**2.1 Materials and method**

Seeds of Ilsan radish (*Raphanus sativus*) soaked for 6 h allowed to germinate on paper towels were soaked in water in the dark. After 5 days the seedlings were transferred to 50 mL plastic tubes containing 10 mL inorganic nutrient solution. The nutrient solution was renewed every day. The composition of the inorganic nutrient solution is given in Table 2. Iron (Fe-EDTA), boron (H3BO3), manganese (MnCl24H2O), zinc (ZnSO47H2O), copper (CuSO45H2O) and molybdenum (H2MoO4H2O) were supplied to all treatments at rates of 40, 460, 90, 7.7, 3.2 and 0.1 M, respectively. Seedlings were grown in a growth chamber maintained at 25°C, 70–80% relative humidity, with a 14 h light/10 h dark cycle and a light intensity of 300 mol m–2s–1.


Table 2. The main compositions of the nutrient solution for hydroponic experiment (mM)

The mixed amino acids (MAA) solution contained 7 equal concentrations of amino acids were as follows: alanine (Ala), β–alanine (β–Ala), aspartic acid (Asp), asparagine (Asn), glutamic acid (Glu), glutamine (Gln) and glycine (Gly). After 10 days, radish seedlings were placed in 10 ml inorganic nutrient solution containing 5.0 mM NO3– and 0, 0.3 or 3.0 mM MAA, as indicated in Table 3. The pH of the nutrient solutions were maintained between 6.0–6.1 by adding 1.0 M KOH appropriately. The nutrient solutions were renewed at 4, 8 and 16 h, respectively. The choice of the levels of MAA and renewed time of the nutrient solutions were according to the study of Kim (2002)

acids (MAA) under the conventional fertilization. These two plants were selected because

in this experiment were alanine (Ala), β–alanine (β–Ala), aspartic acid (Asp), asparagine (Asn), glutamic acid (Glu), glutamine (Gln) and glycine (Gly). These amino acids were selected for the reasons include: (1) their structural role in proteins, (2) significant effect on NO3– uptake which was found in many works, and (3) considerable amounts in plant phloem and xylem (Caputo and Barneix, 1997; Lohaus et al., 1997; Peeters and Van Laere,

In the frame of the studies on the effect of the mixed amino acids (MAA) on nitrate uptake and assimilation, the pot experiments were focused on the role of MAA in process of NO3– uptake and assimilation. In order to distinguish the origin of N in radish, 15N labeled nitrate

In order to develop an approach for more efficient N fertilizer use and to prevent environmental pollution due to nitrate leaching, the aim of the study presented here, is to investigate the effect of amino acid fertilizer (AAF) on nitrate removal in high nitrate soils.

Seeds of Ilsan radish (*Raphanus sativus*) soaked for 6 h allowed to germinate on paper towels were soaked in water in the dark. After 5 days the seedlings were transferred to 50 mL plastic tubes containing 10 mL inorganic nutrient solution. The nutrient solution was renewed every day. The composition of the inorganic nutrient solution is given in Table 2. Iron (Fe-EDTA), boron (H3BO3), manganese (MnCl24H2O), zinc (ZnSO47H2O), copper (CuSO45H2O) and molybdenum (H2MoO4H2O) were supplied to all treatments at rates of 40, 460, 90, 7.7, 3.2 and 0.1 M, respectively. Seedlings were grown in a growth chamber maintained at 25°C, 70–80% relative humidity, with a 14 h light/10 h dark cycle and a light

**Chemicals K+ NO3– Ca2+ H2PO4– Mg2+ SO42–** 

MgSO4 0.50 0.50 Total 1.50 3.75 1.25 0.25 0.50 0.50 Table 2. The main compositions of the nutrient solution for hydroponic experiment (mM)

The mixed amino acids (MAA) solution contained 7 equal concentrations of amino acids were as follows: alanine (Ala), β–alanine (β–Ala), aspartic acid (Asp), asparagine (Asn), glutamic acid (Glu), glutamine (Gln) and glycine (Gly). After 10 days, radish seedlings were placed in 10 ml inorganic nutrient solution containing 5.0 mM NO3– and 0, 0.3 or 3.0 mM MAA, as indicated in Table 3. The pH of the nutrient solutions were maintained between 6.0–6.1 by adding 1.0 M KOH appropriately. The nutrient solutions were renewed at 4, 8 and 16 h, respectively. The choice of the levels of MAA and renewed time of the nutrient

+ preferred crop. The amino acids used

radish is NO3– preferred crop and red pepper is NH4

**2. Hydroponic experiment of radish** 

1994; Winter et al., 1992).

**2.1 Materials and method** 

intensity of 300 mol m–2s–1.

KNO3 1.25 1.25

Ca(NO3)2 2.50 1.25

solutions were according to the study of Kim (2002)

KH2PO4 0.25 0.25

was used.


Table 3. The compositions of the treatment solutions for radish in hydroponic experiment (mM)

Plants were harvested 24 h after treatment and separated into roots and shoots for enzymes assay and N content analysis. Net NO3– uptake rates were determined by amount of NO3– disappeared from the initially treated solution.

#### **2.2 Results and discussion**

#### **2.2.1 Effect on NO3 – uptake**

The MAA treatments showed different effect on nitrate uptake depending on the concentrations (Fig. 2). The NO3– uptake in treatment A1 was similar to that of A0 after 8 h exposure to NO3–. However, exposure for longer hours (16 or 24 h) to 0.3 mM MAA inhibited the NO3– uptake by 38% compared with A0. In contrast, the highest NO3 – uptake was found in treatment A2 that showed 305% higher than A0.

Fig. 2. Effect of mixed amino acids on the nitrate uptake in radish supplied with 5.0 mM NO3–. Values are means ± SD (n=5).

Several authors reported that free amino acids could down regulate NO3 – uptake. It was found that exogenously supplied amino acids and amides could decrease the uptake of NO3– by soybean (Muller and Touraine, 1992); wheat (Rodgers and Barneix, 1993); maize (Ivashikian and Sokolov, 1997; Padgett and Leonard, 1996; Sivasankar et al., 1997); barley (Aslam et al., 2001). In this experiment, the effectiveness of the MAA treatments on NO3– uptake was similar to above references at low MAA treatment rate (0.3 mM MAA, Fig. 2). However, contrary result was found at high MAA treatment rate (3.0 mM MAA, Fig. 2), in

Effect of Mixed Amino Acids on Crop Growth 127

As interim product of NO3– assimilation procedure, the concentration of NO2– depended on

(Table 4) was due to high NR activity (Fig. 3), and the lowest concentration of NO2– in shoots in A1 (Table 4) was due to low NR activity (Fig. 3) too. However, low NiR activity (Fig. 4) led to a blocking of the reduction of NO2– to NH4+ in roots in A1, so that

concentration and high concentration of MAA treatments led to different effects on NR activity (Fig. 3). Both in the shoots and in the roots NR activities were inhibited slightly in A1. Significant increases of NR activities were found in A2 treatment, with 75% in shoots

Shoot Root

<sup>b</sup> <sup>b</sup> <sup>a</sup>

– is reduced to NO2– by catalysis of NR. In this experiment, low

a

– found in A2

the reduction rate of nitrate and nitrite. The highest concentration of NO2

A0 A1 A2

b b

Fig. 3. Effect of mixed amino acids on nitrate reductase activity in radish at 24 h after

There are contradictory results for the possible regulation of NR activity by amino acids for higher plants. For example, Radin (1975, 1977) has shown that the reduction of NO3– to NO2– in cotton roots is inhibited by specific amino acids. On the other hand, Oaks et al. (1979) have found that amino acids inhibited minor levels of NR in both intact and excised corn roots using an *in vitro* assay. Aslam *et al.,* (2001) reported that the amino acids partially inhibited the increase of NR activity in barley roots where most NO3– uptake was facilitated via high affinity transport system (HATS) but had little effect where low affinity transport system (LATS) is operative. It has been suggested that glutamate inhibited NR activity in roots, but no inhibition in shoots (Ivashikian and Sokolov, 1997). Sivasankar et al. (1997) observed that Gln and asparagine (Asn) inhibited the induction of NR activity in corn roots at both 250 M and 5 mM of external NO3– supply. They concluded that inhibition was not the result of altered NO3– uptake, and tissue nitrate accumulation was reduced at 250 M external nitrate in the presence of 1 mM Asn, but

concentration of NO2– showed higher than A0 (Table 4).

**2.2.3 Effect on NRA, NiRA and GSA** 

NRA (

0.0

treatment. Values are means ± SD (n=5).

not at 5 mM Asn.

0.2

0.4

0.6

0.8

1.0

1.2

mol NO2


g-1 FW h-1)

and 340% in roots respectively, relative to A0.

For NO3– assimilation, NO3

which NO3– uptake was 4–fold higher than the control. This result was similar to rice, pea, cucumber and red pepper, which were treated with 5.0 mM MAA (Kim, 2002).

The effect on nitrate uptake seems to respond to kinds and concentration of amino acids. Muller and Touraine (1992) had examined the effect of 14 different amino acids on nitrate uptake in soybean seedlings supplied with 0.5 mM nitrate. After 10 mM single amino acid pretreatment, about half of the tested amino acids had a substantial inhibitory effect on nitrate uptake, mainly Ala, Glu (almost 100% inhibition), Asn and Arg (about 80%), and Asp, Ala, Scr, and Gln (from 70% to 48%). However, when supplied at 100 mM amino acid to the tip−cut cotyledons, only eight of fourteen amino acids had inhibitory effect, and four amino acids had enhanced nitrate uptake.

#### **2.2.2 Effect on NO3 – and NO2 – accumulation**

The application of MAA increased the NO3– concentrations both in shoots and in roots regardless of application rates (Table 4), resulting in the highest concentration in A1 and the lowest concentration in A0. The high concentration of NO3 – in A2 was attributed to the high NR activity (Fig. 3). Although A1 treatment showed the lowest uptake of NO3– (Fig. 2), the highest concentration of NO3– was found by the reason of that low NR activity in A1 (Fig. 3) led to a blocking of the reduction of NO3 – to NO2 –. With respect to the NO2 – values (Table 4), in our experiments, the highest NO2– concentrations in both shoots and roots were found in the A2. In shoots, the lowest NO2– concentration was found in A1 and the lowest in A0 in roots.


Data are means ± SD (n=5). Analysis of variance (ANOVA) was employed followed by Duncan's new multi range test. Values with similar superscripts are not significantly different (P>0.05).

Table 4. Effect of mixed amino acids on NO3 – and NO2– concentration in fresh weight of radish at 24 h after treatment

Although many authors agree that amino acid can negatively regulate nitrate content in higher plants (Chen and Gao, 2002; Gunes et al., 1994, 1996; Wang et al., 2004), the results in the present experiment do not support this interpretation. Both in shoots and in roots, the MAA used in this study led to little increase of NO3 – concentrations (Table 4). The contradiction may reside in treatment method and treatment period of amino acids. It was demonstrated in other studies that amino acid pretreatment decreased NO3– accumulation slightly, but Gln and Asn increased the NO3– concentration in barley roots when they were used together with nitrate (Aslam et al., 2001). The reason of difference between this experiment and others is that NO3– content of shoots includes portion of NO3 – in xylem sap. Concentrations of NO3– in xylem sap can be quite high, especially in plants that transport most of the NO3– taken up to the shoot for reduction (e.g., maize 10.5 mM, Oaks, 1986; barley 27 to 34 mM, Lews et al., 1982).

The effect on nitrate uptake seems to respond to kinds and concentration of amino acids. Muller and Touraine (1992) had examined the effect of 14 different amino acids on nitrate uptake in soybean seedlings supplied with 0.5 mM nitrate. After 10 mM single amino acid pretreatment, about half of the tested amino acids had a substantial inhibitory effect on nitrate uptake, mainly Ala, Glu (almost 100% inhibition), Asn and Arg (about 80%), and Asp, Ala, Scr, and Gln (from 70% to 48%). However, when supplied at 100 mM amino acid to the tip−cut cotyledons, only eight of fourteen amino acids had inhibitory effect, and four

regardless of application rates (Table 4), resulting in the highest concentration in A1 and the lowest concentration in A0. The high concentration of NO3– in A2 was attributed to the high NR activity (Fig. 3). Although A1 treatment showed the lowest uptake of NO3– (Fig. 2), the highest concentration of NO3– was found by the reason of that low NR activity in A1 (Fig. 3)

in our experiments, the highest NO2– concentrations in both shoots and roots were found in the A2. In shoots, the lowest NO2– concentration was found in A1 and the lowest in A0 in

A0 62.47 ± 4.06 a 16.30 ±1.88 b 6.76 ± 0.62 b 11.43 ± 1.67 c A1 67.73 ± 7.49 a 22.99 ±2.23 a 3.77 ± 0.34 c 17.14 ± 2.10 b A2 63.37 ± 3.58 a 17.41 ±1.92 b 29.70 ± 2.78 a 30.39 ± 4.13 a Data are means ± SD (n=5). Analysis of variance (ANOVA) was employed followed by Duncan's new

Although many authors agree that amino acid can negatively regulate nitrate content in higher plants (Chen and Gao, 2002; Gunes et al., 1994, 1996; Wang et al., 2004), the results in the present experiment do not support this interpretation. Both in shoots and in roots, the MAA used in this study led to little increase of NO3– concentrations (Table 4). The contradiction may reside in treatment method and treatment period of amino acids. It was demonstrated in other studies that amino acid pretreatment decreased NO3– accumulation

used together with nitrate (Aslam et al., 2001). The reason of difference between this experiment and others is that NO3– content of shoots includes portion of NO3– in xylem sap. Concentrations of NO3– in xylem sap can be quite high, especially in plants that transport most of the NO3– taken up to the shoot for reduction (e.g., maize 10.5 mM, Oaks, 1986;

**Shoot Root Shoot Root** 

**Treatments NO3– (mol g–1) NO2– (nmol g–1)** 

multi range test. Values with similar superscripts are not significantly different (P>0.05).

cucumber and red pepper, which were treated with 5.0 mM MAA (Kim, 2002).

 **accumulation** 

– uptake was 4–fold higher than the control. This result was similar to rice, pea,

– concentrations both in shoots and in roots

– to NO2–. With respect to the NO2– values (Table 4),

– and NO2– concentration in fresh weight of

– concentration in barley roots when they were

which NO3

**2.2.2 Effect on NO3**

roots.

amino acids had enhanced nitrate uptake.

**–**

led to a blocking of the reduction of NO3

Table 4. Effect of mixed amino acids on NO3

slightly, but Gln and Asn increased the NO3

barley 27 to 34 mM, Lews et al., 1982).

radish at 24 h after treatment

The application of MAA increased the NO3

 **and NO2**

**–**

As interim product of NO3– assimilation procedure, the concentration of NO2 – depended on the reduction rate of nitrate and nitrite. The highest concentration of NO2 – found in A2 (Table 4) was due to high NR activity (Fig. 3), and the lowest concentration of NO2– in shoots in A1 (Table 4) was due to low NR activity (Fig. 3) too. However, low NiR activity (Fig. 4) led to a blocking of the reduction of NO2– to NH4+ in roots in A1, so that concentration of NO2– showed higher than A0 (Table 4).

#### **2.2.3 Effect on NRA, NiRA and GSA**

For NO3– assimilation, NO3– is reduced to NO2 – by catalysis of NR. In this experiment, low concentration and high concentration of MAA treatments led to different effects on NR activity (Fig. 3). Both in the shoots and in the roots NR activities were inhibited slightly in A1. Significant increases of NR activities were found in A2 treatment, with 75% in shoots and 340% in roots respectively, relative to A0.

Fig. 3. Effect of mixed amino acids on nitrate reductase activity in radish at 24 h after treatment. Values are means ± SD (n=5).

There are contradictory results for the possible regulation of NR activity by amino acids for higher plants. For example, Radin (1975, 1977) has shown that the reduction of NO3– to NO2– in cotton roots is inhibited by specific amino acids. On the other hand, Oaks et al. (1979) have found that amino acids inhibited minor levels of NR in both intact and excised corn roots using an *in vitro* assay. Aslam *et al.,* (2001) reported that the amino acids partially inhibited the increase of NR activity in barley roots where most NO3– uptake was facilitated via high affinity transport system (HATS) but had little effect where low affinity transport system (LATS) is operative. It has been suggested that glutamate inhibited NR activity in roots, but no inhibition in shoots (Ivashikian and Sokolov, 1997). Sivasankar et al. (1997) observed that Gln and asparagine (Asn) inhibited the induction of NR activity in corn roots at both 250 M and 5 mM of external NO3– supply. They concluded that inhibition was not the result of altered NO3– uptake, and tissue nitrate accumulation was reduced at 250 M external nitrate in the presence of 1 mM Asn, but not at 5 mM Asn.

The NH4

and the NO3

shoots, presumably NO3

GSA (

0

**3. Hydroponic experiment of red pepper** 

treatment. Values are means ± SD (n=5).

with hydroponic experiment of radish.

nutrient solutions were renewed at 4, 8, and 16 h, respectively.

**3.1 Materials and methods** 

mM NO3

10

20

30

40

50

mol C5H10

N

O2

4 g-1 FW h-1)

Effect of Mixed Amino Acids on Crop Growth 129

primarily by the enzyme GS. In the present experiment, GS activity was inhibited by 0.3 mM MAA treatment in radish roots, whereas 3.0 mM of MAA treatment enhanced the activity (Fig.

was regulated by MAA in radish, especially at high concentration of MAA treatment. In conclusion, the application of high MAA rates (principally A2) could be the direct cause of

Shoot Root

Fig. 5. Effect of mixed amino acids on glutamine synthetase activity in radish at 24 h after

Seeds of Chongok red pepper (*Capsicum annuum*) were sown in February 2005. The seedlings were grown in individual pots filled with commercialized artificial soil in an experimental greenhouse for 35 days and then transferred to 50 mL plastic tubes containing 20 mL inorganic nutrient solution. The nutrient solution was renewed every day. The composition of the inorganic nutrient solution and the cultural condition were the same

The mixed amino acids (MAA) solution was the same with that used in hydroponic experiment of radish which contained 7 equal concentrations of amino acids. At 7 days after transferring, red pepper seedlings were placed in inorganic nutrient solution containing 1.0

solutions were maintained between 6.0–6.1 by adding 1.0 M KOH appropriately. The

– and 0, 0.3 or 3.0 mM MAA, as indicated in Table 5. The pH of the nutrient

– uptake was enhanced when supplied with LATS range of NO3

A0 A1 A2

a a a

– was more available and the MAA content was higher in the roots.

b

c

– reduction is incorporated into an organic form

– assimilation in the roots was higher than in the

a

– uptake and NO3

– assimilation

<sup>−</sup> assimilatory pathway

–.

+ originating in the plant from NO3

The results of the present experiment clearly indicated that NO3

increased activities of the three enzymes (NR, NiR and GS) of the NO3

5). It is also striking that effect of MAA on NO3

In the studies of the possible regulation of NR activity by multiple amino acids in higher plants, the conclusions are also contradictory. The inhibition on NR activity by glycine, asparagines, and glutamine could be partially or wholly prevented by the presence of other amino acids during the induction (Radin, 1977). However when glutamine and asparagines were included along with the "corn amino acid mixture", the inhibition on the induction of NR in corn roots was more severe (Oaks et al., 1979). Chen and Gao (2002) have applied different mixture of glycine, isoleucine and proline to Chinese cabbage and lettuce in hydroponic experiment. They found the amino acids treatment enhanced NR activity in Chinese cabbage, while decreasing it slightly in lettuce.

In this experiment, at 5.0 mM NO3– which is facilitated by LATS, the presence of 0.3 mM MAA partially inhibited NR activity, as observed in other works, whereas the 3.0 mM MAA increased the NR activity more than 4 times (Fig. 3). In addition, the very high NO2– content was found in A2 (Table 4). These results suggest that high concentration MAA can increase NO3– uptake by enhancing NR activity in radish, especially in roots.

The next step in NO3– assimilation is the conversion of NO2– to NH4+ by the action of NiR. Both enzymes, NR and NiR, are induced by the same factors, and therefore the response of NiR to the MAA treatments resembled that of NR in roots, but was a little different with that of the NR in shoots (Fig. 3 and Fig. 4). NiR activities in shoots and roots in A1 were inhibited by 17% and 52% respectively in relation to A0. In A2, NiR activity was inhibited by 15% in shoots and enhanced 8 times in roots. In the present study, the decrease of NiR activity in shoots in A2 might be attributed to the low concentration of amino acids in shoots, too. The increase of NiR activity in roots in A2 was due to the same reason with NR.

Fig. 4. Effect of mixed amino acids on nitrite reductase activity in radish at 24 h after treatment. Values are means ± SD (n=5).

The principal NH4+ pathway is the glutamine synthetase (GS)/glutamate synthase (GOGAT) cycle. The behavior of GS activities in shoots was not affected by MAA treatments (Fig. 5). However differences were found in roots between treatments, showing 22% inhibition in A1 and 17% increase in A2 in relation to A0.

In the studies of the possible regulation of NR activity by multiple amino acids in higher plants, the conclusions are also contradictory. The inhibition on NR activity by glycine, asparagines, and glutamine could be partially or wholly prevented by the presence of other amino acids during the induction (Radin, 1977). However when glutamine and asparagines were included along with the "corn amino acid mixture", the inhibition on the induction of NR in corn roots was more severe (Oaks et al., 1979). Chen and Gao (2002) have applied different mixture of glycine, isoleucine and proline to Chinese cabbage and lettuce in hydroponic experiment. They found the amino acids treatment enhanced NR activity in

MAA partially inhibited NR activity, as observed in other works, whereas the 3.0 mM MAA increased the NR activity more than 4 times (Fig. 3). In addition, the very high NO2– content was found in A2 (Table 4). These results suggest that high concentration MAA can increase

The next step in NO3– assimilation is the conversion of NO2– to NH4+ by the action of NiR. Both enzymes, NR and NiR, are induced by the same factors, and therefore the response of NiR to the MAA treatments resembled that of NR in roots, but was a little different with that of the NR in shoots (Fig. 3 and Fig. 4). NiR activities in shoots and roots in A1 were inhibited by 17% and 52% respectively in relation to A0. In A2, NiR activity was inhibited by 15% in shoots and enhanced 8 times in roots. In the present study, the decrease of NiR activity in shoots in A2 might be attributed to the low concentration of amino acids in shoots, too. The increase of NiR activity in roots in A2 was due to the same

Shoot Root

The principal NH4+ pathway is the glutamine synthetase (GS)/glutamate synthase (GOGAT) cycle. The behavior of GS activities in shoots was not affected by MAA treatments (Fig. 5). However differences were found in roots between treatments, showing 22%

– which is facilitated by LATS, the presence of 0.3 mM

A0 A1 A2

b

c

a

Chinese cabbage, while decreasing it slightly in lettuce.

NO3– uptake by enhancing NR activity in radish, especially in roots.

a

b ab

Fig. 4. Effect of mixed amino acids on nitrite reductase activity in radish at 24 h after

In this experiment, at 5.0 mM NO3

reason with NR.

NiRA (

0.00

treatment. Values are means ± SD (n=5).

inhibition in A1 and 17% increase in A2 in relation to A0.

0.05

0.10

0.15

0.20

0.25

0.30

mol NO2


g- FW h-1

)

The NH4 + originating in the plant from NO3 – reduction is incorporated into an organic form primarily by the enzyme GS. In the present experiment, GS activity was inhibited by 0.3 mM MAA treatment in radish roots, whereas 3.0 mM of MAA treatment enhanced the activity (Fig. 5). It is also striking that effect of MAA on NO3 – assimilation in the roots was higher than in the shoots, presumably NO3 – was more available and the MAA content was higher in the roots.

The results of the present experiment clearly indicated that NO3 – uptake and NO3 – assimilation was regulated by MAA in radish, especially at high concentration of MAA treatment. In conclusion, the application of high MAA rates (principally A2) could be the direct cause of increased activities of the three enzymes (NR, NiR and GS) of the NO3 <sup>−</sup> assimilatory pathway and the NO3 – uptake was enhanced when supplied with LATS range of NO3 –.

Fig. 5. Effect of mixed amino acids on glutamine synthetase activity in radish at 24 h after treatment. Values are means ± SD (n=5).
