m–1) (mg
Kg–1)
NP 5.4
1485
878
214.7
A0 5.7
482
384
68.2
A1 5.7
515
303
62.2
A2 5.7
349
264
70.3
A3 5.7
503
277
68.6
Table 15. Chemical properties of soil at the end of pot experiment for radish with low NO3– soil The commercialized artificial soil used in this experiment was with lower soil density (about 0.4 g cm–3) and higher water–holding capacity. Although the NO3– contents of 62.2~70.3 mg Kg–1 are not low in ordinary soil, available NO3– for plants is very poor in soil solution in this experiment. This might be the probable reason, that effects of MAA on N assimilation in the present experiment were different from that of radish which was planted in high NO3–
soil. Whether in our experiments or in other researches, different effects of amino acids on NO3– reduction and assimilation were observed (Aslam et al., 2001).
In conclusion, the results of the present experiment suggest that application of MAA can
decrease activities of three enzymes of N assimilation (NR, NiR and GS). However, except N
utilization, the application of MAA did not have significant effects on growth, and
concentrations of proteins, amino acids, total N and NO3– content in plant shoots. The
difference in the results were found in both the present experiment and pot experiment
which radish was planted in high NO3– soil may be due to different levels of NO3– content in soil solution. The hypothesis that effect of amino acids on NO3– uptake, reduction and
assimilation depends on concentration of NO3– was justified.
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.
Organic Available Total
Soils pH
EC
matter
P2O5
N
NO3––N
(1:5)
(mS
m–1) (g
Kg–1) (mg
Kg–1) (g
Kg–1) (mg
Kg–1)
Before fertilization
6.0
122
15.6
170
0.81
80.2
After fertilization
6.0
191
15.8
279
0.87
191.2
Table 16. Chemical properties of soils used in field experiment of radish
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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
NU NAAF . (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.
Treatments
Classification (%)
NP*
A0 A1 A2 A3
(mg m–2)
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
* 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.
Effect of Mixed Amino Acids on Crop Growth
145
1.6
)-1
a
) h
ab
1.2
b
b
(FW-1
- g 2 0.8
l NO
0.4
(mo
NRA 0.0
A0
A1
A2
A3
Treatments
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).
) 4.0
-1 h
a
ab
3.2
b
b
(FW) -1 2.4
- g 2
1.6
mol NO 0.8
NiRA ( 0.0
A0
A1
A2
A3
Treatments
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).
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Agricultural Science
)-1
) h 40
a
b
b
(FW-1 30
b
g 4O2N 2010
H 5
10
mol C
A (
0
GS
A0
A1
A2
A3
Treatments
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.
Effect of Mixed Amino Acids on Crop Growth
147
6.2.2 Effect of AAF on biomass and utilization of N and P
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 increase of 17% in relation to A0.
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) Table 18. Effect of amino acid fertilizer on radish yield and utilization of nitrogen and phosphorus 35 day after sowing (g m–2)
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
increased in AAF treatments due to the increase of biomass production (Table 18).
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
could enhance the ability of uptake and assimilation of inorganic N by plants.
Vegetation cover (%)
Net N uptake (g m–2)
Treatments
25 DAS
35 DAS
35 DAS
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
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) Table 19. Net nitrogen uptake and vegetation cover of radish
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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 cabbage growth. However, among the
treatments of AAF, the growth responses were decreased by increasing the application rate of AAF. This may probably be related to the feed–back inhibition of high rate application of amino acids.
6.2.3 Effect of AAF on contents of N and P
The data in Table 20 showed that N contents of the plants were affected by using amino acid fertilizer. The NO3– content of radish was decreased by application of AAF ( P < 0.05) compared with the reference treatment. Among the treatments, A1 gave the best result in
reducing the nitrate to 1.16 mg g–1 (FW), with a decrease of 24% in relation to the highest NO3– content found in A0. This result agrees with the interpretation that amino acid can
negatively regulate nitrate content in higher plants (Gunes et al., 1994, 1996; Chen and Gao, 2002; Wang et al., 2004). But this interpretation was not supported in all cases. It was
observed that the mixed amino acids increased NO3– content slightly in radish when the
plants growing in nutrient solution. The contradiction may reside in amino acids treatment method. It was demonstrated in other studies that amino acid pretreatment decreased NO3–
accumulation slightly, but Gln and Asn led to NO3– concentration increase in barley roots when they were used together with nitrate (Aslam et al., 2001).
With respect to the main products of NO3– assimilation, amino acids and proteins (Table 20), the plants in treatment A1 gave the highest contents of these compounds ( P < 0.01). In the A1 treatment, high activities of main enzymes of NO3– assimilation could explain the
predominance of these nitrogenous compounds in radish. Under treatments of A2 and A3,
the increases of amino acids and proteins derived from the direct uptake of amino acids
from AAF.
NO3–
Amino acids
Proteins
Total-N
Total-P
Treatments
(mg g–1 FW)
(mg g–1 DW)
A0
1.53 ± 0.11 a
1.29 ± 0.02 b
1.28 ± 0.08 c
27.2 ± 1.6 c
6.8 ± 0.3 a
A1
1.16 ± 0.17 b
1.38 ± 0.02 a
1.98 ± 0.16 a
35.9 ± 1.6 a
7.1 ± 0.4 a
A2
1.32 ± 0.20 ab 1.33 ± 0.02 ab
1.74 ± 0.06 b
33.0 ± 0.9 ab
6.8 ± 0.4 a
A3
1.48 ± 0.08 a
1.32 ± 0.04 ab
1.70 ± 0.09 b
31.2 ± 1.2 bc
6.9 ± 0.5 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) Table 20. Effect of amino acid fertilizer on contents of nitrogen and phosphorus in radish 23
day after sowing
The total N content of the plants was also affected significantly by the use of AAF ( P < 0.01).
Treatments of A1, A2 and A3 showed to increase the total N to 32%, 21% and 15% relative to the control, respectively. These increases were due to the positive adjusting of AAF on uptake and assimilation of N, and attributing to the increases of N utilization and net N uptake. The P
content of radish was not affected significantly by the application of AAF (Table 20).
Effect of Mixed Amino Acids on Crop Growth
149
6.2.4 Effect of AAF on chemical properties of soil
The chemical properties of soil in middle growth period and at the end of experiment were showed in Table 21 and Table 22. The planting of radish affected total N of soil clearly, except at 35 days after sowing, with a fall of 10% compared with non planting treatment.
However, there were no differences in total N of soil among treatments planted with radish.
On the other hand, either planting treatment or AAF treatment showed effect on nitrate in soil.
Organic
Available
Total
Treatments
pH EC
matter
P2O5
N
NO3––N
(1:5) (mS
m–1) (g
Kg–1) (mg
Kg–1) (g
Kg–1) (mg
Kg–1)
NP
6.3 81 15.4 267 0.70 75.3
A0
6.3 57 15.2 297 0.67 52.5
A1
6.4 55 15.6 285 0.67 55.7
A2
6.5 65 15.1 310 0.67 58.9
A3
6.4 54 15.3 305 0.66 60.0
Table 21. Chemical properties of soil in the middle of growth period (25 day after sowing) for radish
In the soil of non planting, nitrate was decreased by leaching and runoff by rain. Compared with the non planting treatment, the treatments of planting showed 20~30% decrease at 25
days after sowing and 23~42% decrease at 35 days after sowing in the nitrate content of soil.
Although with the lowest net N uptake, the lowest concentration of nitrate in soil was found in A0 treatment both at two sampling times. This was due to the fact that the vegetation
covers of AAF treatments were higher than treatment of A0, and could effectively prevent
nitrate of soil from leaching or runoff. The planting treatments showed lower values of EC
than non planting treatment, but all were in the range of general soil. There were no
significant differences among all treatments in pH and organic matter of soil. Moreover,
very small differences were observed in available P due to different growth rate of the
plants.
Organic
Available
Total
pH EC
Treatments
matter
P2O5
N
NO3––N
(1:5) (mS
m–1) (g
Kg–1) (mg
Kg–1) (g
Kg–1) (mg
Kg–1)
Before
6.0 191 15.8 279 0.87 191.2
experiment
NP 6.4
99
15.9
308
0.70
94.3
A0 6.4
41
16.0
283
0.63
55.0
A1 6.4
42
15.8
263
0.63
65.9
A2 6.4
49
15.2
270
0.63
65.0
A3 6.4
45
15.3
272
0.63
73.2
Table 22. Chemical properties of soil at the end of field experiment (35 day after sowing) for radish
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Agricultural Science
NO3– removal
Removal rate
Removal rate by
Treatments
(0~20cm)
by plant
leaching
(g m–2) (%)
NP 25.2
–
100.0
A0 35.4
26.3
73.7
A1 32.6
44.4
55.6
A2 32.8
39.7
60.3
A3 30.7
41.8
58.2
Table 23. Effect of amino acid fertilizer on nitrate removal from the soil
The data of NO3– removal are showed in Table 23. Even though the highest NO3– removal
was found in treatment A0, the most removed NO3– was leached (73.7%) and would lead to
pollution for groundwater. The application of AAF can enhance NO3– removal rate by
planting, and avoid N losses through leaching and runoff due to increases of N utilization (Table 18) and vegetation cover (Table 19).
In conclusion, the results of the present experiment suggest that application of amino acid fertilizer can affect activities of three enzymes of N assimilation (NR, NiR and GS) and
increase the growth and N assimilation in radish. However, the exact reason for t