed coat and cotyledon. The cotyledon cells absorb amino acids from the apoplast and they synthesize storage proteins and accumulate them into protein bodies.

absorbed from the roots is immediately reduced in the roots, and transported


A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen


in the form of amino acids, especially asparagine. Another part of NO3

**Figure 18.** A model of N flow in soybean plant originated from N2 (left) and NO3

**3.6. Assimilation of nitrogen in pods and seeds**

treatment [41]. Based on the results obtained, we proposed a

in soybean plants as shown in Figure 18 [42].


is transported to

absorbed from the roots. On the other hand, some


(Right). AA: amino acids, P: protein

ing stage by 15N2 or 15NO3


nutrition.

part of NO3

Relationships

130

NO3 - -

model of N flow derived from N2 and NO3

tively lower than N derived from NO3

The storage protein of soybean seeds mainly consists of glycinin and β-conglycinin. β-con‐ glycinin is comprised of three subunits, designated as α', α, and β−subunits [46]. We hap‐ pened to discover the lack of β-subunit of β-conglycinin in several non-nodulated soybean lines, although an electrophoretic protein band due to this protein was clearly detected in the corresponding nodulated isolines [47]. The suppression of the β−subunit in the non-nod‐ ulating isoline T201 is regulated at the level of mRNA accumulation. The α'−and α−subunits mRNAs were actively expressed in both line. Nitrogen regulation for storage protein in soy‐ bean seeds was evaluated using T201 and T202 with solution culture in the greenhouse [48]. The results indicated that non-nodulated T201 has a normal, non-defective, β−subunit genes, and that limited N availability decreases the accumulation of β−conglycinin.
