**3. Hydroponic experiment of red pepper**

### **3.1 Materials and methods**

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 with hydroponic experiment of radish.

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 mM NO3– and 0, 0.3 or 3.0 mM MAA, as indicated in Table 5. 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.

Effect of Mixed Amino Acids on Crop Growth 131

A0 9.12±0.58 a 6.20±0.23 a 0.036±0.003 b 0.596±0.032 c

A1 7.54±0.34 b 3.99±0.36 b 0.046±0.006 a 1.164±0.046 b

A2 8.31±0.43 ab 2.66±0.19 c 0.024±0.004 c 2.371±0.085 a

– and NO2

– concentration in fresh weight of red

a

b

c

Values are means ± SD (n=5). Analysis of variance (ANOVA) was employed followed by Duncan's new

For NO3– assimilation, NO3– is reduced to NO2– by catalysis of NR. In this experiment, MAA treatments led to different effects on NR activity in leaves and in roots (Fig. 7). In the roots, treatment A1 and treatment A2 showed increases of 35% and 212% respectively in relation to A0 (*P* < 0.01). In contrast, NR activities were inhibited slightly in leaves by MAA

The response of NiR to the MAA treatments resembled that of NR in roots, but was different with that of the NR in leaves (Fig. 8). NiR activities in leaves and roots in A1 were increased by 18% and 60% respectively in relation to A0 (leaves: *P* < 0.05; roots: *P* < 0.01). In A2, NiR activities were the same with A0 in leaves and enhanced 138% in roots (leaves: *P* > 0.05;

Leaf Root

Fig. 7. Effect of mixed amino acids on nitrate reductase activity in red pepper at 24 h after

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

treatments, showing 8.2% in A1 and 10.5% in A2, respectively (*P* > 0.05).

A0 A1 A2

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

**– NO2–**

**Leaf Root Leaf Root** 

**NO3**

Table 6. Effect of mixed amino acids on NO3

pepper at 24 h after treatment (mol g–1)

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

NRA (

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

0.0

0.1

0.2

0.3

0.4

0.5

mol NO2


g-1 FW h-1)

**Treatments** 

roots: *P* < 0.01).


Table 5. The compositions of the treatment solutions for red pepper in hydroponic experiment (mM)

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

#### **3.2 Results and discussion**

#### **3.2.1 Effect on NO3 – uptake**

The MAA treatments showed different effect on nitrate uptake depending on the concentrations (Fig. 6). Application of MAA at both 0.3 mM and 3.0 mM concentrations increased NO3– uptake in red pepper (*P* < 0.001) and the highest NO3 – uptake was found in treatment A2 showing 7 fold increases over A0.

Fig. 6. Effect of mixed amino acids on the nitrate uptake in red pepper supplied with 10.0 mM NO3–. Values are means ± SD (n=5).

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

The highest NO3– concentration both in the roots and leaves were found in treatment A0 (Table 6), with respect to the lowest NO3– content found in treatment A1 in the leaves (*P* < 0.05) and A2 in the roots (*P* < 0.01). With respect to the NO2– values (Table 6), in this experiment, the highest NO2 – concentrations in roots were found in the A2 and the lowest in A0 (*P* < 0.001). In leaves, the lowest NO2– concentration was found in A2 and the lowest in A1 (*P* > 0.05).

Plants were harvested 24 h after treatment and separated into roots and leaves for enzymes assay and N content analysis. Net NO3– uptake rates were determined by amount of NO3–

The MAA treatments showed different effect on nitrate uptake depending on the concentrations (Fig. 6). Application of MAA at both 0.3 mM and 3.0 mM concentrations

> Time (h) 0 5 10 15 20 25

– concentration both in the roots and leaves were found in treatment A0

– content found in treatment A1 in the leaves (*P* <

Fig. 6. Effect of mixed amino acids on the nitrate uptake in red pepper supplied with 10.0

0.05) and A2 in the roots (*P* < 0.01). With respect to the NO2– values (Table 6), in this experiment, the highest NO2– concentrations in roots were found in the A2 and the lowest in A0 (*P* < 0.001). In leaves, the lowest NO2– concentration was found in A2 and the lowest in

 **accumulation** 

– uptake was found in

– uptake in red pepper (*P* < 0.001) and the highest NO3

A0 A1 A2

**Treatments K+ NO3– Ala β–Ala Asp Asn Glu Gln Gly**  A0 10.25 10.0 ─ ─ ─ ─ ─ ─ ─ A1 11.78 10.0 0.3 0.3 0.3 0.3 0.3 0.3 0.3 A2 18.10 10.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0

Table 5. The compositions of the treatment solutions for red pepper in hydroponic

experiment (mM)

**3.2.1 Effect on NO3**

increased NO3

**3.2 Results and discussion** 

NO

**3.2.2 Effect on NO3**

The highest NO3

A1 (*P* > 0.05).

3

0

mM NO3–. Values are means ± SD (n=5).

**–**

(Table 6), with respect to the lowest NO3

 **and NO2**

**–**

20

40

60

80

100


mol g- FW)

disappeared from the initially treated solution.

 **uptake** 

treatment A2 showing 7 fold increases over A0.

**–**


Values 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 6. Effect of mixed amino acids on NO3 – and NO2– concentration in fresh weight of red pepper at 24 h after treatment (mol g–1)

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

For NO3– assimilation, NO3– is reduced to NO2– by catalysis of NR. In this experiment, MAA treatments led to different effects on NR activity in leaves and in roots (Fig. 7). In the roots, treatment A1 and treatment A2 showed increases of 35% and 212% respectively in relation to A0 (*P* < 0.01). In contrast, NR activities were inhibited slightly in leaves by MAA treatments, showing 8.2% in A1 and 10.5% in A2, respectively (*P* > 0.05).

The response of NiR to the MAA treatments resembled that of NR in roots, but was different with that of the NR in leaves (Fig. 8). NiR activities in leaves and roots in A1 were increased by 18% and 60% respectively in relation to A0 (leaves: *P* < 0.05; roots: *P* < 0.01). In A2, NiR activities were the same with A0 in leaves and enhanced 138% in roots (leaves: *P* > 0.05; roots: *P* < 0.01).

Fig. 7. Effect of mixed amino acids on nitrate reductase activity in red pepper at 24 h after treatment. Values are means ± SD (n=5).

Effect of Mixed Amino Acids on Crop Growth 133

also striking that effect of MAA on NO3– assimilation in the roots was higher than in the leaves, presumably NO3– was more available and the MAA content was higher in the roots. Ammonium assimilation in higher plants was long thought to begin with the synthesis of glutamate by glutamate dehydrogenase (GDH). It is now believed that the major pathway of NH4+ assimilation is the GS-GOGAT pathway, and GDH generally acts in a deaminating direction (Milflin and Habash, 2001). However, a role in NH4+ detoxification would explain the increase in GDH expression under conditions that provoke high tissue NH4+ levels

Two possible effect ways of amino acids on N assimilation process had been suggested: direct effect on mRNA of NR (Deng et al., 1991; Li et al., 1995; Vincentz et al., 1993) and

1997; Sivasankar et al., 1997). The hypothesis is that these two effect ways can collectively influence N assimilation in higher plant. This might probably be the main reason for differential effects on NO3– uptake observed in different studies. In the present experiment, GS activity was inhibited slightly by MAA treatments in roots, whereas irregular results

With respect to the main products of NO3– assimilation, amino acids and proteins (Table 7), the plants treated with MAA did not show increase in these compounds as being supposed apart from amino acids in roots (*P* < 0.05). In contrast, the concentration of proteins in the roots (*P* < 0.05) and leaves (*P* > 0.05) decreased with the MAA rate. Amino acids in leaves (*P*

Amino acids are the building blocks for proteins and also the products of their hydrolysis (Barneix and Causin, 1996). In the present experiment, amino acids concentrations (Table 7) were higher in the roots than in leaves. This is normal since the N assimilation occurs primarily in the roots than in the leaves. In roots, proteins concentrations (Table 7) were decreased by MAA treatment due to the possibility that amino acids content had effect on

**Leaf Root Leaf Root** 

**Treatments Amino acids Proteins** 

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

A0 0.93±0.03 a 2.35±0.12 b 5.03±0.27 a 1.92±0.12 a A1 0.78±0.06 b 2.81±0.16 a 4.53±0.18 b 1.90±0.10 a A2 0.67±0.03 b 3.05±0.08 a 4.35±0.24 b 1.45±0.11 b Data are means ± SD (n=5). Analysis of variance (ANOVA) was employed followed by Duncan's new

Table 7. Effect of mixed amino acids on level of amino acids and proteins in fresh weight of

In conclusion, the results of the present experiment clearly indicated that NO3– uptake and NO3– assimilation were regulated by MAA in red pepper. The application of MAA rates could be the direct cause of increased activities of the enzymes (NR and NiR) of the NO3– assimilatory pathway and the NO3– uptake was enhanced when supplied with

– reduction systems (King et al., 1993; Ivashikian and Sokolov,

(Lancien et al., 2000).

feed–back inhibition on NO3

were obtained in leaves (Fig. 9).

> 0.05) showed the same tendency too.

red pepper at 24 h after treatment (mg g–1)

protein breakdown.

**3.2.4 Effect on amino acids and proteins accumulation** 

Fig. 8. Effect of mixed amino acids on nitrite reductase activity in red pepper 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 leaves was increased by 16% in A1 but not affected in A2 (Fig. 9; *P* > 0.05). However, slight inhibitions were found in roots, showing 7% in A1 and 17% in A2 in relation to A0 (*P* < 0.05).

Fig. 9. Effect of mixed amino acids on glutamine synthetase activity in red pepper at 24 h after treatment. Values are means ± SD (n=5).

The first step in nitrate assimilation is the reduction of NO3 – to NO2 – by NR, the main and most limiting step, in addition to being the most prone to regulation (Sivasankar et al., 1997; Ruiz et al., 1999). The next step in NO3– assimilation is the conversion of the NO2– to NH4+ by the action of NiR. Both enzymes, NR and NiR, are induced by the same factors (Oaks, 1994). In our experiment, at 10 mM NO3– which is facilitated by LATS, the presence of MAA could increase the activities of NR and NiR in roots (Fig. 7 and Fig. 8). In addition, the very high NO2 – content was found in MAA treatments in roots (Table 6). These results suggest that MAA can increase NO3 – uptake by enhancing NR activity in roots of red pepper. It is

a

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

A0 A1 A2 a

c

b

A0 A1 A2

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

– uptake by enhancing NR activity in roots of red pepper. It is

Leaf Root

Leaf Root

Fig. 9. Effect of mixed amino acids on glutamine synthetase activity in red pepper at 24 h

The first step in nitrate assimilation is the reduction of NO3– to NO2– by NR, the main and most limiting step, in addition to being the most prone to regulation (Sivasankar et al., 1997; Ruiz et al., 1999). The next step in NO3– assimilation is the conversion of the NO2– to NH4+ by the action of NiR. Both enzymes, NR and NiR, are induced by the same factors (Oaks, 1994). In our experiment, at 10 mM NO3– which is facilitated by LATS, the presence of MAA could increase the activities of NR and NiR in roots (Fig. 7 and Fig. 8). In addition, the very high NO2– content was found in MAA treatments in roots (Table 6). These results suggest

Fig. 8. Effect of mixed amino acids on nitrite reductase activity in red pepper at 24 h after

b

The principal NH4+ pathway is the glutamine synthetase (GS)/glutamate synthase (GOGAT) cycle. The behavior of GS activities in leaves was increased by 16% in A1 but not affected in A2 (Fig. 9; *P* > 0.05). However, slight inhibitions were found in roots, showing 7%

NiRA (

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

GSA (

that MAA can increase NO3

0

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

5

10

15

20

25

b

a

30

mol C5H10

N

O2

4 g-1 FW h-1)

0.0

in A1 and 17% in A2 in relation to A0 (*P* < 0.05).

0.1

0.2

0.3

0.4

0.5

0.6

mol NO2


g- FW h-1

) also striking that effect of MAA on NO3– assimilation in the roots was higher than in the leaves, presumably NO3– was more available and the MAA content was higher in the roots.

Ammonium assimilation in higher plants was long thought to begin with the synthesis of glutamate by glutamate dehydrogenase (GDH). It is now believed that the major pathway of NH4+ assimilation is the GS-GOGAT pathway, and GDH generally acts in a deaminating direction (Milflin and Habash, 2001). However, a role in NH4+ detoxification would explain the increase in GDH expression under conditions that provoke high tissue NH4+ levels (Lancien et al., 2000).

Two possible effect ways of amino acids on N assimilation process had been suggested: direct effect on mRNA of NR (Deng et al., 1991; Li et al., 1995; Vincentz et al., 1993) and feed–back inhibition on NO3 – reduction systems (King et al., 1993; Ivashikian and Sokolov, 1997; Sivasankar et al., 1997). The hypothesis is that these two effect ways can collectively influence N assimilation in higher plant. This might probably be the main reason for differential effects on NO3– uptake observed in different studies. In the present experiment, GS activity was inhibited slightly by MAA treatments in roots, whereas irregular results were obtained in leaves (Fig. 9).

#### **3.2.4 Effect on amino acids and proteins accumulation**

With respect to the main products of NO3– assimilation, amino acids and proteins (Table 7), the plants treated with MAA did not show increase in these compounds as being supposed apart from amino acids in roots (*P* < 0.05). In contrast, the concentration of proteins in the roots (*P* < 0.05) and leaves (*P* > 0.05) decreased with the MAA rate. Amino acids in leaves (*P* > 0.05) showed the same tendency too.

Amino acids are the building blocks for proteins and also the products of their hydrolysis (Barneix and Causin, 1996). In the present experiment, amino acids concentrations (Table 7) were higher in the roots than in leaves. This is normal since the N assimilation occurs primarily in the roots than in the leaves. In roots, proteins concentrations (Table 7) were decreased by MAA treatment due to the possibility that amino acids content had effect on protein breakdown.


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 7. Effect of mixed amino acids on level of amino acids and proteins in fresh weight of red pepper at 24 h after treatment (mg g–1)

In conclusion, the results of the present experiment clearly indicated that NO3– uptake and NO3– assimilation were regulated by MAA in red pepper. The application of MAA rates could be the direct cause of increased activities of the enzymes (NR and NiR) of the NO3– assimilatory pathway and the NO3– uptake was enhanced when supplied with

Effect of Mixed Amino Acids on Crop Growth 135

ab

A0 A1 A2 A3

Treatments

+ by the action of

Fig. 10. Effect of mixed amino acids on nitrate reductase activity of radish leaves 28 day after

NiR. The MAA treatments showed different effects on NiR activity depending on the applied concentrations and times of MAA (Fig. 11). The highest activity of NiR was found in treatment A2, showing an increase of 7% compared with A0 (*P* < 0.1). However, the activity

> <sup>8</sup> ab <sup>a</sup> b

> > A0 A1 A2 A3

Treatments

Fig. 11. Effect of mixed amino acids on nitrite reductase activity of radish leaves 28 day after

sowing in pot experiment with high NO3– soil. Values are means ± SD (n=4).

sowing in pot experiment with high NO3– soil. Values are means ± SD (n=4).

The next step in NO3– assimilation is the conversion of the NO2– to NH4

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

a

0

of NiR showed a decrease of 11% in A1 (*P* < 0.05).

0

NiRA (

mol NO2

g


)


2

4

6

ab

NRA (

mol NO2

g


)


1

2

3

LATS range of NO3–. In addition, NO3– uptake by red pepper in unit weight plant was less than that of radish due to the different preference on N form between these two plants.

#### **4. Pot experiment of radish with high NO3 – soil**
