50
-1 h
A0
a
FW
40
A1
-1
b
g
A2
4O
c
2
30
N
a
a
a
10H5 20
mol C
10
GSA (
0
Shoot
Root
Fig. 5. Effect of mixed amino acids on glutamine synthetase activity in radish at 24 h after treatment. Values are means ± SD (n=5).
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.
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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)
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 NO –
3 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.
) 100
A0
- FW
80
A1
l g
A2
60
mo
40
ake (
- upt
20
3
NO
0
0
5
10
15
20
25
Time (h)
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 NO –
–
3 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).
Effect of Mixed Amino Acids on Crop Growth
131
NO3– NO2–
Treatments
Leaf Root Leaf Root
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
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).
) 0.5
a
-1 h
A0
0.4
A1
FW-1
A2
- g 2 0.3
a
a
a
b
0.2
c
mol NO
A ( 0.1
NR
0.0
Leaf
Root
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).
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Agricultural Science
0.6
)
a
1- h 0.5
A0
b
b
A1
- FW 0.4
A2
a
- g 2 0.3
b
l NO
mo 0.2
c
(A 0.1
NiR
0.0
Leaf
Root
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).
) 30
-1 h
a
25
A0
b
FW
b
A1
-1 g
A2
4
20
O 2N10 15
H 5
l C 10
a
ab
b
mo
5
GSA (
0
Leaf
Root
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
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
(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.
Amino acids
Proteins
Treatments
Leaf Root Leaf Root
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 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
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Agricultural Science
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 NO –
3 soil
4.1 Materials and methods
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
were sown into 100 mL pots filled with the incubated soil and grown in a glasshouse.
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
KOH appropriately.
Composition of treated solutions (mM)
Applied time
Treatments
K+
Ala
β–Ala Asp Asn Glu Gln Gly
DAS
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
* Same amount of distilled water sprayed
Table 8. Composition of the treated solutions and application times for radish in pot
experiment
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
yield and N assimilation. After harvest the soils were collected for chemical analysis.
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.
Effect of Mixed Amino Acids on Crop Growth
135
3
)-1
a
) h
ab
b
b
(FW 2
-1
- g 2
1
mol NO
NRA ( 0
A0
A1
A2
A3
Treatments
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).
)-1 8
a
ab
) h
ab
b
(FW 6
-1
- g 2
4
l NO
mo 2
RA (
Ni 0
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).
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Agricultural Science
)-1 15
) h
a
ab
(FW
ab
-1
b
g 10
4
O 2N10H5 5 C
mol
A (
0
GS
A0
A1
A2
A3
Treatments
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.
Amino acids
Proteins
NO
Treatments
3–
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
sowing in pot experiment with high NO3– soil
Effect of Mixed Amino Acids on Crop Growth
137
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).
Fresh weight
Dry weight
N utilization
Treatments
(g/plant) (mg/plant)
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 sowing i