**4.2 Results and discussion**

#### **4.2.1 Effect of MAA on enzyme activities**

Nitrate reductase is the first enzyme involved in the metabolic route of NO3– assimilation in higher plants. Significant differences were found in the NR activity between the treatments (*P* < 0.01) (Fig. 10). The highest activity was attained with A2, showing an increase of 30% compared with the activity attained with A0. Treatment A1 and A3 were less effective in increasing the activity of NR than A2, with increase of 21% and 7%, respectively.

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

Commercialized artificial soil (pH, 5.2; EC, 1240 mS m–1; NO3––N, 280 mg Kg–1; available P2O5, 1020 mg Kg–1) was mixed with 15N labeled potassium nitrate (10 atom % 15N) and incubated at room temperature for 14 days at 60% of their maximum water–holding capacity. Finally, the high nitrate soil (pH, 5.0; EC, 3230 mS m–1; NO3––N, 1906 mg Kg–1; available P2O5, 1060 mg Kg–1) was obtained and used for this experiment. Seeds of radish

The mixed amino acids (MAA) solution contained equal concentrations of amino acids viz., alanine (Ala), β–alanine (β–Ala), aspartic acid (Asp), asparagines (Asn), glutamic acid (Glu), glutamine (Gln) and glycine (Gly). From 17 or 24 days after sowing, seedlings of radish were sprayed with 0.2 or 0.5 mM MAA solution for 2 or 4 times, as indicated in Table 8. The pH of the MAA solutions was maintained between 6.0–6.1 by adding 1.0 M

**Treatments Composition of treated solutions (mM) Applied time** 

A0\* ─ ─ ─ ─ ─ ─ ─ ─ ─ A1 0.78 0.2 0.2 0.2 0.2 0.2 0.2 0.2 17, 20, 24, 27 A2 2.10 0.5 0.5 0.5 0.5 0.5 0.5 0.5 17, 20, 24, 27 A3 0.78 0.2 0.2 0.2 0.2 0.2 0.2 0.2 24, 27

Table 8. Composition of the treated solutions and application times for radish in pot

yield and N assimilation. After harvest the soils were collected for chemical analysis.

Fresh leaves were collected at 28 days after sowing to determine the NO3– content and enzyme activities and at 30 days after sowing to determine the NO3–, amino acids and protein contents. Plant shoots were harvested at 30 days after sowing to determine crop

Nitrate reductase is the first enzyme involved in the metabolic route of NO3– assimilation in higher plants. Significant differences were found in the NR activity between the treatments (*P* < 0.01) (Fig. 10). The highest activity was attained with A2, showing an increase of 30% compared with the activity attained with A0. Treatment A1 and A3 were less effective in increasing the activity of NR than A2, with increase of 21% and 7%,

**K+ Ala β–Ala Asp Asn Glu Gln Gly DAS** 

were sown into 100 mL pots filled with the incubated soil and grown in a glasshouse.

**– soil** 

plants.

**4. Pot experiment of radish with high NO3**

**4.1 Materials and methods** 

KOH appropriately.

experiment

respectively.

\* Same amount of distilled water sprayed

**4.2 Results and discussion** 

**4.2.1 Effect of MAA on enzyme activities** 

Fig. 10. Effect of mixed amino acids on nitrate reductase activity of radish leaves 28 day after 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 + by the action of 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 of NiR showed a decrease of 11% in A1 (*P* < 0.05).

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).

Effect of Mixed Amino Acids on Crop Growth 137

the plants treated with MAA showed a little increase of these compounds (*P* > 0.05) and the

The total N content of the plants was affected significantly by using MAA (*P* < 0.01). Treatments of A1, A2 and A3 showed to decrease the total N content to 14%, 7% and 10%

The result of NO3– content agrees with the interpretation that amino acid can negatively regulate nitrate content in higher plants (Chen and Gao, 2002; Gunes et al., 1994, 1996; Wang

after MAA treating (the data were not shown). This was probably due to the different response of individual plant to the complex mechanism of MAA in NO3– assimilation process in short period. However, 3 days after MAA application, regular result of NO3

The predominance of amino acids and proteins were attributed to high activities of main

The result of total N content was opposite from that of field experiment in which total N content was increased by applying amino acid fertilizer. These contradictory results were due to different stage of amino acids treatment. Possibly, young plants may lack a complete functional system for NO3– uptake and assimilation (Pessarakli, 2002). Wang et al. (2004) reported that application of amino acids in autumn could increase total N in pakchoi but no

The plant production in terms of fresh weight was found to be significantly higher (*P* < 0.05) in treatment A1 and A2, with increases of 13% and 12% compared with the control, respectively (Table 10). The response of production in dry weight to MAA treatments was more sensitive than that of fresh weight (Table 10), with significant influences in MAA application (*P* < 0.01). The highest yield in dry weight was found in A2, with an increase of 44% in relation to A0. The results of N utilization (Table 10) were similar to dry yield described above, again registering the highest value in A2, with an increase of 34% compared with A0 (*P* < 0.01). Furthermore, significant effects were also observed in A1 and

enzymes of NO3– assimilation and the direct uptake of amino acids from MAA.

– assimilation, amino acids and proteins (Table 9),

– content was also found at 24 h

–

With respect to the main products of NO3

compared with the control, respectively.

content in shoots of radish was found.

et al., 2004). In the present experiment, surged value of NO3

significant effect was observed when treated in summer.

**4.2.3 Effect of MAA on radish yield and N utilization** 

sowing in pot experiment with high NO3– soil

A3, with increase of 27% and 13% respectively, relative to A0 (*P* < 0.01).

**Treatments Fresh weight Dry weight N utilization** 

A0 13.32 ± 0.71 b 0.86 ± 0.10 b 37.60 ± 2.87 c A1 14.99 ± 1.01 a 1.22 ± 0.13 a 47.60 ± 4.11 ab A2 14.86 ± 0.57 a 1.23 ± 0.11 a 50.72 ± 2.53 a A3 13.01 ± 0.71 b 1.06 ± 0.07 ab 42.43 ± 3.67 bc Data are means ± SD (n=4). 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 10. Effect of mixed amino acids on radish yield and nitrogen utilization 30 day after

**(g/plant) (mg/plant)** 

highest contents were found in A2.

Fig. 12. Effect of mixed amino acids on glutamine synthetase activity of radish leaves 28 day after sowing in pot experiment with high NO3– soil. Values are means ± SD (n=4).

The response of GS to MAA treatments is showed in Fig. 12. The greatest activity was observed in treatment A2, with an increase of 7% over the reference treatment (*P* > 0.1). On the contrary, the least activity of GS was found in A1, with a 12% decrease compared with A0 (*P* < 0.05).

The results of activities of enzymes are similar to those of other research, which indicated that treatment of MAA (Section 3.1) and amino acid fertilizer (Section 3.7) could enhance activity of NR in radish when supplied with high rate NO3 –. In the present experiment, the treatments of MAA led to different rates of increase in NR activity and also affect NiR and GS activities depending on applied rates. Higher activities of three enzymes were found in A2 for the reason that the positive effect on NR was stronger than the feed–back inhibition. However, decrease of NiR and GS was observed in A1 due to the feed–back inhibition on NO3– reduction systems which affected GS first.

#### **4.2.2 Effect of MAA on N contents**


The data in Table 9 showed that N contents of the plants were affected by using MAA. The NO3– content of radish was decreased by 24–38% by applying MAA (*P* < 0.001) compared with the reference treatment.

Data are means ± SD (n=4). 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 9. Effect of mixed amino acids on nitrogen contents of radish leaves 30 day after sowing in pot experiment with high NO3– soil

b

a

A0 A1 A2 A3

Treatments

Fig. 12. Effect of mixed amino acids on glutamine synthetase activity of radish leaves 28 day

The response of GS to MAA treatments is showed in Fig. 12. The greatest activity was observed in treatment A2, with an increase of 7% over the reference treatment (*P* > 0.1). On the contrary, the least activity of GS was found in A1, with a 12% decrease compared with A0 (*P* < 0.05).

The results of activities of enzymes are similar to those of other research, which indicated that treatment of MAA (Section 3.1) and amino acid fertilizer (Section 3.7) could enhance activity of NR in radish when supplied with high rate NO3–. In the present experiment, the treatments of MAA led to different rates of increase in NR activity and also affect NiR and GS activities depending on applied rates. Higher activities of three enzymes were found in A2 for the reason that the positive effect on NR was stronger than the feed–back inhibition. However, decrease of NiR and GS was observed in A1 due to the feed–back inhibition on

The data in Table 9 showed that N contents of the plants were affected by using MAA. The NO3– content of radish was decreased by 24–38% by applying MAA (*P* < 0.001) compared

**Treatments Amino acids Proteins NO3– Total N (mg g–1 FW) (mg g–1 DW)**  A0 2.91 ± 0.10 a 9.05 ± 0.58 a 5.79 ± 0.59 a 44.9 ± 1.9 a A1 2.93 ± 0.07 a 9.57 ± 0.46 a 3.85 ± 0.44 bc 38.4 ± 1.3 b A2 3.03 ± 0.07 a 9.77 ± 0.54 a 3.57 ± 0.45 c 41.9 ± 1.8 ab A3 2.99 ± 0.09 a 9.19 ± 0.69 a 4.38 ± 0.18 b 40.2 ± 1.7 ab Data are means ± SD (n=4). 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 9. Effect of mixed amino acids on nitrogen contents of radish leaves 30 day after

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

ab

0

GSA (

NO3– reduction systems which affected GS first.

sowing in pot experiment with high NO3– soil

**4.2.2 Effect of MAA on N contents** 

with the reference treatment.

mol C

H5 10

N

O2

4 g-1 (FW) h-1

)

5

10

ab

15

With respect to the main products of NO3 – assimilation, amino acids and proteins (Table 9), the plants treated with MAA showed a little increase of these compounds (*P* > 0.05) and the highest contents were found in A2.

The total N content of the plants was affected significantly by using MAA (*P* < 0.01). Treatments of A1, A2 and A3 showed to decrease the total N content to 14%, 7% and 10% compared with the control, respectively.

The result of NO3– content agrees with the interpretation that amino acid can negatively regulate nitrate content in higher plants (Chen and Gao, 2002; Gunes et al., 1994, 1996; Wang et al., 2004). In the present experiment, surged value of NO3– content was also found at 24 h after MAA treating (the data were not shown). This was probably due to the different response of individual plant to the complex mechanism of MAA in NO3– assimilation process in short period. However, 3 days after MAA application, regular result of NO3 – content in shoots of radish was found.

The predominance of amino acids and proteins were attributed to high activities of main enzymes of NO3– assimilation and the direct uptake of amino acids from MAA.

The result of total N content was opposite from that of field experiment in which total N content was increased by applying amino acid fertilizer. These contradictory results were due to different stage of amino acids treatment. Possibly, young plants may lack a complete functional system for NO3– uptake and assimilation (Pessarakli, 2002). Wang et al. (2004) reported that application of amino acids in autumn could increase total N in pakchoi but no significant effect was observed when treated in summer.

#### **4.2.3 Effect of MAA on radish yield and N utilization**

The plant production in terms of fresh weight was found to be significantly higher (*P* < 0.05) in treatment A1 and A2, with increases of 13% and 12% compared with the control, respectively (Table 10). The response of production in dry weight to MAA treatments was more sensitive than that of fresh weight (Table 10), with significant influences in MAA application (*P* < 0.01). The highest yield in dry weight was found in A2, with an increase of 44% in relation to A0. The results of N utilization (Table 10) were similar to dry yield described above, again registering the highest value in A2, with an increase of 34% compared with A0 (*P* < 0.01). Furthermore, significant effects were also observed in A1 and A3, with increase of 27% and 13% respectively, relative to A0 (*P* < 0.01).


Data are means ± SD (n=4). 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 10. Effect of mixed amino acids on radish yield and nitrogen utilization 30 day after sowing in pot experiment with high NO3– soil

Effect of Mixed Amino Acids on Crop Growth 139

The chemical properties of soil at the end of experiment are showed in Table 12. The planting of radish affected these chemical properties of soil clearly. However, there were no differences in pH of soil among treatments planted with radish. On the other hand, either planting treatment or MAA treatment showed effect on nitrate in soil. Compared with the non planting treatment, the treatments of planting showed a decrease of 65~81% and 35~47% of nitrate and available P at 30 days after sowing, respectively. The different rates of

**pH EC\* Available P2O5 NO3––N** 

**(1:5) (mS m–1) (mg Kg–1)** 

NP 5.0 1985 599.7 1008.6 A0 5.7 981 390.6 339.4 A1 5.7 902 344.5 298.5 A2 5.7 674 316.5 192.0 A3 5.7 894 356.0 355.0

\* The soil used in these experiments was commercialized artificial soil with lower soil density (about 0.4 g cm–3) and higher water–holding capacity. Since determination of soil chemical properties is based on dry weight, the determined values of EC and NO3––N are quite high relative to ordinary soil. However,

Table 12. Chemical properties of soil at the end of pot experiment for radish with high NO3–

In conclusion, the results of the present experiment suggest that application of MAA can affect activities of three enzymes of N assimilation (NR, NiR and GS). However, the exact reason for this observation is unknown and further investigation is necessary. Furthermore, the application of MAA can enhance growth, N utilization, and concentrations of proteins and amino acids, and reduce the NO3– content in plant shoots. Considerable increase of N uptake from soil was indicated by the increased 15N recovery by applying MAA compared with the control. These results suggest that the main role of MAA on nitrate uptake and

> **– soil**

Commercialized artificial soil (pH, 5.2; EC, 1240 mS m–1; NO3––N, 280 mg Kg–1; available P2O5, 1020 mg Kg–1) was used for this experiment. Plant culture and MAA treatment were

analysis of plant and soil also were according to procedures adopted for radish with high

– uptake and assimilation, but not

– soil. The sampling and

**4.2.5 Effect of MAA on chemical properties of soil** 

**Treatments** 

soil

these are not very higher in soil solution.

as sources of reduced nitrogen.

**5.1 Materials and methods** 

NO3– soil.

assimilation might be relation with the regulation of NO3

the same with that of pot experiment of radish with high NO3

**5. Pot experiment of radish with low NO3**

decrease were due to the different growth rates led by MAA treatment.

For responses of growth, the application of MAA showed enhanced effects obviously. These results are in agreement with those observed by Chen et al. (1997), who reported that application of amino acids led to positive effects on Chinese cabbage growth. Among the treatments of MAA, the growth responses were increased by increasing the application rate of MAA. The increases of yield were due to the positive adjusting of MAA on growth of plants, thus contributing to the increases of N utilization (Table 10) even though the total N content was decreased in MAA treatments (Table 9).
