**6. Field experiment of radish**

## **6.1 Materials and methods**

The study was conducted in summer of 2005 at the experimental farm of the Chungnam National University, Daejeon, Korea. The average chemical properties of the soil of the field are described in Table 16. The fertilizer mixture was uniformly broadcasted onto the soil surface and incorporated before ridging. The seeds of radish were sown at the end of May 2005 and arranged in a completely randomized block design, with three replications. The plots were 5 m × 2 m consisting of 2 rows.

At 15 and 22 days after sowing, AAF was applied 2 times to plots by spraying to leaves after diluting 500, 1000 and 2000 times by water, respectively. The main chemical contents of the AAF and application quantities are shown in Table 17.


Table 16. Chemical properties of soils used in field experiment of radish

Effect of Mixed Amino Acids on Crop Growth 145

ab <sup>a</sup>

A0 A1 A2 A3

Treatments

b b

+ assimilation (Oaks, 1994). The response of

A0 A1 A2 A3

Treatments

Fig. 17. Effect of amino acid fertilizer on nitrite reductase activity in leaves of radish 23 day

The reversible amination of 2–oxoglutarate to glutamic acid via GDH has long been considered as a major route of NH4+ assimilation (Srivastava and Singh, 1987). However the discovery of the enzyme GS–GOGAT system altered this point of view, and the incorporation of NH4+ to glutamine via GS and subsequently into glutamic acid by GOGAT

GS (Fig. 18) to AAF treatments was similar to that of the NiR (Fig. 17). The greatest activity was reached in treatment A1, with an increase of 20% over the reference treatment (*P* < 0.001). On the contrary, the activity of GS was the lowest in A3, with a decline of 11%

Fig. 16. Effect of amino acid fertilizer on nitrate reductase activity in leaves of radish 23 day

a ab

b

0.0

0.0

0.8

NiRA (

after sowing. Values are means ± SD (n=3).

is now widely accepted as the main route of NH4

compared with A0 (*P* < 0.05).

mol NO2

g


)


1.6

2.4

3.2

4.0

NRA (

after sowing. Values are means ± SD (n=3).

mol NO2

g


)


0.4

0.8

1.2

b

1.6

Fresh leaves were collected at 23 days after sowing to determine the NO3 –, amino acids and protein contents and enzyme activities. The plots were harvested at 35 days after sowing to determine crop yield and N assimilation. The topsoil samples (0–20 cm) were collected at 25 and 35 days after sowing for chemical analysis.

In order to compare the different AAF treatments for their N uptake, net N uptake was estimated by balancing N utilization and N input by applying AAF thus:

$$N\_N = N\_{\mathcal{U}} - N\_{AAF} \,. \tag{1}$$

where *NN* is the net N uptake by plant; *NU* is the total N utilization at harvest; *NAAF* is N input by applying AAF.

It was assumed that N would have been either taken up by the plants or lost from the soil– plant system. In our experiment, leaching was the main way of N loss. Furthermore, N loss attributable to soil erosion and runoff was considered for our site with 2~5% slope. Since these losses may be influenced by protecting of the plants from the rain, the vegetation cover was observed at 25 and 35 days after sowing.


\* NP: No-planting

Table 17. Amino acid fertilizer applied to radish in the field experiment

#### **6.2 Results and discussion**

#### **6.2.1 Effect of AAF 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. 16). The highest activity was obtained with A1, showing an increase of 16% in relation to the activity obtained with A0. A2 was less effective in increasing the activity of NR than A1; whereas no increase of NRA occurred in A3, even treated with fourfold AAF than A1.

The next step in NO3– assimilation is the conversion of the NO2 – to NH4 + by the action of NiR. The AAF treatments showed different effect on NiR activity depending on the applied rate of AAF (Fig. 17). The highest activity of NiR was found in treatment A1, showing an increase of 4% compared with A0 (*P* < 0.05). However, the activities of NiR were inhibited by 12 and 13% in A2 and A3, respectively.

Fresh leaves were collected at 23 days after sowing to determine the NO3–, amino acids and protein contents and enzyme activities. The plots were harvested at 35 days after sowing to determine crop yield and N assimilation. The topsoil samples (0–20 cm) were collected at 25

In order to compare the different AAF treatments for their N uptake, net N uptake was

where *NN* is the net N uptake by plant; *NU* is the total N utilization at harvest; *NAAF* is N

It was assumed that N would have been either taken up by the plants or lost from the soil– plant system. In our experiment, leaching was the main way of N loss. Furthermore, N loss attributable to soil erosion and runoff was considered for our site with 2~5% slope. Since these losses may be influenced by protecting of the plants from the rain, the vegetation

AAF application — — 750 1500 3000 Essential amino acid 2.22 — — 16.7 33.3 66.6 Total amino acid 5.14 — — 38.6 77.2 154.4 Total–N 3.80 — — 28.5 57.0 114.0 Soluble P 3.12 — — 23.4 46.8 93.6 Soluble K 4.97 — — 37.3 74.6 149.2 Soluble B 0.13 — — 0.98 1.95 3.90

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. 16). The highest activity was obtained with A1, showing an increase of 16% in relation to the activity obtained with A0. A2 was less effective in increasing the activity of NR than A1; whereas no increase of NRA occurred in A3, even treated with fourfold AAF

NiR. The AAF treatments showed different effect on NiR activity depending on the applied rate of AAF (Fig. 17). The highest activity of NiR was found in treatment A1, showing an increase of 4% compared with A0 (*P* < 0.05). However, the activities of NiR were inhibited

Table 17. Amino acid fertilizer applied to radish in the field experiment

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

*N NN N U AAF* . (1)

**Treatments NP\* A0 A1 A2 A3 (mg m–2)** 

+ by the action of

estimated by balancing N utilization and N input by applying AAF thus:

and 35 days after sowing for chemical analysis.

cover was observed at 25 and 35 days after sowing.

input by applying AAF.

**Classification (%)** 

\* NP: No-planting

than A1.

**6.2 Results and discussion** 

**6.2.1 Effect of AAF on enzyme activities** 

by 12 and 13% in A2 and A3, respectively.

Fig. 16. Effect of amino acid fertilizer on nitrate reductase activity in leaves of radish 23 day after sowing. Values are means ± SD (n=3).

Fig. 17. Effect of amino acid fertilizer on nitrite reductase activity in leaves of radish 23 day after sowing. Values are means ± SD (n=3).

The reversible amination of 2–oxoglutarate to glutamic acid via GDH has long been considered as a major route of NH4+ assimilation (Srivastava and Singh, 1987). However the discovery of the enzyme GS–GOGAT system altered this point of view, and the incorporation of NH4+ to glutamine via GS and subsequently into glutamic acid by GOGAT is now widely accepted as the main route of NH4 + assimilation (Oaks, 1994). The response of GS (Fig. 18) to AAF treatments was similar to that of the NiR (Fig. 17). The greatest activity was reached in treatment A1, with an increase of 20% over the reference treatment (*P* < 0.001). On the contrary, the activity of GS was the lowest in A3, with a decline of 11% compared with A0 (*P* < 0.05).

Effect of Mixed Amino Acids on Crop Growth 147

The plant biomass production in fresh weight was found to be significantly higher (*P* < 0.01) in the AAF treatments (mean biomass in fresh weight of A1, A2 and A3 are 5.056, 4.738 and 4.653 Kg m–2, respectively) compared with the control (mean biomass in fresh weight is 4.026 Kg m–2) (Table 18). Among AAF treatments, the treatment with low concentration of AAF (A1) had a higher (*P* > 0.05) biomass production than the treatment with high concentration of AAF (A3). The response of biomass production in dry weight to AAF treatments resembled that in fresh weight (Table 28), with significant influence by applying AAF (*P* < 0.01). The highest biomass production in dry weight was found in A1, with an

**Treatments Fresh weigh Dry weight N utilization P utilization**  A0 4026 ± 227 c 345.7 ± 14.2 c 9.33 ± 0.87 c 2.35 ± 0.09 b A1 5056 ± 213 a 404.4 ± 11.6 a 14.48 ± 0.89 a 2.87 ± 0.11 a A2 4738 ± 183 ab 394.8 ± 12.1 ab 13.04 ± 0.53 ab 2.68 ± 0.12 a A3 4653 ± 189 b 382.0 ± 14.5 b 12.83 ± 0.67 b 2.64 ± 0.07 a Values are means ± SD (n=3). 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)

increased in AAF treatments due to the increase of biomass production (Table 18).

could enhance the ability of uptake and assimilation of inorganic N by plants.

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

Table 19. Net nitrogen uptake and vegetation cover of radish

Table 18. Effect of amino acid fertilizer on radish yield and utilization of nitrogen and

The result of N utilization (Table 18) was similar to biomass production as described above, again registering the highest value in A1 (14.48 ± 0.89 g m–2), with an increase of 55% compared with A0 (9.33 ± 0.87 g m–2) (*P* < 0.01). Furthermore, significant effects were observed in A2 and A3 too, with increases of 40% and 37% respectively, in relation to A0 (*P* < 0.01). Even though P content was not influenced by the application of AAF (Table 20), P utilization

The observed result of vegetation cover and calculated values of net N uptake are showed in Table 19. The treatments of AAF showed higher vegetation cover than the control. Besides the N input by applying of AAF, the treatments of AAF showed significant increase of 36~55%net N uptake compared with the control. Gunes et al. (1996) suggested that plants probably preferred amino acids as sources of reduced nitrogen, but they did not distinguish origin of the N contents in the plants. In our experiment, the increase of N uptake is about 200 times (Table 19) more than N supplied by applying AAF, indicating application of AAF

**Treatments Vegetation cover (%) Net N uptake (g m–2)** 

Values are means ± SD (n=3). Analysis of variance (ANOVA) was employed followed by Duncan's new

A0 63 ± 3 c 91 ± 2 b 9.33 ± 0.87 b A1 85 ± 5 a 100 ± 0 a 14.45 ± 0.89 a A2 79 ± 6 ab 100 ± 0 a 12.98 ± 0.53 a A3 76 ± 3 b 100 ± 0 a 12.72 ± 0.67 a

**25 DAS 35 DAS 35 DAS** 

**6.2.2 Effect of AAF on biomass and utilization of N and P** 

increase of 17% in relation to A0.

phosphorus 35 day after sowing (g m–2)

Fig. 18. Effect of amino acid fertilizer on glutamine synthetase activity in leaves of radish 23 day after sowing. Values are means ± SD (n=3).

The reduction of NO3– to NO2– by NR, is the main and most limiting step, in addition to being the most prone to regulation (Sivasankar et al., 1997; Ruiz et al., 1999). The synthesis of this enzyme is induced by nitrate (Oaks, 1994), but although its activity is known to be repressed by ambient ammonium, there are evidences that this enzyme can be regulated by certain amino acids. The results for the possible regulation of NR activity by amino acids for higher plants are contradictory. Many authors agree with that amino acids can inhibit the activity of NR in higher plants (Radin, 1975, 1977; Oaks et al., 1979; Ivashikian and Sokolov, 1997; Sivasankar et al., 1997; Aslam et al., 2001). But Aslam et al. (2001) reported that inhibition did not occur when the concentration of NO3– in the external solutions had been increased to 10 mM. This result is consistent with the other research, which indicates that radish treated with mixed amino acids containing 5.0 mM NO3– in growth medium showed significant increase of NR activity (Liu et al., 2005). The effect of amino acids on NR activity seems to be depended on plant materials, age of plants, growth conditions, nitrate concentration, kinds of amino acids, amino acids concentration and other factors. In this experiment, the positive effect on NR activity by applying AAF was due to high NO3– content in soil.

In the present experiment, the treatments of AAF led to different levels of increase of NR activity and inhibition on GS activity depending on applied rates. The high activities of three enzymes were found in A1 due to the positive effect of AAF on process of NO3– assimilation. However, inhibition on NiR and GS was observed in A2 and A3 for the reason that high rates of AAF application had high feed–back inhibition on NO3– reduction systems which affected GS first. This is probably the main reason why different effects on the enzymes were observed in this study.

a

A0 A1 A2 A3

Treatments

Fig. 18. Effect of amino acid fertilizer on glutamine synthetase activity in leaves of radish 23

The reduction of NO3– to NO2– by NR, is the main and most limiting step, in addition to being the most prone to regulation (Sivasankar et al., 1997; Ruiz et al., 1999). The synthesis of this enzyme is induced by nitrate (Oaks, 1994), but although its activity is known to be repressed by ambient ammonium, there are evidences that this enzyme can be regulated by certain amino acids. The results for the possible regulation of NR activity by amino acids for higher plants are contradictory. Many authors agree with that amino acids can inhibit the activity of NR in higher plants (Radin, 1975, 1977; Oaks et al., 1979; Ivashikian and Sokolov, 1997; Sivasankar et al., 1997; Aslam et al., 2001). But Aslam et al. (2001) reported that inhibition did not occur when the concentration of NO3– in the external solutions had been increased to 10 mM. This result is consistent with the other research, which indicates that radish treated with mixed amino acids containing 5.0 mM NO3– in growth medium showed significant increase of NR activity (Liu et al., 2005). The effect of amino acids on NR activity seems to be depended on plant materials, age of plants, growth conditions, nitrate concentration, kinds of amino acids, amino acids concentration and other factors. In this experiment, the positive effect on NR activity by applying AAF was due to high NO3–

In the present experiment, the treatments of AAF led to different levels of increase of NR activity and inhibition on GS activity depending on applied rates. The high activities of three enzymes were found in A1 due to the positive effect of AAF on process of NO3– assimilation. However, inhibition on NiR and GS was observed in A2 and A3 for the reason that high rates of AAF application had high feed–back inhibition on NO3– reduction systems which affected GS first. This is probably the main reason why different effects on the

b

b

0

day after sowing. Values are means ± SD (n=3).

content in soil.

enzymes were observed in this study.

GSA (

mol C

H5 10

N

O2

4 g-1 (FW) h-1

)

10

20

30

b

40
