Mechanism Of Oxidative Stress In Plants And Its Regulation
RESULTS
In the present study, an attempt has been made to study the mechanism of oxidative stress
in plants and its regulation through various factors. For this a total of six experiment has
been included comprising both petridish and pot culture. Light, 2,4-D and Cd have been
analyzed as oxidative stress causing agent and their effects have been mentioned on
various plants such as black gram, pea maize and wheat. In order to study oxidative stress
a number of parameter has been analyzed. For studying regulation or remediation of
oxidative stress certain mineral nutrients like copper and phosphorus were also given to
study any positive alteration they cause in plants. In the present investigations
morphological parameters such as germination percentage, plumule length, radical
length, fresh and dry matter yield, and moisture percentage were recorded after 15 days in
petridish experiments and 45 days in pot experiments in pea, black gram. To study the
oxidative stress, stress indicator enzymes like catalase, peroxidase, superoxide dismutase,
ascorbate peroxidase, and glutathione reductase and stress indicator components like
proline, cystiene and non-protein thiol group were studied. For studying membrane
permeability, two important parameters i.e. electrolyte leakage percentage and membrane
lipid peroxidation were also studied. Different pigment contents (chlorophyll a & b, total
chlorophyll and carotenoid) were also studied. Accumulation of Fe and Zn in root and
shoot estimated through spectroscopic as well as through deposition method whereas Cd
accumulation was studied only through spectroscopic method. All the results are shown
in the table 4.1.1 to 4.9.4, figures 1 to 11 and photo plates 1 to 9, all data represent mean
± SE of triplicates in appropriate units. Least significant difference was used to observe
the level of significance at P<0.05 and **- value significant at P<0.01 levels.
4.1 PETRIDISH CULTURE:
4.1.1 Effect of light on seedling growth pigment contents, antioxidative enzymes,
amino acids and lipid peroxidation in black gram seedlings.
The results obtained in this experiment are given in table 4.1.1, figure 1 and 2, and photo
plate 1. Table 4.1.1 shows the effect of different levels of light on plumule and radical
length, FW, DW, pigment contents, enzymes activity, accumulation of proline, cysteine,
50
Mechanism Of Oxidative Stress In Plants And Its Regulation
non-protein thiol group and lipid peroxidation in the black gram seedlings. In this study,
photosynthetic efficiency measured in the term of photosynthetic yield FW and DW of
seedlings have been found to be very low which was also comparable to photosynthetic
pigment contents. As the light intensity increased the pigment synthesis together with
biomass also increased. The FW, DW and moisture percentage in black gram seedling
treated with 75μmol m-2s-1 were 1100mg, 89mg and 92% compared to control seedlings
where values were 623mg, 61.3mg and 90%. In response to increasing light intensity
pigment synthesis increased in black gram leaves. Results have shown that Chl T, Chl a,
Chl b and carotenoids contents were significantly (P<0.01) increased to 80, 67, 70 and
123% in leaves of black gram exposed to higher irradiance levels 75μmol m-2s-1
compared to control seedlings 4 m-2s-1. Table 4.1.1 exhibited the change in activity of
various enzymes such as CAT, POX, and SOD in black gram seedling under various
level of light. In this study, CAT activity increased as the light intensity increased that
protects plants from damaging effect of H2O2. The activity of catalase in shoot for control
plants was 653 which was significantly (p<0.01) increased by 114 and 99% for 40 and 50
μmol m-2s-1. The activity of POX in control seedling was 18.2 mg-100 FW tissue which
was reduced by 19.5, 28, 37.8 and 51.3% for 30, 40, 50 75 μmol m -2s-1 (figure 1). The
value of SOD for control seedling (4μmol m-2s-1.) was 12.5 mg-100 FW tissue which was
change to 23.5, 18.9, 16.7 and 15.8 mg-100 FW tissue under 30, 40, 50 75 μmol m-2s-1. On
increasing light intensity SOD activity increased to 87% in the leaves of black gram
plants exposed to 30 μmol m-2s-1. Variation in the synthesis of SOD under fluctuating
light environment could be adaptive features of plants to light. Table 4.1.1 shows the
effect of various level of light on synthesis of proline cysteine and NP-SH in black gram
seedlings. Compare to control seedlings synthesis of proline, cysteine and non-protien
thiol group increased with increase in light intensity. The value of proline, cysteine and
non-protien thiol group for control seedling was 0.70, 1.65 and 0.85 respectively which
was significantly (P<0.01) increased to 57, 42 and 53% in black gram seedlings exposed
to 75 μmol m-2s-1. Membrane integrity or permeability is responsible for the lives of cell.
In this case, Lipid peroxidation increased with increase in light intensity. The value of
MDA content in control seedlings was observed as 69 units which were reduced by 15.9,
26, 30.4 and 71% in black gram seedlings exposed to 30, 40, 50 and 75μmol m-2s-1.
51
Mechanism Of Oxidative Stress In Plants And Its Regulation
Table 4.1.1
52
Mechanism Of Oxidative Stress In Plants And Its Regulation
Chl T,
Chl a,
** Chl b,
Carotenoids
3
*
*
mg g-1 FW
2.5
2
1.5
**
**
*
*
*
*
1
**
**
*
20
ΔOD mg-100 FW
3.5
0.5
POX
25
*
15
*
**
10
5
0
0
4
30
40
50
4
75
30
40
50
75
Light intensity (μmol m-2 s-1)
Light intensity(μmol m-2 s-1)
Figure 1: Effect of light on pigment content and peroxidase activity in the leaves of black gram
seedlings at 15 days.
1.4
1.2
1
0.8
0.6
0.4
0.2
0
MDA
**
*
nmol g-1 FW
μmol g-1 FW
Proline
4
30
40
50
Light intensity (μmol m-2 s-1)
75
80
70
60
50
40
30
20
10
0
**
4
30
40
50
75
Light intensity (μmol m-2s-1)
Figure 2: Effect of light on proline and MDA content in the leaves of black gram seedlings at 15 days.
53
Mechanism Of Oxidative Stress In Plants And Its Regulation
4.1.2 Effect of 2,4-D on seedling growth pigment contents, antioxidative enzymes,
amino acids and lipid peroxidation in black gram seedlings.
In present study, toxicity imposed by 2,4-D on black gram seedling were observed in
petridish culture. Leaves of thirteen days old seedlings were exposed to different doses of
2,4-d and for the evaluation of their toxic responses, physiological and biochemical
parameters were analyzed in leaves after 48 hour treatment. The results obtained in this
experiment are tabulated in table 4.2.1, figure 3, and photo plate 2. Table 4.2.1 shows that
exposure to 2,4-D significantly (P<0.01) reduced the shoot and root length. Shoot length
decreased by 16 and 30% exposed to 50 and 100ppm 2,4-D respectively, while root
length decreased by 45 and 69% at the similar 2,4-d exposure. After 48 hours of
treatment fresh weight (FW) of whole seedling significantly increased by 45% (P<0.05)
and 53.7% (P<0.01) at 10 and 30ppm of 2,4-D respectively then declining trend was
observed at higher dose but not less than control seedlings. As the FW, DW was not
significantly increased at 10 and 30 ppm of 2,4-D, despite its significantly (P<0.05)
decreased by 28.6% at 100ppm of 2,4-D. Result of moisture % obtained was similar to
that of FW of seedlings. Moisture % significantly (P<0.05) and (P<0.01) increased at 10
and 30ppm 2,4-D concentration respectively. . Result of moisture % obtained was similar
to that of FW of seedlings. Moisture % significantly (P<0.05 and P<0.01) increased both
at 10 and 30ppm 2,4-D concentration. After 48 hour of treatment pigment concentration
significantly (P<0.05) reduced only at higher dose of 2,4-D. Concentration of chlorophyll
and carotenoids increased in the leaves of black gram exposed to lowest dose of 10ppm
2,4-D while, concentration of Chl T, Chl a, Chl b and Carotenoid significantly (P<0.01)
reduced by 16.2, 39, 15 and 39% at 100ppm 2,4-D, respectively table 4.2.1. In respect to
study defense mechanism of black gram seedlings against toxicity of 2,4-D, antoxidative
enzymes have been studied. The activity of CAT, POX and SOD was found to be varied
with different doses of 2,4-D. Activity of CAT was found to be increased at all doses of
2,4-D treatment while POX activity decreased at all doses of 2,4-D treatment (table 1.1).
In response to that CAT activity significantly (P<0.01) increased by 88 and 102% in the
leaves of black gram exposed to 30 and 50ppm of 2,4-D then slightly declining trend in
CAT activity was observed at higher dose but was not less than that of control value. In
this work, POX activity was significantly (P<0.01) inhibited by 53.8 and 50.5% in leaves
54
Mechanism Of Oxidative Stress In Plants And Its Regulation
of black gram seedling exposed to 10 and 30ppm of 2,4-D respectively. Table 4.2.1
Result showed that activity of SOD was found to be gradually increased as the
concentration of 2,4-D increases and significant (P<0.05) increment was observed at
50ppm and then decreased by 12% at higher dose(100ppm) compared to control
seedlings. Rather than antioxidative enzymes, antioxidant compound such as NP-SH,
cysteine and proline also plays vital role in detoxification of 2,4-D induced toxicity.
Synthesis of all these antioxidant was found to be increased under 2,4-D toxicity, which
suggest their participation in detoxification of 2,4-d mediated toxicity in plants. NP-SH
synthesis was found to be significantly (P<0.05) increased by 88% in the leaves of black
gram seedling exposed to 100ppm of 2,4-D while proline synthesis significantly (P<0.01)
increased by 85% at similar exposure of 2,4-D. 100ppm 2,4-D treated leaves of black
gram accumulate 100% more proline content than control seedlings. Membrane was the
main site of 2,4-D action so their evaluation was necessary. Membrane damage was
measured in the term of malondialdehyde content. MDA content was found to be
increased in the leaves of black gram on 2,4-D exposure. MDA content significantly
(P<0.01) increased by 49% in leaves exposed to 100ppm of 2,4-D.
55
Mechanism Of Oxidative Stress In Plants And Its Regulation
Table 4.1.2
56
Mechanism Of Oxidative Stress In Plants And Its Regulation
4.1.3 Effect of Cadmium on black gram and wheat seedling.
(A) Effect of Cadmium on seedling growth pigment contents, antioxidative enzymes,
amino acids and lipid peroxidation in black gram seedlings.
The results obtained in this experiment are tabulated in table 4.3.1, figure 4, and photo
plate 3. This experiment was performed to explore the detrimental effect of cadmium on
black gram seedlings. The petridish culture experiment was conducted for 15 days. Table
4.3.1 shows the effect of cadmium on plumule and radical length, number of lateral roots,
fresh and dry matter weight, MP, pigment contents. Cd treatment reduces plumule and
radical length, fresh and dry weight and number of lateral roots. Compared to control
seedlings, Plumule length and radical length was significantly (P<0.01) decreased to
18.6% and 62% in black gram seedlings exposed to 1.0mM Cd. The number of lateral
roots in control seedlings was 6.66 which were reduced to 3.00 in black gram seedlings
exposed to 1.0 mM Cd. The FW of control seedlings was 913 mg which was reduced to
810, 748, 671 and 566 mg in black gram seedlings exposed to 0.1, 0.2, 0.4 and 1.0 mM
Cd. Dry weight of black gram seedlings significantly (P<0.01) reduced by 40% as
compared to control seedlings. The chlorophyll (a, b and total) contents were declined
with increasing concentration of Cd. In control the total chlorophyll contents were 1.83
mg/g which was slightly increase in 0.1 mM Cd (2.00 mg/g) and then it significantly
(P<0.05, P<0.05) decreased to 1.48 mg/g and 1.37 mg/g at 0.4 and 1.0 mM Cd
respectively. As compared to control (1.23 mg/g), Chl a, was decreased to 0.83 mg/g in
leaf of black gram exposed to 1.0mM Cd. In control the Chl a, was 0.71 mg/g recorded
which was reduced to 0.53 mg/g in black gram seedlings exposed to 1.0 mM Cd. In
control the carotenoids contents were 0.56 mg/g which was slightly increase in 0.1 and
0.2 mM Cd treated seedlings and then it significantly (P<0.01) reduced to 0.40 mg/g at
1.0 mM Cd( table 4.3.1). The activity of antioxidative enzymes- CAT, POX and SOD
was slightly increased at low dose of Cd while at higher dose it was decreased. The CAT
activity was 1100 in control which significantly (P<0.01) increased to 1800 in seedlings
exposed to 1.0 mM Cd. Compared to control (14.1) seedling activity of POX significantly
(P<0.01) increased to 24.0 at 0.1 mM Cd then significantly decreased to 12.5 at 1.0mM
of Cd. Similarly activity of SOD also increased at low dose of Cd then decreased at
higher dose. The value of SOD activity in control seedlings was 24.5 which were
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Mechanism Of Oxidative Stress In Plants And Its Regulation
decreased to 6.4 in seedlings exposed 1.0 mM of Cd. The Proline content was
significantly higher in the seedlings treated with Cd than in the control. Contents of
proline gradually increase on increasing Cd concentration (table 4.3.1). The value of
proline contents for control seedlings was 0.7 which increased to 0.85, 1.1, 1.7 and 1.9 in
black gram seedlings exposed to 0.1, 0.2, 0.4 and 1.0 mM of Cd. Similarly cystiene
values were also increased to 1.9, 2.25, 2.65 and 3.2 in black gram seedlings compared to
control (1.65). The value of NP-SH in control plants was 0.85 which was significantly
(p<0.01) increased by 12, 29, 47 and 65% for 0.1, 0.2, 0.4 and 1.0 mM Cd concentrations
(table 1.3). The value of MDA content for control plants was 35.6 nmol g-1 FW tissue
which were significantly (p<0.01) increased by 100, 135, 163 and 180% for 0.1, 0.2, 0.4
and 1.0mM Cd concentrations (table 4.3.1).
(B) Effect of cadmium and its interaction with phosphorus and Copper on seedling
growth pigment contents, antioxidative enzymes, amino acids, lipid peroxidation
and metal accumulation in wheat seedlings.
The results obtained in this experiment are given in table 4.4.1 to 4.4.6, figure 5 to 6, and
photo plate 4. In the present study, significant decrease was observed in shoot length, on
increasing doses of cadmium in the treatment. Shoot length for the control plants was 14
cm. which reduced to 11, 10.8, 9.0 and 8.0 cm. for 0.1, 0.2, 0.4 and 1.0mM Cadmium
concentrations (table 4.4.1). On rectification with P and Cu at 0.4 mM Cd treatment shoot
length showed increase and became 13.8 and 11cm. respectively. At 1.0 mM Cd
concentration shoot length increased by 25 and 12.5 % for P and Cu rectification
respectively. Relative to control plants, growth of the root was drastically reduced due to
Cd at all tested concentration. A significant decrease was found in root length, on
increasing doses of Cd in treatment. Roots length showed two times reduction than the
shoot length, this could be due to direct contact of Cd of root to toxic metals. Table 4.4.1
shows that the FW for shoot in controlled group was 0.94 g. decrease percentage was
10.6, 31.0, 35 and 36 % for 0.1, 0.2, 0.4 and 1.0mM Cd concentrations. On rectification
with P and Cu at 0.4 mM Cd concentration, FW significantly increased by 42.6 and
32.8% respectively. While there was no significant increase in FW of shoot at 1.0 mM Cd
reified with P and Cd. The value of FW of root for control plants was 0.67 which was
significantly reduced by 70, 70, 73 and 74% for 0.1, 0.2, 0.4 and 1.0mM Cd
concentrations. On rectification with P and Cu FW of root increase 50 and 11% was
58
Mechanism Of Oxidative Stress In Plants And Its Regulation
observed at 0.4mM Cd concentration. DW for shoot in controlled group was 0.14 g.
which was significantly decreased at all tested Cd concentration. Decrease percentage
was 44.3, 54, 60 and 79 % for 0.1, 0.2, 0.4 and 1.0mM Cd concentrations. On
rectification with P and Cu at 0.4mM Cd concentration, shoot DW significantly increased
at p<0.05 level. At 1.0mM Cd concentration DW increase of 55.5 and 9.25 % was
observed for P and Cu respectively. The value of DW of root for control plants was 0.045
which was significantly reduced by 56, 62, 62 and 64% for 0.1, 0.2, 0.4 and 1.0mM Cd
concentrations. On rectification with P and Cu DW of root increase 35 and 18% was
observed at 0.4 mM Cd concentration. Moisture percentage for shoot in controlled group
was 85% (table 4.4.1). Increase percentage was 6.9, 5.8, 6.6 and 7.2% for 0.1, 0.2, 0.4
and 1.0mM Cd concentrations. On rectification with P and Cu at 0.4 mM Cd
concentration, shoot moisture percentage decrease to 2.5 and 0.77% respectively. At 1.0
mM Cd concentration, moisture % corresponding decrease of 4.7 and 2.8 % was
observed for P and Cu respectively. Moisture percentage for root in controlled group was
93.3%. Significantly decrease percentage was 3.5, 2.4, 2.3 and 2.2 % for 0.1, 0.2, 0.4 and
1.0.0mM Cd concentrations. On rectification with P and Cu at 0.4 mM Cd concentration,
root moisture percentage decreased to 0 .5 and 1.0 respectively. The value of total
chlorophyll for control plants was 1.85 which was significantly reduced by 19, 23, 38 and
66% for 0.1, 0.2, 0.4 and 1.0mM Cd concentrations (table 4.4.2). However, increase of
25.7 and 55.5% was observed for 0.4 and 1.0mM Cd treatment on rectification with P.
The value of Chl a, for control plants was 1.18 which reduced by 20, 20, 37 and 66% for
0.1, 0.2, 0.4 and 1.0mM Cd concentrations. However, significant increase of 57% was
observed for 1.0mM Cd treatment on rectification with P. On rectification with Cu
increase of 11 and 30% was observed at 0.4 and 1.0 mM Cd concentration. The value of
Chl b, for the control plants was 0.70 which significantly reduced to 23, 30, 40 and 65 for
0.1, 0.2, 0.4 and 1.0mM Cd concentrations. On rectification with P and Cu at 0.4 mM Cd
treatment Chl b, showed increase and value became 0.48 and 0.34 respectively. At 1.0
mM Cd concentration value of Chl b, increased of 41 and 20 % for P and Cu rectification
respectively. Synthesis of carotenoids significantly (p<0.01) reduced with increasing
concentration of Cd (table 4.4.2). As compared to control plant carotenoid content
decreased by 8.0, 27, 30 and 51% at 0.1, 0.2, 0.4 and 1.0 mM Cd levels. On rectification
with P and Cu increase of 6 and 20% was observed at 0.4 mM Cd concentration and 12.5
59
Mechanism Of Oxidative Stress In Plants And Its Regulation
and 8.3 at 1.0 mM. The Cd accumulation in the shoots and roots of wheat grown at
different Cd concentrations are shown in table 4.4.3 and figure 8. The accumulation of
Cd in shoots increased with the increase in Cd concentrations (P < 0.01), peaked and
reached a maximum of 94.6 and 97.4 µg g-1 DW at 0.4 and 1.0mM Cd treatment
respectively. The value of Cd accumulation for shoot in controlled group was 2.09 µg g-1
DW, which increased to 35, 54, 54 and 97 µg g-1 DW at 0.1, 0.2, 0.4 and 1.0 mM Cd
concentrations respectively. On rectification with P and Cu at 0.4 mM Cd concentration,
Cd content significantly (0.05) decreased by 29.6 and 28.3%. Accumulation of Cd
decreased by 24.3 and 18.8% at 1.0mM Cd treatment rectified with P and Cu
respectively. The value of Cd accumulation in root for control plants was 11.7 which was
significantly (p<0.01) increased by 872, 907, 940 and 981% for 0.1, 0.2, 0.4 and 1.0 mM
Cd concentrations (table 4.4.3 and figure 8).On rectification with P and Cu at 0.4 mM Cd
concentration, Cd content decreased by 2.3 and 3.9% in root, while at 1.0 mM decreased
% was 1.8 and 4.64% respectively. The value of Fe content in shoot for control plants
was 19.8 which was significantly (p<0.01) increased by 63% at 0.1 mM Cd treatment.
Iron accumulation decreased by 5.1 and 11.5% at 0.4 and 1.0 Cd treatment respectively
(table 4.4.3 and figure 8). On rectification with P and Cu at 0.4 Cd level significant
increased accumulation of iron was observed in wheat shoot. A significant decrease in
iron accumulation was noticed at 1.0mM Cd level rectified with Cu as compared to
treated plants. The value of Fe content in root for control plants was 20.76 which was
significantly (p<0.01) increased by 159% at 0.4 mM Cd treatment. Iron accumulation
decreased by 43 and 21% at 0.1 and 0.2 Cd treatment respectively. On rectification with
P and Cu at 0.4 Cd level significant increased accumulation of iron was observed in
wheat root. Accumulation of Iron content increased at 0.4 mM Cd level rectified with P.
A significant (p<0.01) increase in iron accumulation was noticed at 1.0 mM Cd level
rectified with copper as compared to treated plants. The value of Zn accumulation in
shoot for control plants was 12.44 which were increased by 35, 13.7 and 2.8% for 0.1, 0.2
and 0.4 mM Cd concentrations (table 4.4.3 and figure 8). A significant (p<0.01) increase
in Zn accumulation at 0.1 mM Cd concentration was observed in wheat shoot. On
rectification with P and Cu at 0.4 mM Cd concentration, Zn content in case of P increased
by 14.5% while in case of Cu significantly decreased by 37.5%.similar results were
observed at 1.0 mM treatment. The value of Zn accumulation in root for control plants
60
Mechanism Of Oxidative Stress In Plants And Its Regulation
was 15.5 which were decreased by 6, 26, 31, and 33% for 0.1, 0.2, 0.4 and 1.0 mM Cd
concentrations. A significant (p<0.05) decrease in Zn accumulation at 0.4 and 1.0 mM Cd
concentration was observed in wheat root. On rectification with P and Cu at 0.4 mM Cd
concentration, Zn content in case of P increased by (p<0.05), 14.5% while in case of Cu
significantly increased by (p<0.01) 37.5%. Similar results were observed at 1.0 mM
treatment. The value of MDA content for control plants was 21.1 which were
significantly (p<0.01) increased by 41.7, 51.6, 78 and 96% for 0.1, 0.2, 0.4 and 1.0 mM
Cd concentrations (table 4.4.4 and figure 9). On rectification with P and Cu at 0.4 mM Cd
concentration, MDA content decreased by 5.7 and increased by 17.2% respectively.
However, significantly (p<0.05) increase of 21.2% was observed for 1.0 mM Cd
treatment on rectification with Cu. The value of MDA content for control plants was 6.5
which were significantly (p<0.01) increased by 146, 153 and 158% for 0.2, 0.4 and 1.0
mM Cd concentrations. On rectification with Cu at 0.4 and 1.0 mM Cd concentration,
MDA content significantly increased by 63.6 and increased by 80% respectively. The
activity of CAT was measured in term of H2O2 decomposed/g fresh weight tissue. The
activity of CAT in shoot for control plants was 226 which was significantly (p<0.01)
increased by 377 and 400% for 0.1, 0.2 mM Cd concentrations while at 0.4 mM Cd
treatment increased (p<0.05) percent was 182 (table 4.4.5 and figure 10). On rectification
with P and Cu at 0.4 mM Cd treatment activity of CAT, showed significant (p<0.01)
increase and value became 1260 and 1320 respectively. However P rectification showed
significant increase in CAT activity at 1.0mM Cd treatment. The activity of CAT in root
for control plants was 231 which was significantly (p<0.01) increased by 71 and 125%
for 0.1, 0.2 mM Cd concentrations while at 1.0 mM Cd treatment decreased (p<0.05)
percent was 54. On rectification with P at 0.4 mM Cd treatment activity of CAT, showed
significant (p<0.05) increase and value became 461. However at 1.0 mM both
phosphorus and copper rectification showed significant (p<0.01) increase in CAT
activity. The activity of POX was measured in term of H2O2 decomposed/g fresh weight
tissue. The activity of POX in shoot for control plants was 10.74 which was significantly
(p<0.01) increased by 14.7 and 29.6% for 0.2, 0.4 mM Cd concentrations while at 1.0
mM Cd treatment significantly decreased (p<0.01) percent was 40.7 (table 4.4.5). On
rectification with Phosphorus and copper at 0.4 mM Cd treatment activity of POX,
showed significant (p<0.05) and (p<0.01) decrease and value became 12 and 10.6
61
Mechanism Of Oxidative Stress In Plants And Its Regulation
respectively. However at 1.0 mM Cd treatment both rectification showed significant
increased at p<0.01 level. The activity of POX in root for control plants was 17 which
were increased by 2.9, 11 and 18% for 0.1, 0.2 and 0.4 mM Cd concentrations while at
1.0 mM Cd treatment decreased percent was 11. On rectification with P and Cu at 0.4
mM Cd treatment activity of POX, showed significant (p<0.05) and (p<0.01) decrease
and value became 12 and 10.1 respectively. Similarly at 1.0 mM Cd treatment both
rectification showed significant decreased at p<0.01 level. The activity of SOD was
measured in term of unit mg-1 protein. The activity of SOD in shoot for control plants
was 154.16 which was significantly (p<0.01) increased by 114% for 0.2 mM Cd
concentrations while at 0.4 and 1.0 mM Cd treatment decreased percent was 28 and 43
respectively (table 4.44). On rectification with P and Cu at 0.4 mM Cd treatment activity
of SOD, showed significant (p<0.05) decrease and value became 25.2 and 32.2
respectively. However, on rectification with P and copper SOD activity decrease 55 and
75% was observed at 1.0 mM Cd concentration. The activity of SOD was measured in
term of unit mg-1 protein. The activity of SOD in root for control plants was 56.5 which
was significantly (p<0.01) increased by 60 and 165% for 0.1 and 0.2 mM Cd
concentrations while at1.0 mM Cd treatment decreased percent was 45.2. On rectification
with P and Cu at 0.4 mM Cd treatment activity of SOD, showed significant (p<0.01) and
(p<0.05) increase and value became 116 and 92.5 respectively. However, on rectification
with P and copper SOD activity significantly (p<0.01) increase 185 and 173% was
observed at 1.0 mM Cd concentration. The activity of APX was measured in term of unit
g-1 fresh weight tissue. The activity of APX in shoot for control plants was 48.1 which
was significantly (p<0.01) increased by 45% for 0.4mM Cd concentration (table 4.4.4).
On rectification with P and Cu at 0.4 mM Cd treatment activity of APX, showed
significant (p<0.05) decrease and value became 25.2 and 32.2 respectively compared to
plant treated alone Cd. However, on rectification with P and Cu APX activity
significantly (p<0.01) decreased 70% was observed at 1.0mM Cd concentration. The
activity of APX in root for control plants was 53 which was significantly (p<0.05)
decreased by 15.5% at 0.2mM Cd concentrations while at 0.04 and 1.0mM Cd treatment
decreased percent was 32 and 40.8 respectively. On rectification with Phosphorus and
copper at 0.4 mM Cd treatment activity of APX, showed significant (p<0.05) decrease by
copper and value became 43 compared to plant treated alone with Cd. However, on
62
Mechanism Of Oxidative Stress In Plants And Its Regulation
rectification with P and Cu APX activity significantly (p<0.01) increase 11.5 and 52.8%
was observed at 1.0 mM Cd concentration. The Proline content was significantly higher
in the seedlings treated with Cd than in the control. Proline synthesis was different in
different plant tissue. Therefore, the Proline contents in shoot were respectively 0.63, 1.1,
1.36, 1.53, and 2.2 at 0, 0.1, 0.2, 0.4, and 1.0 mM of Cd which, in comparison to the
control, increased by respectively 75, 115, 142 and 249% (table 4.4.6 and figure 7).
However, in root were respectively 0.50, 0.85, 1.16, 1.25, and 1.6 at 0, 0.1, 0.2, 0.4, and
1.0 mM of Cd which, in comparison to the control, increased by respectively 70, 132, 150
and 220%. On rectification with P and Cu at 0.4 mM Cd concentration, shoot proline
content decrease to 16.3 and 18.3% respectively. At 1.0 mM Cd concentration, proline
concentration corresponding decrease of 38.6 and 25 % was observed for Phosphorus and
copper respectively. Cysteine involved in the synthesis of heavy metal-binding peptides
which involved in buffering cytosolic metal concentration. The accumulation of cystiene
in the shoots and roots significantly (p<0.01) increased with the increase in Cd
concentrations (table 4.4.6 and figure 7). Therefore, the cystiene contents in shoot were
respectively 3.25, 5.30, 5.95, 6.21, and 8.75 at 0, 0.1, 0.2, 0.4, and 1.0 mM of Cd which,
in comparison to the control, increased by respectively 63, 83, 91 and 169%. On
rectification with Phosphorus and copper at 1.0 mM Cd concentration, root cystiene
content significantly (p<0.01) increased to 20 and 23% respectively. However, in root
were respectively 2.65, 4.65, 5.12, 5.65, and 6.65 at 0, 0.1, 0.2, 0.4, and 1.0 mM of Cd
which, in comparison to the control, increased by respectively 75, 93, 113 and 150%. On
rectification with P at 0.4 and 1.0 mM Cd concentration, root cystiene content decrease to
18 and 20% respectively. While application of copper at 0.4 mM Cd concentration,
significantly (p<0.05) enhanced the cystiene accumulation in the root. The value of NPSH in shoot for control plants was 1.17 which was significantly (p<0.01) increased by 23,
32, 68 and 45% for 0.1, 0.2, 0.4 and 1.0 mM Cd concentrations (table 4.4.6 and figure 7).
However, significantly (p<0.01) decrease of 15% was observed for 0.4 mM Cd treatment
on rectification with Cu. %. On rectification with P at 1.0 mM Cd concentration, thiol
content significantly (p<0.01) increase to 19% in shoot. However NP-SH in root for
control plants was 0.63 which was significantly (p<0.01) increased by 307, 439 and
492% for 0.2, 0.4 and 1.0 mM Cd concentrations.
63
Mechanism Of Oxidative Stress In Plants And Its Regulation
Table 4.3.1
64
Mechanism Of Oxidative Stress In Plants And Its Regulation
Table 4.4.1
65
Mechanism Of Oxidative Stress In Plants And Its Regulation
Table 4.4.2
66
Mechanism Of Oxidative Stress In Plants And Its Regulation
Table 4.4.3
67
Mechanism Of Oxidative Stress In Plants And Its Regulation
Table 4.4.4
68
Mechanism Of Oxidative Stress In Plants And Its Regulation
Table 4.4.5
69
Mechanism Of Oxidative Stress In Plants And Its Regulation
Table 4.4.6
70
Mechanism Of Oxidative Stress In Plants And Its Regulation
Proline
MDA
1.4
**
*
1
nmol g-1 FW
μmol g-1 FW
1.2
0.8
0.6
0.4
0.2
0
4
80
70
60
50
40
30
20
10
0
30
40
50
75
Concentration 2,4-D (ppm)
4
30
40
50
Concentration 2,4-D (ppm)
75
Shoot length
Root length
20
18
16
14
12
10
8
6
4
2
0
FW mg seedling-1
Length (cm)
Figure-3 : Effect of 2,4-D on proline and MDA contents in leaves of black gram seedling at 15 days.
0
0.1
0.2
0.4
Treatments
1
1000
900
800
700
600
500
400
300
200
100
0
0
0.1
0.2
0.4
Treatments
1
Figure 4: Effect of Cd on shoot and root length and FW of black gram seedlings at 15 days.
71
Mechanism Of Oxidative Stress In Plants And Its Regulation
50 μmol m-2s-1
75 μmol m-2s-1
40 μmol m-2s-1
30 μmol m-2s-1
4 μmol m-2s-1
PHOTOPLATE 1. Effect of light on black gram seedlings growth.
Control
10 ppm
30 ppm
50 ppm
100 ppm
PHOTOPLATE 2. Effect of 2,4-D on black gram seedling growth.
Control
0.1 mM Cd
0.2 mM Cd
0.4 mM Cd
1.0 mM Cd
PHOTOPLATE 3. Effect of Cadmium on black gram seedling growth.
72
10
9
8
7
6
5
4
3
2
1
0
Cadmium
Iron
Zinc
120
Proline
Cysteine
NP-SH
100
80
Cd (µg g-1 DW)
Fe (µg g-1 DW)
Zn (µg g-1 DW)
Proline (µmol g-1 FW)
Cysteine ( mM g-1 FW)
NP-SH (mM g-1 FW)
Mechanism Of Oxidative Stress In Plants And Its Regulation
60
40
20
0
Treatments
Treatments
MDA contents (nmol g-1 FW)
60
shoot
root
50
40
30
20
10
0
Treatments
Catalase activity (H2O2 decompose
mg-100 FW)
Figure 5: Changes in accumulation of proline, cystiene and non protein thiol and accumulation of
cadmium, Iron and non zinc content of shoot of wheat seedlings exposed to Cd, Cd+P and Cd+Cu
observed at 15 days. ± Represent SE. *- value significant at P<0.05 and **- value significant at
P<0.01 levels.
shoot
root
1400
1200
1000
800
600
400
200
0
Treatments
Figure 6: Effect of Cd on MDA contents and activity of CAT in shoots and roots of wheat seedlings
observed at 15 days. ± Represent SE. *- value significant at P<0.05 and **- value significant at
P<0.01 levels.
73
Mechanism Of Oxidative Stress In Plants And Its Regulation
PHOTO PLATE - 4
Control
0.1 mM Cd
0.2 mM Cd
0.4 mM Cd
1.0 mM Cd
4a- Effect of Cadmium on wheat seedlings.
Control
1.0 mM Cd
0.1 mM Cd+P
0.1 mM Cd+Cu
4b- Effect of Cadmium on wheat seedlings.
74
Mechanism Of Oxidative Stress In Plants And Its Regulation
4.2 POT CULTURE:
4.2.1 Effect of light on seedling growth pigment contents, antioxidative enzymes,
amino acids and lipid peroxidation in black gram and maize plants observed at 45
days.
This experiment was performed in soil under different shades of sun light to study the
role of light intensity in generating oxidative stress in plants.
Light intensity was
controlled by shading plant pot with 0, 1, 2, 3 and 4 layer of muslin cloth to achieve (343,
185, 78, 46 and 40μmolm-2s-1). After 45 days of sowing the leaves of plants were
collected to analyze physiological parameter like chlorophyll contents, enzymes activity
lipid peroxidation etc. the results of experiments are depicted in tables 4.5.1 to 4.5.2,
figure 7 and photo plate 5. Most common etiolating growth was observed in black gram
than maize exposed to low shades of sun irradiance. In black gram, plants height
significantly (P<0.01) increased by 42 and 33% exposed to 78 and 46% µmolm -2s-1,
while in maize, plants height significantly (P<0.01) increased by 66.4% exposed to
185µmolm-2s-1and decreased by 23% at 40 µmolm-2s-1 compared to plant exposed to
normal sun irradiance 343 µmol m-2 s-1 (table 4.5.2 and figure 11). Biomass yields were
expressed in the term of FW and DW of plants. FW of black gram plant decreased by
43.5% exposed to 185µmol m-2 s-1, while at the same exposure FW of maize increased by
3.0% compared to plants exposed to 343µmol m-2 s-1. DW was reduced in both plants
exposed to different shades of irradiance. But reduction in DW in black gram was so high
compared to maize. Decrease percent of DW in black gram was 51.9, 77.4, 80.2 and
86.0% exposed to 185, 78, 46 and 40 µmolm-2s-1 levels of sun irradiance while in maize it
was 31.0, 76.3, 85.8 and 84.4% at same exposure. The contents of total chlorophyll, Chl
a, Chl b and carotenoids were significantly modified under low light irradiance. The
effect of low irradiance on pigments synthesis in black gram and maize plants was given
in figure (table 4.5.1 to 4.5.2 figure 11). In black gram, pigment synthesis was higher
under low irradiance levels. The value of total chlorophyll in leaves of black gram
increased by 17, 44.0, 18.0 and 10.0% exposed to 185, 78, 46 and 40 µmolm-2s-1 of sun
irradiance, while in maize it was decreased by 15.0, 22.0, 29.0 and 38.0% at same
exposure of sun irradiance. Similar pattern of Chl a, Chl b and carotenoids synthesiswas
observed as the irradiance level decreased. Total chlorophyll, Chl a, Chl b and
carotenoids synthesis significantly (p<0.01) increased in leaves of black gram exposed to
75
Mechanism Of Oxidative Stress In Plants And Its Regulation
78µmol m-2 s-1 compared to fully exposed (343µmol m-2 s-1) plants. While in maize plant
synthesis of these pigments significantly (p<0.01) decreased by 38, 22, 48 and 34% at
40µmol m-2 s-1 sun irradiance respectively. Lipid peroxidation is the measurement of
membrane damage obtained by calculating malondialdehyde content. It is believed that
light intensity more than optimal/normal causes membrane damage but in this study,
increase in membrane damage was observed on decreasing irradiance levels of sun light.
MDA content, significantly (p<0.01) increased in leaves of black gram plants by 62% at
46µmol m-2 s-1, while in maize maximum value of MDA content 36.6 nmol g-1 FW was
observed at 78µmol m-2 s-1 sun irradiance. ELP is another parameter of measurement of
membrane damage. ELP also increases on decreasing irradiance levels in both plants. In
black gram, ELP increased by 18.4% at 48µmol m-2s-1 while, in maize leaves its value
gradually increases on decreasing irradiance and significantly (p<0.05) increased by 38%
at 48 µmol m-2 s-1. The activity of CAT was measured in term of H2O2 decomposed g-1
FW tissue. The activity of CAT in the leaf tissue of both plants decreased as the
irradiance levels decreased. The activity of CAT for control plants was 1560 in black
gram and 1360 in maize which was significantly (p<0.01) decreased by 51.0 and 39.0%
for 78 µmolm-2s-1 light concentrations respectively. The activity of POX in two plants
was inversely expressed in response to different irradiance of light. Hydrogen peroxides
and lipid peroxides are the main substrate for the POX enzymes. Increasing trend of POX
activity was observed with decreasing sun irradiance and reached significantly (p<0.01)
up to 59% at lowest 40µmolm-2s-1sun irradiance in black gram. In spite in case of maize,
activity of POX decreased with decreased in sun irradiance and reached to 4.3 unit mg-1
which was 38% less than activity of POX in plant exposed 343µmolm-2s-1. SOD activity
responded to light treatments, but response was more significant (p˂0.05) in leaf of maize
than black gram. In maize plants SOD activity gradually decreases as the intensity of
light decreased. But in black gram lowest value of SOD was noticed at 185 µmol m -2s-1,
then begin to increase and reach to significant (p<0.05) at 40 µmol m-2s-1. In present
study, NP-SH synthesis showed decreasing trend under decreasing levels of sun
irradiance. In black gram, NP-SH synthesis significantly (p<0.01) decreased by 26.6 and
25.0% at 185 and 78µmol m-2 s-1 compared to plant exposed normal sun irradiance while
in maize significant (P<0.05) reduction was observed at 78 µmolm-2s-1.
76
Mechanism Of Oxidative Stress In Plants And Its Regulation
Table 4.5.1
77
Mechanism Of Oxidative Stress In Plants And Its Regulation
Table 4.5.2
78
Mechanism Of Oxidative Stress In Plants And Its Regulation
Plant height
Maize
**
70
60
50
40
30
20
10
0
cm.plant-1
Black gram
*
**
343
185
* **
**
78
43
40
Light intensity (µmol m-2s-1
Black gram
Fresh weight
Maize
50
g.plant-1
40
30
*
20
*
10
*
**
*
**
0
343
185
78
43
40
Light intensity (µmol m-2s-1
Black gram
Total chlorophyll
4
**
3
mg g-1 fw
Maize
*
*
**
2
1
0
343
185
78
Light intensity (µmol
43
40
m-2s-1
79
Mechanism Of Oxidative Stress In Plants And Its Regulation
Black gram
µmol H2O2 decompose fw-100
Catalase
1600
1200
*
**
800
*
**
*
**
**
400
0
343
185
78
43
Light intensity (µmol
40
m-2s-1
Black gram
4
mM g-1 fw
Maize
Maize
NP-SH
3
*
**
**
*
**
* 2.7
2
1
0
343
185
78
43
Light intensity (µmol
40
m-2s-1
Black gram
Maize
ELP
60
percentage value
50
*
40
*
30
20
10
0
343
185
78
43
40
Light intensity (µmol m-2s-1
Figure 7. Effect of light intensity on plant height, fresh weight, total chlorophyll, catalase, peroxidase,
non-protein thiol group and electrolyte leakage % in mature leaves of two different plants viz. black
gram and maize observed at 45 days. Bars represent SE. *- value significant at P<0.05 and **- value
significant at P<0.01 levels.
80
Mechanism Of Oxidative Stress In Plants And Its Regulation
PHOTO PLATE - 5
343μmolm-2s-1
185μmolm-2s-1
78μmolm-2s-1
46μmolm-2s-1
40μmolm-2s-1
Effect of light on maize plants growth. Light intensity was controlled by shading plant pot with 0, 1, 2, 3 and 4
layer of muslin cloth to achieve (343, 185, 78, 46 and 40 μmol m -2 s-1).
343μmolm-2s-1
185μmolm-2s-1
78μmolm-2s-1
46μmolm-2s-1
40μmolm-2s-1
Effect light on black gram plants growth. Light intensity was controlled by shading plant pot with 0, 1, 2, 3 and
4 layer of muslin cloth to achieve (343, 185, 78, 46 and 40 μmol m-2 s-1).
81
Mechanism Of Oxidative Stress In Plants And Its Regulation
4.2.2 Effect of 2,4-D and its interaction with phosphorus and copper on plant height,
fresh and dry weight, pigments content, antioxidative enzymes, certain amino acids,
thiol contents, electrolyte leakage % and lipid peroxidation in pea and maize plant
observed at 45 days.
This experiment was conducted to evaluate the effect of 2,4-D in Pea and maize plants. P
and Cu were used to ameliorate the toxic effect of 2,4-D in plants. The plants were raised
in soil in earthen pots. Visual symptoms were observed day to day after treatment.
Various morphological and physiological parameters were recorded after 15 days of
treatments. Results obtained in this experiment are depicted in table 4.6.1 to 4.6.4, Figure
8 and 9 and photo plate 6. 2,4-D treatment promote stem curvature in both plants while
leaflets of pea leaves were observed to convert into tendrils and fruit become seedless.
Table 4.6.1 shows the data of various morphological parameters such as plants height,
FW, DW and MP. Plant height decreased with increasing the level of 2,-D from 62 cm in
control to 17.7, 19 and 22% in pea plants exposed to 500, 2000 and 4000ppm of 2,4-D
while in maize its were10, 16 and 20.6 % at same exposure. Two doses of 2,4-D i.e. 2000
ppm and 4000 ppm were recovered by 40 ppm P and 10 ppm of Cu through foliar
application. In pea, application of P improve plant height by 12 and 8% at 2000 and 4000
ppm of 2,4-D while at same exposure, Cu application enhance plant height by 8 and 4%.
2,4-D treatment declined the FW of pea as well as maize plants compared to control
plants. In pea FW for control plants was 14.8 g plant-1 which was reduced to 14.2, 14,
13.0 and 12 g plant-1 exposed to 100, 500, 2000 and 4000 ppm of 2,4-D. But P
application at 2000 ppm and 4000 ppm significantly increased FW of pea plant by 13.9
and 13% respectively while Cu has not significant mitigatory effect. While for maize
plants FW was significantly (p<0.01) decreased to 6.25, 7.89 and 11.2% for 500, 2000
and 4000 ppm 2,4-D concentrations. On rectification with P at 2000 ppm and 4000 ppm
2, 4-D concentration, plant FW significantly increased by 2.85 and 3.70 % respectively.
While on rectification with Cu, FW of maize plants treated with 2000 and 4000 ppm of
2,4-D increased to 1.7 and 0.0% respectively. Compared to control, at all doses of 2,4-D
treatment, DW of both plant was decreased while P application raised DW about 13.6%
and 10% respectively in pea and 3.58 and 1.40% in maize at 2000 and 4000 ppm 2,4-D
respectively. While Cu application raised DW to 6.8 and 5.0% at 2000 and 4000 ppm
2,4-D in pea. In pea plant, moisture % was increased with increase in 2,4-D concentration
82
Mechanism Of Oxidative Stress In Plants And Its Regulation
(table 4.6.1) While in maize moisture % decreased with increase in 2,4-D concentration.
A decrease in the total chlorophyll content was found at all concentration of 2,4-D in pea
leaves. At higher dose (4000 ppm) it was declined by 23%. The total chlorophyll content
significantly enhanced of about 11% at 4000 ppm of 2,4-D with P application. The value
of total chlorophyll in maize was significantly (p<0.05) increased by 6.95% at 100 ppm
while further increment of 2,4-D it was reduced to 2.17, 3.04, and 13.5% at 500, 2000
and 4000 ppm of 2,4- D concentrations respectively. However, increase of 6.72 and
15.6% was observed for 2000 and 4000 ppm 2, 4-D treatment on rectification with P,
while Cu not sowing mitigatory effect in respect to total chlorophyll. The Chl a content in
pea plant was significantly decreased with increasing 2,4-D concentration. Contents of
Chl a, for control was 1.79 which was reduced to 1.73, 1.67, 1.65 and 1.48 mg g-1 tissue
in the leaves treated with 100, 500, 2000 and 4000 ppm of 2,4-D. But P application at
two doses of 2,4-D ( 2000 and 4000 ppm) increase the Chl a by 10 and 13 % respectively
while Cu application at both doses of 2,4-D increase by 3%. The Chl a content in maize
plant was significantly decreased with increasing 2,4-D concentration. Contents of Chl a,
for control was 1.53 mg g-1 FW tissue which was reduced to 1.55, 1.50, 1.45 and 1.38 mg
g-1 tissue in the leaves treated with 100, 500, 2000 and 4000 ppm of 2,4-D. But P
application at two doses of 2,4-D (2000 and 4000 ppm) increase the Chl a by 8.2 and 0.0
% respectively while Cu application at 2000ppm increased the chlorophyll contents 4.8%
and at 4000ppm decreased by 11%. Contents of Chl b, for control pea leaves was 1.51
mg g-1 tissue which was reduced to 1.35, 1.18, 1.15 and 1.06 mg g-1 tissue in the leaves
treated with 100, 500, 2000 and 4000 ppm of 2,4-D. P and copper application at 2000
ppm of 2,4-D significantly improved the Chl b content by 20 and 13 %. The value of Chl
b was significantly (P<0.05) increased about 18.4% at 100 ppm and again it was reduced
to 13.1, 14.5 and 21% for 500, 2000, and 4000 ppm 2,4-D concentrations in maize leaves.
On rectification with P at 2000 ppm and 4000 ppm 2,4-D treatment Chl b, showed
increase and value became 1.08 and 0.92 mg g-1 respectively compared to unrectified
plants (table 4.7.3). The carotenoids content in pea plant was significantly decreased with
increasing 2,4-D concentration. Contents of carotenoids, for control was 0.92 mg g-1
tissue which was reduced to 0.72, 0.90, 0.85 and 0.79 mg g-1 tissue in the leaves treated
with 100, 500, 2000 and 4000 ppm of 2,4-D. But P application at two doses of 2,4-D (
2000 and 4000 ppm) increase the carotenoids by 2 and 1 % respectively while Cu
83
Mechanism Of Oxidative Stress In Plants And Its Regulation
application at both doses of 2,4-D increase by 30 and 8.8% (table 4.6.3 figure 12). In
maize, results shows contents of carotenoids significantly increased by 20.3, 14.5 and
4.34 % at 500, 2000 and 4000 ppm with increasing concentration of 2,4-D. On
rectification with P increase of 13.9 and 5.55% was observed at 2000 and 4000 ppm 2, 4D concentration respectively. While Cu application at 2000 and 4000 ppm of 2,4-D
carotenoid contents decreased. Compare to control, CAT activity was decreased at lower
dose of 2,4-D treatment while further enhancement in the activity of CAT was noticed as
2,4-D concentration increased (table 4.6.2). CAT activity in control leaves of pea plants
was 1340 which was reduced 1300, 1200 and 1270 at 100, 500, and 2000 ppm 2,4-D
treatment while increased to 1490 at 4000 ppm of 2,4-D treatment. At higher dose (4000
ppm) of 2,4-D treatment P significantly increase activity of CAT by 11.2% while at the
same Cu reduced by 27.5%. The activity of CAT in maize leaves for control plants was
290 which was significantly (p<0.01) decreased by 1.72, 46.5, 51.7% for 100, 500 and
2000 ppm 2,4-D while at 4000 ppm 2,4-D activity increased to 36% . However, P
rectification showed significant increase in CAT activity by 7.14 % at 2000 ppm 2, 4-D
treatment (table 4.7.2 and figure 14). In pea leaves, activity of POX for control was 5.2
which was 6.8, 5.9, 5.5and 4.7 for the plant exposed to 100, 500, 2000 and 4000 ppm of
2,4 –D. Application of P at 2000 ppm have not any change in POX activity but at 4000
ppm it was increased by 71%. Application of Cu at two doses of 2,4-D (2000 and 4000
ppm) increase the POX activity by 15 and 52 % respectively. While POX activity in
maize increased by 37.2% at 100 ppm 2,4-D which was decreased by 18% at 4000 ppm
2,4-D treatment (table 4.7.2). On rectification with P at 4000 ppm 2,4-D treatment, POX
activity showed significant (p<0.05) increased and value became 8.25 compared 5.44 in
2,4-D alone. In pea SOD activity was declined by 35.7% at 100 ppm whereas further
enhancement in 2,4-D doses it was
significantly increased. But P application
significantly decreased the activity of SOD about 10.5% at 4000 ppm while at same dose
of 2,4-D copper increase the SOD activity by 12.5%. While in maize, activity of SOD for
control was 22.0 which was increased to 43.0, 45.0, 48.6 and 51.7 unit in the leaves
treated with 100, 500, 2000 and 4000 ppm of 2,4-D. P application at two doses of 2,4-D (
2000 and 4000 ppm) also increase the SOD activity by 32 and 21 % respectively while
copper application at 2000 ppm increased the activity by 43.0% and at 4000ppm
decreased by 31.0% ( table 4.7.2).
84
Mechanism Of Oxidative Stress In Plants And Its Regulation
The NP-SH content in pea plant was significantly increased with increasing 2,4-D
concentration (table 4.6.4). Contents of NP-SH, for control was 1.10 mM g-1 FW tissue
which was increased to 1.30, 1.40, 1.50 and 1.78 mM g-1 tissue in the leaves treated with
100, 500, 2000 and 4000 ppm of 2,4-D. At 2000 ppm of 2,4-D P and Cu reduced the NPSH content by 13 and 10% while at 4000 ppm it was 27 and 13%. The value of NP-SH in
maize leaves was decreased by 2.15% at 100 ppm and then increased by 18.3, 59.1 and
80.6% for 500, 2000 and 4000 ppm 2,4-D concentrations. On rectification with P NP-SH
synthesis decreased by 14.4 and 20.2% at 2000 and 4000 ppm 2,4-D. While on
rectification with Cu at 2000 and 4000ppm 2, 4-D NP-SH contents was found to reduce
by 34.5 and 7.73% (table 4.7.4). The MDA content in pea plant was significantly
increased with increasing 2,4-D concentration. Contents of MDA, for control was 17.5
nmol g-1 which was increased to 19.8, 21.4, 22.3 and 23 nmol g-1 tissue in the pea leaves
treated with 100, 500, 2000 and 4000 ppm of 2,4-D (table 4.6.4). At 2000 ppm of 2,4-D P
and Cu reduced the MDA content by 18 and 18% while at 4000 ppm it was 16 and 13%.
While in maize, MDA content was increased by 10.83, 17.34, 29.5 and 40.4% for 100,
500, 2000 and 4000 ppm 2,4-D concentrations. On rectification with Phosphorus at 2000
and 4000 ppm 2,4-D concentration, MDA content was significantly increased by 32.3%
and 31.0%. While on rectification with copper it was increased by 63 and 61% (table
4.7.4). ELP for control pea plant was 29.5 % which was change to 28, 32, 38 and 45% in
plants exposed to 100, 500, 2000 and 4000 ppm of 2,4-D. Phosphorus and copper
application at 2000ppm of 2,4-D reduced the ELP by 18 and 11% while at 4000 ppm of
2,4-D it was increased by 16 and 11 % respectively (table 4.6.4). The ELP in the maize
leaves was decreased by 4 % at 100 ppm and then increased by 6.89, 21.7 and 54.1% at
500, 2000 and 4000ppm 2,4-D concentrations. On rectification with Phosphorus at 2000
ppm 2, 4-D concentration, it was decrease 15% (table 4.7.4).
85
Mechanism Of Oxidative Stress In Plants And Its Regulation
Table 4.6.1
86
Mechanism Of Oxidative Stress In Plants And Its Regulation
Table 4.6.2
87
Mechanism Of Oxidative Stress In Plants And Its Regulation
Table 4.6.3
88
Mechanism Of Oxidative Stress In Plants And Its Regulation
Table 4.6.4
89
Mechanism Of Oxidative Stress In Plants And Its Regulation
Table 4.7.1
90
Mechanism Of Oxidative Stress In Plants And Its Regulation
Table 4.7.2
91
Mechanism Of Oxidative Stress In Plants And Its Regulation
Table 4.7.3
92
Mechanism Of Oxidative Stress In Plants And Its Regulation
Table 4.7.4
93
4
3.5
3
2.5
2
1.5
1
0.5
0
** **
Chl T
Chl a
Chl b
* **
Carotenoids
**
** ** **
** ** **
* **
**
**
* ** **
* **
* * ** **
**
Pea
Maize
35
30
25
20
15
10
5
0
Plant height plant-1 (cm)
mg g-1 FW tissue
Mechanism Of Oxidative Stress In Plants And Its Regulation
1
2
3
4
5
6
7
8
9
Treatment
Treatments
Figure 8: Effect of 2,4-D and its interaction with Phosphorus and copper on total chlorophyll,
chlorophyll a, chlorophyll b and carotenoids contents in pea plants and plant height of pea and maize
plants.
NP-SH (mM g-1 FW tissue)
30
MDA( nmol g-1 FW tissue)
Pea
Maize
Pea
Maize
25
20
15
10
5
0
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
Treatments
Treatments
Figure 9: Effect of 2,4-D and its interaction with Phosphorus and copper on MDA and NP-SH
contents in leaves of Pea and maize plants.
PHOTO PLATE - 6
Effect of different concentration of 2,4-D on pea plants.
94
Mechanism Of Oxidative Stress In Plants And Its Regulation
4.2.3 Effect of Cd and its interaction with phosphorus and copper on plant height,
fresh and dry weight, pigments content, antioxidative enzymes, certain amino acids,
thiol contents, electrolyte leakage % and lipid peroxidation in black gram and maize
plant observed at 45 days.
This experiment was conducted to evaluate the toxic effect of Cd in black gram and
maize plants. Phosphorus and Cu were used to ameliorate the plants growth and yield in
plants. The plants were raised in soil in earthen pots. Visual symptoms were observed and
recorded day to day after treatment. Various morphological and physiological parameters
were recorded after 15 days of treatments. Results obtained in this experiment are
depicted in table 4.8.1 to 4.8.4, Figure 10 to 11 and photo plate 7 and 8. Plant height for
control black gram plants was observed as 64 cm. which were decreased to 60, 57, 54 and
51 cm at 0.1, 0.2, 0.4 and 1.0 mM Cd treatments. While on amelioration with P and Cu at
1.0 mM Cd treatment plants height increased to 58 and 54 cm compared to 51 cm at 1.0
mM Cd alone. Plant height for control maize plant was 23 cm. which was 26, 23.4, 23
and 21 cm for 0.1, 0.2, 0.4 and 1.0mM Cd treatments. On rectification with P and Cu at
1.0 mM Cd plant height increase to 24 and 23 respectively. Compared to control, FW of
black gram plants decreased to 5.6, 10.4, 34.0 and 55.0% for 0.1, 0.2, 0.4 and 1.0 mM Cd
treatments while at 1.0 mM Cd treatment P and Cu alleviate FW by 7 and 8%. Similarly
DW also decreased with increased in Cd concentration. Whereas FW for control maize
plant was 22.6 g plant-1 which was increase to 25 and 24 g plant-1 at 0.1 and 0.2 mM Cd
while decreased to 18 and 17 g plant-1 respectively. Similar results for DW were also
obtained. Moisture percentage was decreased with increase in Cd concentration. Moisture
percentage for control maize plant was 93% which were decreased to 92, 88, 86 and 83 %
at 0.1, 0.2, 0.4 and 1.0 mM Cd treatments. Similar pattern of moisture percentage was
observed in case of black gram plants. Compared to control black gram plant contents of
all the pigments decreased with increased in Cd concentration. Contents of total
chlorophyll for control black gram plant was observed as 2.42 mg g-1 FW tissue which
was increased to 58% at 0.1 mM Cd treatments while decreased to 21 % in the leaves of
black gram exposed to 1.0 mM Cd. Contents of Chl a and Chl b was also increased at
lower dose (0.1mM) of Cd by 40 and 44% while both were decreased by 35 and 66% at
1.0 mM of Cd. Carotetoid contents for control plants was 0.53 mg g-1 FW tissue which
were 0.55, 0.49, 0.35 and 0.26 mg g-1 FW tissue for 0.1, 0., 0.4 and 1.0 mM Cd
95
Mechanism Of Oxidative Stress In Plants And Its Regulation
treatments respectively. Table 4.9.2 shows the effect of Cd on different pigment contents
in maize plants. Total chlorophyll for control was 3.19 mg g-1 FW tissue which was 3.7,
3.22, 3.00 and 2.70 at 0.1, 0.2, 0.4 and 1.0 mM Cd treatment respectively. Compared to
Chl T content at 1.0 mM Cd (2.7 mg g-1 FW) it was increase to 3.36 and 3.2 mg g-1 FW
tissue on rectification with phosphorus and copper. Similar results for Chl a & b were
obtained in maize leaves under different levels of Cd treatment. Carotenoids contents for
control was 0.89 mg g-1 FW tissue which were 0.97, 0.86, 0.84 and 0.66 mg g-1 FW at
0.1, 0.2, 0.4 and 1.0 mM Cd treatments respectively. In black gram CAT activity for
control plants was 634 which were increase to 701, 740, 762 and 770 in plants treated
with 0.1, 0.2, 0.4 and 1.0 mM Cd, While interaction of P and Cu at 1.0 mM of Cd
reduced the CAT activity by 32 and 29 % respectively in comparison to plants treated Cd
alone. Table 4.9.3 shows the results of activity of CAT, POX and SOD under various
levels of Cd in maize plants. Activity of CAT was increase with increase in Cd
concentration except at 1.0mM Cd. It was 328 for control which were 345, 350, 433 and
245 units at 0.1, 0.2, 0.4 and 1.0 mM Cd treatments respectively. POX activity for
control black gram plants was observed as 9.20 which were decreased to 6.0, 5.0, 4.6 and
4.0 at 0.1, 0.2, 0.4 and 1.0 mM Cd treatments. Comparison to POX activity (4.0) in the
leaves of black gram exposed to 1.0 Cd it was increased to 7.6 and 8.8 in plant exposed to
1.0 mM Cd+P and 1.0 mM Cd+Cu respectively. Similar results for SOD were obtained in
maize leaves under Cd treatments. Value of POX activity for control maize plants was
8.0 which were 8.4, 9.3, 12.1, and 9.1 for 0.1, 0.2, 0.4 and 1.0 mM Cd treatments
respectively. Similar results for SOD were obtained in maize leaves under Cd treatments.
Table 4.84 indicated that MDA contents were increased in black gram leaves treated with
Cd. It was 17.1 nmol g-1 FW tissues in control plants which was increase to 18.0, 18.5,
19.5 and 21.0 in plants treated with 0.1, 0.2, 0.4 and 1.0 mM of Cd treatment. Value of
MDA contents for control maize plant was 16.4 nmol g-1 FW tissues which were 14.9,
16.3, 21.5 and 27.0 nmol g-1 FW tissues at 0.1, 0.2, 0.4 and 1.0 mM Cd respectively.
While on rectification with P and Cu at 1.0 mM of Cd value of MDA contents became 26
and 28 nmol g-1 FW tissue. Electrolyte leakage percentage for control black gram plant
was observed as 87% which were increased to 90.0, 98, 104.0, and 112% for treatments
0.1, 0.2, 0.4 and 1.0 mM Cd treatments. On rectification with P and Cu at 1.0 mM Cd
electrolyte leakage percentage were decreased to 11 nd 9% respectively. Value of
96
Mechanism Of Oxidative Stress In Plants And Its Regulation
electrolyte leakage percentage for control maize plants was 58 % which were 56, 60, 63
and 64 % at 0.1, 0.2, 0.4 and 1.0 mM Cd treatments respectively. Value of NP-SH was
found to be increase with increase in Cd concentration in both plants table 11 and 11.
.
97
Mechanism Of Oxidative Stress In Plants And Its Regulation
Table 4.8.1: Effect of cadmium and its interaction with phosphorus and copper on plant height, fresh
weight, dry weight, and moisture % in black gram plants observed at 45 days.
Parameter
Treatments
Plant height(cm)
Fresh weight(gm)
Dry weight(gm)
Moisture %
0.0
64±3.6
14.5±0.8
2.00±0.08
92.33±1.45
0.1
60±3.4*
13.7±1.1
1.96±0.15
90.00±1.73
0.2
57±3.1**
10.4±0.7*
1.41±0.11*
87.00±1.73**
0.4
54±3.1**
9.6±0.8**
1.38±0.20**
86.00±0.57**
1.0
51±3.1**
6.51±0.8**
0.82±0.06**
84.00±1.73**
58±3.4
7.13±0.8
1.42±0.11*
90.00±2.30**
54±3.1**
7.00±1.1
0.95±0.15
88.00±1.73*
4.01
6.20
3.01
4.65
0.41
0.64
2.60
4.02
1.0+P
1.0+Cu
*
**
P= 40ppm phosphprus, Cu= 10ppm copper *- value significant at P<0.05 and **- value significant at P<0.01 levels.
Table 4.8.2: Effect of cadmium and its interaction with phosphorus and copper on total chlorophyll,
chlorophyll a, chlorophyll b carotenoids in mature leaves of black gram plants observed at 45 days.
Treatments
Parameter
Total chlorophyll
Chlorophyll a
Chlorophyll b
Carotenoids
0.0
2.42±0.18
1.56±0.06
0.89±0.03
0.53±0.01
0.1
3.84±0.21
2.19±0.11*
1.28±0.11*
0.55±0.03
0.2
2.57±0.17
1.52±0.10
0.87±0.05
0.49±0.05
0.4
1.82±0.11
1.42±0.06
0.40±0.03*
0.35±0.03**
1.0
1.38±0.04*
1.01±0.04*
0.30±0.02**
0.26±0.02**
1.0+P
1.55±0.13
1.12±0.39
0.45±0.02
0.35±0.04
1.0+Cu
1.48±0.11
1.1±0.03
0.34±0.04
0.30±0.03
0.81
1.25
0.37
0.57
0.34
0.53
0.10
0.16
*
**
P= 40ppm phosphprus, Cu= 10ppm copper *- value significant at P<0.05 and **- value significant at P<0.01 levels.
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Mechanism Of Oxidative Stress In Plants And Its Regulation
Table 4.8.3: Effect of cadmium and its interaction with phosphorus and copper on catalase,
peroxidase and superoxide dismutase activity in mature leaves of black gram plants observed at 45
days.
Treatments
Parameter
Catalase
Peroxidase
SOD
0.0
634±02.96
9.20±0.41
17.3±1.5
0.1
701±4.41
6.00±0.35*
26.2±1.8
0.2
740±2.88*
5.00±0.29**
30.0±1.6*
0.4
762±4.41*
4.60±0.35**
32.0±2.3*
1.0
770±5.78*
10.6±1.2
39.5±1.9**
1.0+P
520±2.89**
7.6±0.23*
45.0±1.73
1.0+Cu
555±2.81**
8.8±0.46
46.5±2.10
*
**
93.7
144.7
2.39
3.69
9.50
14.30
P= 40ppm phosphprus, Cu= 10ppm copper *- value significant at P<0.05 and **- value significant at P<0.01 levels.
Table 4.8.4: Effect of cadmium and its interaction with phosphorus and copper on MDA contents,
electrolyte leakage percentage non-protien thiol-group in mature leaves of black gram plants
observed at 35 days.
Treatments
Parameter
MDA
ELP
NP-SH
0.0
17.1±0.58
87±1.22
0.89±0.13
0.1
15.9±0.19
90±3.73
1.28±0.11
0.2
16.3±0.72
98±1.24*
1.45±0.15
0.4
17.2±0.69
104±2.34**
1.90±0.13
1.0
18.2±0.90
112±1.72**
2.5±0.22
1.0+P
15.6±0.92
104±1.52
1.85±0.32
1.0+Cu
20.6±1.14
108±2.30
1.95±0.14
*
**
1.58
2.45
8.55
13.2
0.35
0.53
P= 40ppm phosphprus, Cu= 10ppm copper *- value significant at P<0.05 and **- value significant at P<0.01 levels.
99
Mechanism Of Oxidative Stress In Plants And Its Regulation
Table 4.9.1: Effect of cadmium on plant height, fresh weight, dry weight, and moisture % in maize
plants observed at 45 days.
Treatments
Parameter
Plant height(cm)
Fresh weight(gm)
Dry weight(gm)
Moisture %
0.0
23.13±0.59
22.6±1.4
3.4±0.1
93±0.7
0.1
26.26±1.75**
25.2±1.1
4.0±0.3
92±1.1
0.2
23.40±0.75
24.2±0.6
3.4±0.2
88±2.8*
0.4
23.00±1.73
18.1±2.3*
2.1±0.1*
86±3.4**
1.0
21.50±2.00*
17.6±2.0*
1.4±0.1**
83±1.1**
1.0+P
26.06±1.15**
29.2±2.3**
4.3±0.1
89±2.8*
1.0+Cu
24.00±1.73**
21.2±1.1
3.0±0.2
86±2.3**
1.59
2.45
3.78
5.41
0.95
1.47
3.27
5.05
*
**
P= 40ppm phosphprus, Cu= 10ppm copper *- value significant at P<0.05 and **- value significant at P<0.01 levels.
Table 4.9.2: Effect of cadmium on total chlorophyll, chlorophyll a, chlorophyll b carotenoids in
mature leaves of maize plants observed at 45 days.
Treatments
Parameter
Total chlorophyll
Chlorophyll a
Chlorophyll b
Carotenoids
0.0
3.19±0.04
1.83±0.09
1.32±0.09
0.89±0.06
0.1
3.70±0.05*
1.82±0.04
1.78±0.08**
0.97±0.04
0.2
3.22±0.04
1.80±0.03
1.39±0.8
0.86±0.03
0.4
3.00±0.06
1.77±0.05
1.22±0.07
0.84±0.06
1.0
2.70±0.06*
1.65±0.05**
1.13±0.07
0.66±0.05**
1.0+P
3.36±0.12**
1.88±0.04**
1.39±0.05**
0.82±0.09**
2.40±0.08
1.65±0.12
0.88±0.05*
0.73±0.10
0.39
0.61
0.08
0.12
0.25
0.39
0.09
0.14
1.0+Cu
*
**
P= 40ppm phosphprus, Cu= 10ppm copper *- value significant at P<0.05 and **- value significant at P<0.01 levels.
100
Mechanism Of Oxidative Stress In Plants And Its Regulation
Table 4.9.3: Effect of cadmium on catalase, peroxidase and superoxide dismutase activity in mature
leaves of maize plants observed at 45 days.
Treatments
Parameter
Catalase
Peroxidase
SOD
0.0
328±7.3
8.0±0.29
3.22±0.09
0.1
345±2.9
8.4±0.23
6.00±0.08
0.2
350±2.9
9.3±0.52*
7.88±0.8**
0.4
433±8.8**
12.1±0.58**
5.6±0.07
1.0
245±2.9*
9.1±0.15
4.3±0.07
1.0+P
290±5.8
9.2±0.18
12.20±0.05**
1.0+Cu
230±5.8
9.6±0.35
10.3±0.05**
64.36
99.42
1.22
1.89
3.00
4.64
*
**
P= 40ppm phosphprus, Cu= 10ppm copper *- value significant at P<0.05 and **- value significant at P<0.01 levels.
Table 4.8.4: Effect of cadmium on MDA contents, electrolyte leakage percentage non-protein thiolgroup in mature leaves of maize plants observed at 45 days.
Treatments
Parameter
MDA
ELP
NP-SH
0.0
16.4±0.20
57.7±0.09
1.13±0.09
0.1
14.9±0.57
56.8±0.27
1.22±0.08
0.2
16.3±0.17
60.0±0.57
1.32±0.08
0.4
21.5±0.28*
62.9±0.58
1.39±0.07*
1.0
27.0±0.57**
64.1±0.15*
1.78±0.07**
26.1±0.49
66.2±0.18
1.39±0.05**
19.2±0.58**
76.3±0.35**
1.35±0.05**
4.55
7.04
6.20
9.58
0.25
0.38
1.0+P
1.0+Cu
*
**
P= 40ppm phosphprus, Cu= 10ppm copper *- value significant at P<0.05 and **- value significant at P<0.01 levels.
101
Mechanism Of Oxidative Stress In Plants And Its Regulation
Black gram
Maize
Black gram
Maize
35
FW g plant-1
Plant height plant-1 (cm.)
70
60
50
40
30
25
20
30
15
20
10
10
5
0
0
Treatment
Treatment
Total chlorophyll (mg g-1 FW tissue)
4.5
Black gram
Maize
4
3.5
3
2.5
2
1.5
1
0.5
CAT activity (H2O2 decompose
mg-100 FW tissue)
Figure 10: Effect of cadmium and its interaction with phosphorus and copper on plant height and
fresh weight of black gram and maize plants observed at 45 days.
900
Black gram
800
Maize
700
600
500
400
300
200
100
0
0
0
0
0.1
0.2
0.4
Treatment
1
0.1
0.2
0.4
1
1.0+P 1.0+Cu
1.0+P 1.0+Cu
Treatment
Figure 11: Effect of cadmium and its interaction with phosphorus and copper on total chlorophyll,
and activity of CAT in leaves of black gram and maize plants observed at 45 days.
102
Mechanism Of Oxidative Stress In Plants And Its Regulation
PHOTO PLATE- 7
Control
0.1 mM
0.2 mM
0.4 mM
1.0 mM
Effect of cadmium on black gram plants.
Control
1.0 mM
1.0 mM+P
1.0 mM+Cu
Effect of cadmium and its interaction with phosphorus and copper on black gram plants
103
Mechanism Of Oxidative Stress In Plants And Its Regulation
PHOTO PLATE - 8
Control
0.1 mM Cd
0.2 mM Cd
0.4 mM Cd
1.0 mM Cd
Effect of cadmium on maize plants.
Control
1.0 mM
Cd
1.0 mM Cd+P
1.0 mM Cd+Cu
Effect of cadmium and its interaction with phosphorus and copper on maize plants.
104