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Plant Gene and Trait, 2013, Vol.4, No.20, 109
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of these pathways rendering the plant more tolerant to
abiotic oxidative stress.
The concentration of ROS and cellular membrane
lipid peroxidation as measured by MDA in plant cells
under stress are the key parameter to access the
severity of the stress including drought stress (Hossain
et al., 2013a; 2013b). Here we observed that drought
stress treatment resulted in a robust increase in both
H
2
O
2
and MDA levels. Sharp increase in MDA and
H
2
O
2
levels in response to drought stress has also been
reported (Liu et al., 2010; Hossain et al., 2013a,
2013b). However, the levels of MDA and H
2
O
2
were
significantly lower in H
2
O
2
pre-treated seedling as
compared to the seedlings subjected to drought stress
without pre-treatment. Therefore, we speculated that
initial mild oxidative stress through H
2
O
2
pre-treatment might contribute in alleviating the
drought-induced oxidative stress through the
acclimation of plants that ultimately helps in
membrane stability. Similar to our results, Liu et al
(2010) also found that exogenous H
2
O
2
pre-treatment
significantly reduced the MDA and H
2
O
2
levels in
cucumber. Critical analysis of the phenotypic
appearance of the seedlings showed that H
2
O
2
non
pre-treated drought-stressed seedlings showed severe
wilting (Figure 8), a typical symptom of susceptibility
of water stress, whereas no such wilting was observed
in H
2
O
2
pre-treated drought stressed seedlings.
Figure 8 Phenotypic appearance of control, drought-stressed,
and H
2
O
2
pre-treated drought-stressed seedlings
The findings of the present experiment permit us to
conclude that imposition of drought stress in mustard
seedlings led to severe oxidative damage as mani-
fested by sharp increase in MDA and H
2
O
2
levels due
to insufficient activation or inhibition of MG and ROS
detoxification systems. Surprisingly, pre-treatment of
seedlings with H
2
O
2
stimulated the activities of CAT,
APX, GR, GST and Gly II under drought conditions
associated with a decreased level of oxidative stress
parameter like MDA and H
2
O
2
as compared to the
seedlings subjected drought stress without H
2
O
2
pre-treatment. Our results confirmed the beneficial
effects of exogenous H
2
O
2
in alleviating drought
induced oxidative damage. The results also allow us
also conclude that co-ordinate stimulation of
glyoxalase system and antioxidant is important
determinant for the acquisition of tolerance to drought
stress and the data are of considerable value in
elucidating the biochemical mechanisms of plant
abiotic stress tolerance and in developing appropriate
and efficient methods for crop protection against
abiotic stresses. However, additional key players
involved in H
2
O
2
induced-drought tolerance needs to
be clarified.
3 Materials and Mmethods
3.1 Plant materials and growth conditions
Uniform-sized mustard (
Brassica juncea
cv. Shambal)
seeds collected from the Department of Genetics and
Plant Breeding, Bangladesh Agricultural University,
were selected and surface-sterilized with 70%
ethanol then washed several times with distilled
water. The seeds were then soaked with distilled
water for 15 min and sown in Petri dishes (9 cm)
lined with 4 layers of filter paper moistened with 10
ml of distilled water for germination under dark
conditions for 3 days. Germinated seedlings were
then grown in Petri dishes that contained 10,000-fold
diluted Hyponex solution (Hyponex, Japan) under
controlled conditions (light, 100 μmol photon m
−2
s
−1
;
temp, (25±2)°C; RH, 65%~70%) as previously
described by Hosain et al (2013a).
3.2 Hydrogen peroxide pre-treatment and drought
stress treatment
Eight-day-old seedlings of approximately equal sizes
were employed for H
2
O
2
pre-treatment. Based on the
preliminary experiment, the optimal concentration for
H
2
O
2
pre-treatment was found to be 50 µM. For H
2
O
2
pre-treatment the root portion of the seedlings were
immersed in H
2
O
2
(50 µM H
2
O
2
25 mL solution)
under dark conditions for 24 h. The control seedlings
were kept at 25°C under dark conditions. Afterwards,
the seedlings with or without H
2
O
2
pre-treatment were
subjected to drought stress (induced by 20%
PEG-6000) in Hyponex solution and grown under the