centrifuge tubes, adding 5 cm
3
of 0.05 mol m
− 3
imidazole pH 7.9 buffer containing 0.005 mol m
− 3
ditiothreitol. The homogenates were cen-
trifuged 30 min at 15000 g. The supernatants were collected and centrifuged again for 10 min at
15000 g, kept in ice and used for GS and NADH- GDH assays as well as for the determination of
soluble protein contents according to Bradford 1976.
GS was assayed following Pe`rez-Soba et al. 1994. The standard assay mixture 0.5 cm
3
con- sisted of 0.1 mol m
− 3
imidazole buffer pH 7.5, 0.048 mol m
− 3
NH
2
OH, 0.040 mol m
− 3
MgCl
2
, 0.32 mol m
− 3
glutamate, 0.05 mol m
− 3
ATP. Adding 0.15 cm
3
of crude enzyme extract started the reaction. After incubation for 1 h at 30°C, the
d -glutamyl-hydroxamate GH formed was deter-
mined by adding 1.0 cm
3
of ferric chloride reagent 0.37 mol m
− 3
FeCl
2
, 0.67 mol m
− 3
HCl, and 0.2 mol m
− 3
trichloroacetic acid and measuring ab- sorbance at 540 nm with a spectrophotometer.
Activity of GS was expressed as mmoles of GH formed per g
− 1
fresh weight fr. wt. h
− 1
and specific activity of GS as mmoles GH formed
mg
− 1
protein h
− 1
. GDH was assayed by means NADH oxidation
at 30°C according to Cammaerts and Jacobs 1985. The 3.0 cm
3
assay mixture consisted of 0.1 mol m
− 3
Tris – HCl buffer pH 8.2, 0.15 mol m
− 3
NH
4 2
SO
4
, 0.02 mol m
− 3
a -ketoglutarate,
0.001 mol m
− 3
CaCl
2
, and 0.14 mol m
− 3
NADH. Adding 0.5 cm
3
of the crude enzyme extract started the reaction. NADH consumption was
followed spectrophotometrically at 340nm. The blank did not have a-ketoglutarate. GDH activity
was expressed as mmoles NADH g
− 1
fr. wt. min
− 1
.
2
.
5
. Statistical analysis Each experimental treatment consisted of three
replicates, each one containing nine plants. The experiments described in Section 2.2 were carried
out twice and the results represent the mean of two experiments. Therefore, there were 54 values
per growth parameter per treatment n = 54 and six replicates per biochemical analysis per treat-
ment n = 6 Data were statistically analyzed us- ing the one-way analysis of variance. Tukey’s
multiple range test was used to compare the means of all nitrogen treatments. In the tables and
graphic representations, values marked with dif- ferent letters are different at
P B 0.05.
3. Results
3
.
1
. Plant growth After 30 days of incubation in different nitro-
gen sources, the highest plant growth rates were found in glutamine treated-plants Table 1. Plant
dry matter accumulation in the presence of glu- tamine was about 30 to 63 higher than plants
grown in inorganic nitrogen. Dry matter parti- tioning to plant parts was slightly affected by
glutamine and inorganic nitrogen sources. The shootplant ratio indicated a tendency of dry mat-
ter accumulation mainly in the shoots, which was enhanced by glutamine Table 1.
Table 1 Effects of inorganic and organic nitrogen sources 6 mM on the dry matter accumulation, height, and root length of C. fimbriatum
plants n = 54 after 30 days of incubation NO
3 −
:NH
4 +
ratio = 2:3
a
Length cm Dry matter mg
Root Plant
Nitrogen sources Shoot
Root Shoot
ShootPlant ratio 10.6 9 0.5
b
5.6 9 0.2
b
NO
3 −
5.0 9 0.4
ab
3.4 9 0.3
b
3.1 9 0.2
a
0.53 9 0.02
b
12.9 9 0.8
b
0.52 9 0.02
b
NO
3 −
:NH
4 +
3.5 9 0.2
a
3.5 9 0.2
b
6.2 9 0.5
ab
6.7 9 0.4
b
0.57 9 0.02
ab
3.0 9 0.2
a
3.5 9 0.2
b
4.3 9 0.2
b
NH
4 +
6.0 9 0.4
b
10.3 9 0.5
b
16.8 9 1.1
a
10.5 9 0.6
a
Glutamine 6.4 9 0.6
a
4.2 9 0.2
a
3.1 9 0.2
a
0.63 9 0.01
a
a
bold superscript letters compare values inside the columns.
Fig. 1. Relative growth rates Wt
30
− Wt
Wt of entire
plants, shoot and root of C. fimbriatum plants n = 54 incu- bated in different NO
3 −
:NH
4 +
ratios. Bars represent the stan- dard error.
NH
4 +
concentration was raised Fig. 2C and Fig. 3. The maximum free ammonium level was ob-
served in the root of plants cultivated with glu- tamine Fig. 2C.
3
.
3
. Nitrate tissue contents The methodology used for nitrate ion determi-
nation in this work Cataldo et al., 1975 was not accurate enough to detect the presence of this ion
in shoot and root tissues of C. fimbriatum plants, even in those plants grown with NO
3 −
as sole nitrogen source.
3
.
4
. Chlorophyll contents Higher concentration of NO
3 −
than NH
4 +
in the media reduced shoot chlorophyll content Fig. 4.
The levels of root chlorophyll represented about 20 – 27 of the shoot chlorophyll contents. Maxi-
mum root chlorophyll amounts were observed in plants incubated with a 1:1 NO
3 −
: NH
4 +
ratio.
3
.
5
. Nitrogen assimilating enzymes acti6ity
3
.
5
.
1
. NR In vivo NR activity was detected only in plants
grown in NO
3 −
added media Table 2. NH
4 +
ion inhibited NR activity when its concentration was
higher than the NO
3 −
concentration in the culture media NO
3 −
:NH
4 +
2:3 ratio. In the other three NO
3 −
:NH
4 +
proportions, the activity of NR was quantitatively similar and showed the same pat-
tern of distribution among plant parts Table 2. About 70 of NR activity of C. fimbriatum plants
was observed in the apical root region 2 cm.
3
.
5
.
2
. GS and GDH The highest rates of GS activity and GS specific
activity were found in the shoots of C. fimbriatum plants Tables 3 and 4. According to the treat-
ment, the activity and specific activity of GS in roots ranged from about 5 to 13 of the values
measured in the shoots.
Shoot GS activity was only slightly influenced by nitrogen sources. Increased levels of GS activ-
ity in shoot tissues was found in plants grown in media containing NH
4 +
as the major nitrogen form Table 3. Nevertheless, shoot GS activity
NH
4 +
as the only nitrogen source resulted in relatively low growth rates when compared to
plants grown with different NO
3 −
:NH
4 +
ratios Fig. 1. Root growth appeared to be limited by
NH
4 +
supplied alone. Plants fed with NO
3 −
:NH
4 +
ratio 3:2 showed better vegetative appearance as well as a more vigorous root system than those
grown in the other NO
3 −
:NH
4 +
ratios not shown.
3
.
2
. Free amino-N, soluble sugars and free ammonium contents
Regardless of the nitrogen form used, the levels of free amino-N and soluble sugars in the shoot
were higher than those found in roots Fig. 2A and B. The free amino-N content of the shoot
increased with exogenous NH
4 +
concentration as well as in the presence of glutamine. A significant
high level of free amino-N was observed in the root of glutamine-treated plants. Soluble sugar
contents decreased in the shoot and root of plants supplied with NH
4 +
and glutamine. Fig. 2B. The free NH
4 +
amounts in both shoot and root tissues were relatively low in NO
3 −
treated plants Fig. 2C and Fig. 3. The highest free NH
4 +
contents of shoot tissues were found in plants grown in the presence of glutamine Fig. 2C.
However, the concentration of free ammonium in root tissues increased to the extent that external
B A
B A
A d
dc c
b a
0,4 0,8
1,2 1,6
1:0 3:2
1:1 2:3
0:1 NO
3
-
:NH
4
+
ratio mo
le s
N H
4 +
g
-1
fr w
t.
SHOOT ROOT
2 4
6 8
10
moles amino N g
-1
fr. wt
SHOOT ROOT
10 20
30 40
50
moles soluble sugar g
-1
fr.wt
A C
B D
a b
b c
C B
B A
d c
b a
0,4 0,8
1,2 1,6
NO3- NO3-NH4+
NH4+ Glutamine
Nitrogen sources
moles free NH
4 +
g
-1
fr wt.
A
B
C
a A
C C
c B
b b
a
A AB
A B
B
c cb
a b
cb
50 100
150 200
250 300
1:0 3:2
1:1 2:3
0:1
NO
3 -
:NH
4 +
RATIO mg chlorophyll g
-1
fr. wt
Shoot Root
was unaffected in a second group of experiments Table 4. Shoot GS specific activity was not
affected by the nitrogen sources. Nitrogen sources had no influence on GS activities in roots Tables
3 and 4.
Glutamine and inorganic forms of nitrogen had no effect on GDH activity in the shoots Tables 3
and 4.
However, root
GDH activity
was enhanced by NH
4 +
and by glutamine. A positive
Fig. 3. Free NH
4 +
amounts in shoot and root of C. fimbriatum plants grown for 30 days in different NO
3 −
:NH
4 +
ratios.
Fig. 2. Tissue contents of amino N 1A, soluble sugar 1B and free NH
4 +
1C in C. fimbriatum plants incubated on glutamine and inorganic nitrogen forms 6 mol m
-3
for 30 days. The NO
3 −
:NH
4 +
ratio was 2:3. Fig. 4. Shoot and root chlorophyll contents of C. fimbriatum
plants incubated in different NO
3 −
:NH
4 +
ratios 6 mol m
− 3
. N = 6.
correlation between NH
4 +
concentration in the media and the level of GDH activity in root
tissues was observed.
3
.
6
. pH of culture media C. fimbriatum plants grown for 30 days caused
a substantial decrease in the pH of culture media in all nitrogen sources studied Table 5. This
effect was attenuated by the presence of NO
3 −
ions and intensified by NH
4 +
as isolated nitrogen source Table 5.
Table 2 Nitrate reductase activity nmoles NO
2 −
g
− 1
fr. wt h
− 1
in various parts of C. fimbriatum plants n = 6 grown for 30 days in media with different NO
3 −
:NH
4 +
ratios 6 mol m
− 3
a
NO
3 −
:NH
4 +
Ratio 3:2
Plant part 1:1
1:0 2:3
0:1 65.4 9 11.8
A
62.9 9 12.3
A
Root apice 25.0 9 3.5
B
64.7 9 6.4
A
nd
b
Mature root 6.2 9 2.5
A
9.8 9 3.9
A
5.0 9 1.5
A
3.8 9 1.6
A
nd 12.3 9 4.0
A
13.8 9 3.4
A
9.2 9 1.7
A
nd Bulb
21.0 9 7.7
A
6.0 9 1.3
A
7.0 9 1.6
A
6.7 9 1.7
A
4.6 9 0.9
A
Leaf nd
Total 93.5 9 20.4
A
98.6 9 17.3
A
88.7 9 19.0
A
42.6 9 6.6
B
nd
a
Bold superscript capital letters compare values among the columns.
b
nd means non detected NR activity. Table 3
Ammonium assimilating enzyme activity in shoot and root tissues of C. fimbriatum plants n = 6 grown for 30 days in glutamine and inorganic nitrogen forms 6 mol m
− 3
. In this experiment the NO
3 −
:NH
4 +
ratio was 2:3
a
Root Shoot
GS GDH
GS GDH
Activity
d
Activity
d
Specific activity
c
Nitrogen sources Activity
b
Activity
b
Specific activity
c
0.28 9 0.04
a
0.08 9 0.01
c
NO
3 −
3.3 9 0.1
b
1.01 9 0.02
a
0.08 9 0.02
a
0.39 9 0.03
a
0.36 9 0.02
a
0.24 9 0.02
a
NO
3 −
:NH
4 +
4.2 9 0.2
a
0.96 9 0.02
a
0.10 9 0.01
a
0.14 9 0.01
b
0.25 9 0.03
a
0.46 9 0.05
a
0.19 9 0.01
a
NH
4 +
0.10 9 0.02
a
4.0 9 0.1
a
0.98 9 0.01
a
0.36 9 0.04
a
0.24 9 0.02
a
Glutamine 3.8 9 0.01
ab
1.00 9 0.03
a
0.18 9 0.01
a
0.08 9 0.01
a
a
Superscript bold letters compare values inside the columns.
b
GS activity — moles glutamyl hydroxamate min
− 1
g
− 1
fr. wt.
c
GS specific activity — moles glutamyl hydroxamate min
− 1
mg
− 1
protein.
d
GDH activity — moles NADH oxidized min
− 1
g
− 1
fr. wt. Table 4
Ammonium assimilating enzyme activity in shoot and root tissues of C. fimbriatum plants n = 6 grown for 30 days in five different NO
3 −
:NH
4 +
ratios 6 mol m
− 3
a
Root Shoot
GS GDH
GS GDH
Activity
d
Activity
a
Specific activity
c
NO
3 −
:NH
4 +
ratio Activity
b
Activity
d
Specific activity
c
0.38 9 0.02
a
0.29 9 0.01
a
1:0 3.1 9 0.1
a
1.02 9 0.01
a
0.06 9 0.01
d
0.06 9 0.02
a
0.41 9 0.02
a
0.08 9 0.01
d
0.31 9 0.02
a
3:2 0.08 9 0.03
a
3.7 9 0.2
a
1.05 9 0.01
a
0.36 9 0.01
a
0.28 9 0.01
a
1:1 3.2 9 0.2
a
1.00 9 0.04
a
0.07 9 0.03
a
0.09 9 0.01
c
0.13 9 0.02
b
0.21 9 0.01
a
0.32 9 0.01
a
2:3 0.08 9 0.01
a
3.8 9 0.1
a
1.00 9 0.04
a
0.07 9 0.02
a
0.39 9 0.02
a
0.23 9 0.02
a
0.15 9 0.01
a
0:1 3.3 9 0.1
a
0.94 9 0.01
a
a
Superscript bold letters compare values inside the columns.
b
GS activity — moles glutamyl hydroxamate min
− 1
g
− 1
fr wt.
c
GS specific activity — moles glutamyl hydroxamate min
− 1
mg
− 1
protein.
d
GDH activity — moles NADH oxidized min
− 1
g
− 1
fr. wt.
Table 5 pH values of culture media after 30 days of growth of C.
fimbriatum plants n = 5 in different nitrogen forms 6 mol m
− 3
: the initial pH was 5.5 Nitrogen forms
pH 4.62 9 0.05
a
NO
3 −
3.98 9 0.05
b
NO
3 −
:NH
4 +
NH
4 +
3.55 9 0.04
c
4.60 9 0.03
a
Glutamine pH
Nitrogen forms NO
3 −
:NH
4 +
4.61 9 0.04
a
1:0 3:2
4.05 9 0.04
b
1:1 4.00 9 0.04
b
2:3 3.96 9 0.03
b
0:1 3.70 9 0.03
c
than when
treated with
inorganic nitrogen
Chapin et
al., 1993.
Seedlings of
Hakea Proteaceae metabolize glycine, with rapid trans-
fer of
15
N to serine and other amino acids. Fur- thermore, plant species from the subtropical
heathland of Australia were shown to take up on average three-fold greater quantities of glycine
than NO
3 −
Schmidt and Stewart, 1999. While free amino-N contents increased in both
ammonium and glutamine-treated plants, soluble sugars presented the highest levels in nitrate-fed
plants Fig. 2A and B. In NH
4 +
cultured plants, ammonium is converted to amino acids and
amides in roots, and transported to the leaves Barneix and Causin 1996. Absorbed ammonium
ions are immediately assimilated by GS-GOGAT glutamine synthetase and glutamine-2-oxoglu-
tarate-amino-transferase to form glutamine and glutamate Lea et al., 1990; Sechley et al., 1992.
Besides its role in primary NH
4 +
assimilation, the GS-GOGAT cycle assimilates NH
4 +
from the photorespiratory
nitrogen cycle
and from
catabolism of storage and transport compounds Lea et al., 1990. Ammonia-grown plants often
have higher free amino-N concentration in their tissues than nitrate fed plants Barneix and
Causin, 1996. The availability of carbon skele- tons is necessary to prevent the toxic effects of
NH
4 +
Magalha˜es et al., 1992 and may be re- lated to the depletion of soluble carbohydrates
observed in ammonium and glutamine fed plants Fig. 2B and C. Feng et al. 1998 have shown
that the uptake and assimilation of exogenous ammonium by maize roots is followed by an
intense endogenous production of ammonium from the catabolism of soluble organic N.
Our results
suggest that
glutamine-treated plants accumulate amides glutamine and as-
paragine in their roots Fig. 2A. Glutamine can be used as a substrate in a number of transamida-
tion reactions, e.g. glutamate synthase GOGAT, asparagine synthetase AS and carbamoyl-phos-
phate
synthetase CPS.
Glutamine is
also transaminated or used in the synthesis of proteins
and in the export of carbon and nitrogen to other parts of the plant Sechley et al., 1992. The
transfer of amino nitrogen from glutamine to keto acids or amino acids requires the availability of
4. Discussion
