2
.
3
. Mn and Fe analyses Mn and Fe concentrations mg g FW were
determined in leaves of Mn-tolerant and Mn-sensi- tive plants by atomic absorption spectrophotome-
try Perkin – Elmer 370 after the samples had been rinsed in distilled water, dried at 80°C for 24 h and
wet-ashed in a nitric and perchloric acid mixture 5:2 vv on an electric thermostatic plate at 300 –
350°C.
2
.
4
. Statistical analyses All measurements were made in triplicate. All
values reported have been expressed as means of triplicates 9 SE. The significance of differences be-
tween control and treated mean values have been evaluated by Student’s t-test and reported in the
tables as P 5 0.05 and P 5 0.01.
3. Results
3
.
1
. Mn and Fe uptake Leaf Mn content increased with rising metal
concentrations in the medium for both Mn-toler- ant and Mn-sensitive plants. At 2 mM Mn
medium concentration the Mn-tolerant and Mn- sensitive plants showed approximately the same
leaf metal concentrations, whereas at 5 mM Mn the tolerant plants showed a higher concentration
of Mn almost double than the Mn-sensitive plants treated with the same metal concentration
Table 1. Furthermore to the increase in leaf Mn concentration, a corresponding parallel decrease
of leaf Fe concentration occurred, also shown in Table 1 r = − 0.84, P B 0.01.
3
.
2
. Enzyme acti6ity
3
.
2
.
1
. Stem The GPXs of the extracellular fluid showed a
reduced activity compared to the control either in the Mn-tolerant or Mn-sensitive plants treated
with high concentrations of Mn Table 2. In the Mn-sensitive plants treated with 2 mM Mn the
activity of the POD of the ionically and covalently bound wall fractions was also depressed. By con-
trast, 5 mM Mn tolerant plants showed an en- hanced activity of the covalently bound wall POD
Table 2.
The activity of syringaldazine POD in the extra- cellular fluid and in the ionically bound wall frac-
Table 1 Mn and Fe concentrations in leaves of Mn-tolerant and Mn-sensitive N. tabacum regenerated plants
a
Treatments Mn-sensitive plant
Mn-tolerant plant Metal content
Mn 2 mM Control
C+Mn 5 mM C+Mn 2 mM
Mn 5 mM 422.3 9 49.5
7255.0 9 104.5 11521.6 9 304.7
8021.5 9 630.5 6247.3 9 1040.3
Mn 171.3 9 9.2
Fe 248.0 9 37.0
483.7 9 51.1 156.0 9 15.7
277.7 9 37.7
a
Values are means of three replicates 9 SE. Concentrations are reported in mg g
− 1
DW. Table 2
Guaiacol extracellular peroxidase activity in stems of Mn-tolerant and Mn-sensitive N. tabacum regenerated plants
a
Treatments Mn-sensitive plants
Mn-tolerant plants Guaiacol peroxidase activity
C+Mn 5 mM Mn 2 mM
Control C+Mn 2
Mn 5 mM mM
582 9 32 570 9 130
1518 9 70 Extracellular fluid D
470
min
− 1
mg
− 1
protein 825 9 167
569 9 87 7.0 9 2.2
6.1 9 0.9 4.0 9 0.5
Ionically wall bound D
470
min
− 1
mg
− 1
protein 2.0 9 0.3
7.6 9 1.5 3.6 9 1.2
2.7 9 0.1 6.4 9 0.6
3.9 9 0.6 Covalently wall bound D
470
min
− 1
mg
− 1
FW 3.8 9 0.3
a
Values are means of three replicates 9 SE. P]0.055; P]0.01.
Table 3 Syringaldazine extracellular peroxidase activity in stems of Mn-tolerant and Mn-sensitive N. tabacum regenerated plants
a
Treatments Siringaldazine peroxidase activity
Mn-tolerant plants Mn-sensitive plants
Mn 2 mM Mn 5 mM
Control C+Mn 2
C+Mn 5 mM mM
Extracellular fluid D
530
min
− 1
mg
− 1
protein 5051 9 9380 4134 9 400
4979 9 745 6483 9 880
4162 9 713 Ionically wall bound D
530
min
− 1
mg
− 1
protein 4.6 9 0.5
4.4 9 0.1 3.3 9 0.1
2.3 9 0.5 3.5 9 0.2
6.7 9 0.9 9.3 9 0.9
5.1 9 0.9 6.4 9 0 4
4.9 9 0.9 Covalently wall bound D
530
min
− 1
mg
− 1
FW
a
Values are means of three replicates 9 SE. P]0.05; P]0.01. Table 4
Enzyme activity in leaves of Mn-tolerant and Mn-sensitive N. tabacum regenerated plants
a
Treatments Enzyme activity
Mn-tolerant plants Mn-sensitive plants
Mn 2 mM Control
Mn 5 mM C+Mn 2
C+Mn 5 mM mM
30.56 9 5.67 12.17 9 1.05
11.83 9 2.29 3.80 9 0.70
Guaiacol peroxidase D
470
min
− 1
mg
− 1
9.05 9 2.75 protein
Ascorbate peroxidase D
290
min
− 1
mg
− 1
1.44 9 0.25 0.58 9 0.09
0.24 9 0.09 0.16 9 0.03
0.12 9 0.01 protein
0.25 9 0.01 0.21 9 0.03
0.32 9 0.02 Glutathione reductase D
340
min
− 1
mg
− 1
0.33 9 0.02 0.34 9 0.04
protein 36.87 9 14.79
40.87 9 10.66 Superoxide dismutase U mg
− 1
protein 33.23 9 2.12
91.93 9 3.07 56.50 9 8.68
a
Values are means of three replicates 9 SE. P]0.05; P]0.01.
tion did not vary in the different treatments, ex- cept for the Mn-sensitive plants treated with 2 mM
Mn Table 3. In this case the ionically bound wall activity decreased in comparison to the control.
The effect of Mn on the covalently bound wall syringaldazine POD was identical to that observed
in the guaiacol: the activity was significantly higher in the Mn-tolerant plants grown in the
presence of 5 mM Mn Table 3.
3
.
2
.
2
. Leaf The behavior of the enzymes involved in the
antioxidant defense in the presence of elevated tissue Mn concentrations was practically similar
for the Mn-tolerant and Mn-sensitive plants. The increase of Mn content inhibited the activity of
GPX, ASPX and SOD in the Mn-tolerant as well as in the Mn-sensitive plants Table 4. GPX
activity was reduced to about 40 of the control in Mn-tolerant plants and to 12 in the Mn-sensi-
tive ones. ASPX activity was 11 of the control in the Mn-sensitive plants treated with 2 mM Mn,
whereas it was 40 of the control in the Mn-toler- ant ones. The activity of SOD was depressed to
40 of the control in both Mn-sensitive and Mn- tolerant plants with the 2 mM Mn treatment. At
the highest Mn treatment 5 mM the inhibitory effect remained constant in the Mn-tolerant plants
and Mn-sensitive plants. However, the effect was greater in the Mn-sensitive plants. The activity of
GR did not show significant variation, except for the 2 mM Mn-sensitive plants, where an enhanced
activity was detected.
4. Discussion
