ECOBIOS, Vol. 8.

(1&2), 2015 ISSN: 0972-6446

(International Journal for Biology, Ecology and Allied Sciences)

PEG induced water stress alters growth and antioxidant

responses in rice (Oryza sativa L.)
S Shome1, S Sen1, MK Bhattacharya1, SK Panda2, H Upadhyaya1,3*
1
Department of Botany and Biotechnology, Karimganj College, Karimganj-788710, Assam, India
2
Plant Biochemistry and Molecular Biology Laboratory, School of Life Sciences, Assam, University,
Silchar 788011
3
Department of Botany, Cotton College State University, Guwahati-781001, Assam, India

ABSTRACT

Water stress is a serious impediment to agriculture and food production sector as it

generates abiotic stress that interferes with plant growth and productivity. Water stress
reduces cell growth, cell elongation and cell expansion that results in retarded plant growth.
In the present study water stress significantly reduces root length by 6.7%, 9.8%, and
12.13% at 20%, 25% and 30% concentration of PEG relative to control. Shoot length also
decreases by 16%, 21% and 29% at 20%, 25% and 30% PEG concentration compared to
control. This suggest that both root length and shoot length decreases with increasing PEG
concentration. The fresh mass and dry mass of both root and shoot has decreased with
increasing concentration of PEG. In case of antioxidant enzyme the activity of catalase has
decreased with increasing PEG concentration having maximum decrease of 46% at 20%
PEG concentration relative to the control in the root. Similarly at 20% PEG concentration
there is a maximum decrease of GPX activity by 19% and 36% in root and shoot
respectively relative to the control. The GR activity has decreased by 37% and 60% in root
and shoot compared to control at 30% PEG suggesting the damaging effect of water stress
on enzyme activity.

Keywords: PEG, Water stress, growth, antioxidant enzyme, Oryza sativa L.

*Email:hkupbl_au@rediffmail.com
Published by
The Society for Biometry, Ecology & Econometrics (BEES), Karimganj, Assam, India 73
ECOBIOS, Vol. 8. (1&2), 2015
INTRODUCTION
Water has become a limiting factor in
agricultural and food production and its
deficit causes extensive loss to
agricultural production worldwide, thus
being a severe threat to sustainable
agriculture 1,2. Depleting water supply and
increasing populations require increased
food supply and crop varieties that are
highly adapted to dry environments 3.
Proper growth and development of roots
are required for adaptation of rice to water
stress conditions. Healthy root system is
an important factor for water and nutrient
uptake that helps the plants to withstand
stress. Water stress affects various
physiological process both at cellular and
whole plant level4. During water stress
due to the reduction in turgor pressure,
cell growth is severely impaired5. Water
stress reduces both cell elongation and
cell expansion and inhibits cell
enlargement more than cell division6.
Another important adverse effect of water
stress is the reduction in biomass
production7. Several studies indicate that
water stress cause significant decrease in
fresh and dry weights of shoots and roots
8,9
. Reduced root and shoot fresh mass as
well as their lengths reduce the
photosynthetic rate of physiology and
ECOBIOS, Vol. VIII. (I&II), 2015
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biochemical processes of rice 10. During
water stress the level of various reactive
oxygen species gets elevated which leads
to oxidative damage in rice cultivars. To
ameliorate water stress plants have ROS
scavenging mechanism which is a
common defense response 11. To detoxify
ROS, plants can intrinsically develop
different types of antioxidants reducing
oxidative damage and conferring drought
tolerance. The ROS scavengers are
antioxidant enzymes containing super-
oxide dismutase, peroxidase and
12
catalase .
Polyethylene glycol can be used to induce
water stress in plants in laboratory setup
for testing the drought tolerant genotypes
as polyethylene glycol acts as a non-
penetrating osmotic agent resulting into
increasing solute potential and blockage
of absorption of water by the root system
13
. The aim of the present work was to
study the comparative effects of different
concentrations of PEG (0%, 5%, 10%,
15%, 20%, 25% & 30%) on physiological
(root-shoot length, root-shoot fresh mass,
root-shoot dry mass) and antioxidant
enzymes like catalase (CAT), peroxidase
(POD) and glutathione reductase (GR)
parameters of rice cultivar KMJ-6-1-1 of
Karimganj district to optimise the
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ECOBIOS, Vol. 8. (1&2), 2015
concentration of PEG for use in drought
stress tolerance study in rice cultivars
commonly grown in Karimganj and the
effect of concentration of PEG induced
osmotic damage due to water stress on
growth and enzymes activities in the
growing rice seedlings.
MATERIALS AND METHODS
Germination and treatment procedure
Regional Agricultural Research Station,
Akbarpur, Karimganj , Assam provided
the Rice (Oryza sativa L.) seeds for the
experiment. Rice cultivar KMJ-6-1-1
seeds were used for this experiment. PEG
6000 was taken in appropriate amount to
prepare 0%, 5%, 10%, 15%, 20%, 25%
and 30% PEG solution to develop
osmotic stress in the rice seedlings.
Floatation method was used to perform
the seed viability test. In this method the
rice seeds that were obtained from
Regional Agricultural Research Station,
Akbarpur, Karimganj, were placed in a
beaker containing water and was left
undisturbed for five to ten minutes. Seeds
that sank to the bottom of the beaker were
viable. All the rice seeds used throughout
the experiment were surface sterilized
with 0.1% HgCl2 and was washed
properly with distilled water for three
times. The seeds were then transferred to
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petriplates containing moisten filter paper
and allowed to germinate at 28C for
three days in a seed germinator chamber.
After completion of three days of
incubation the healthy germinated seeds
having uniform plumule length were
transferred to plastic cups (200ml)
containing nutrient Hoagland solution.
The cups containing the germinated seeds
were placed under tube light having
16h/8h light/dark cycle in the growth
chamber at 22300C and the plants were
allowed to grow for five days.
On the 5th day from the day of transfer to
Hoagland solution, the plants were treated
with 0%, 5%, 10%, 15%, 20%, 25% and
30% PEG dissolved in Hoagland nutrient
solution and allowed to grow for another
48h. After 48h of treatment the plants
were then sampled for various
physiological parameters like root length ,
shoot length, root & shoot fresh mass,
root & fresh dry mass were measured and
expressed in g plant-1.
Germination Percentage
The appearance of the plumule at the
filter paper surface was taken as
germination. Germination% was recorded
after 72h of incubation in a seed
germinator at 280C by counting the
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ECOBIOS, Vol. 8. (1&2), 2015
number of germinated seeds out of total
seed plated.
Root, shoot length and fresh mass
After 48h of treatment growing rice
seedlings at least 10 plants per treatment
were sampled and root and shoot length
were measured using centimeter ruler and
were separated into root and shoot and
then oven dried at 700C for 48h to
estimate the drymass and expressed in g
plant-1. Root and shoot ratio was
measured by dividing root length by shoot
length. Total dry mass of plant was
estimated by adding root and shoot dry
mass.
Antioxidant enzyme analysis
The tissues were homogenized with 0.1M
phosphate buffer (pH 6.8) in pre-chilled
mortar and pestle. The extract wass
centrifuged at 4 for 15 min at 13,000
rpm in a cooling centrifuge The
supernatant was used for assay of
guaiacol peroxidase (GPX), catalase
(CAT) and gluthathionereductase (GR)
activity assay. The activity of CAT was
measured by the method of Aebi14 and
was determined by monitoring the
disappearance of H2O2 at 240 nm by
using the extinction coefficient 0.036
mM-1 cm-1. The CAT activity was
expressed as mole H2O2 destroyed min-
1
g fresh weight (FW). GPx activity was
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assayed as per Chance and Maehly15. The
reaction mixture contained 2.1ml of 0.1 M
potassium phosphate buffer (pH 6.8)
followed by 0.3 ml guaiacol (30mM), 0.3
ml H2O2 (12.5mM), 0.3 ml of enzyme
extract and incubated for 5 min after
which the Glutathione reductase (GR)
was assayed by the method of Smith et
al.,16. The reaction mixture contained 1.0
ml of 0.2M potassium phosphate buffer
(pH 7.5) having 1 mM EDTA, 0.5 ml of
3.0 mM DTNB in 0.01 M potassium
phosphate buffer (pH 7.5), 0.1 ml of 2.0
mM NADPH, 0.1 ml tissue extract and
distilled water to make up a final volume
of 2.9 ml. Reaction was initiated by
adding 0.1 ml of 2 mM GSSG (oxidised
glutathione). The increase in Absorbance
at 412 nm was recorded at 250C over a
period of 5 min, spectrophotometrically.
The activity has been expressed as
Absorbance change (A412) g fress mass-1
sec-1.
RESULT AND DISCUSSION
The shoot length of the rice cultivars has
decreased with increasing concentration
of polyethylene glycol suggesting the
inhibitory effect of osmotic stress on rice
plant growth. Rice seedlings treated with
20% PEG showed 15.93% decrease in
shoot length compared to control (Fig.1.).
At higher concentration of 25% and 30%
PEG the shoot length decreased by
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ECOBIOS, Vol. 8. (1&2), 2015
21.11% and 29.38% respectively.
Similarly the root length also decreased as
the concentration of PEG was increased
with 12.13% decrease in root length at
30% PEG treatment (Fig.1.). There was
3.36%, 6.7% and 9.8% decrease in root
length at 15%, 20% and 25% PEG
concentration respectively indicating the
damaging effect of water stress on the
growth of rice seedlings. The data suggest
that water stress significantly retarded the
shoot growth more than the root growth
which may be due to the poor uptake and
translocation of water and nutrient
towards the shoot of the rice plant.
Nutrient uptake process and water
Fig.1. Changes in length and fresh mass of root and shoot of growing rice seedlings
subjected to different concentration of PEG (0, 5, 10, 15, 20, 25 and 30%)
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availability is related to each other.
Farooq et al 7. Reported that during water
stress the transpiration rate decreases
which reduces the nutrient absorption
processes and efficiency of their
utilization. During water stress the
optimum turgor pressure within cell
required for cell growth and enlargement
decreases which results in poor growth of
plant cells 17. PEG induced water stress in
rice significantly reduces the root and
shoot growth compared to the control due
to reduced cellular turgor pressure and
nutrient uptake under water deficit
conditions.
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ECOBIOS, Vol. 8. (1&2), 2015
PEG induced water stress caused
significant decrease in root and shoot
fresh mass relative to the control with
increase in concentration of PEG (Fig.1.).
The data suggest that at 15%
concentration of PEG there is 29% and
10% decrease in root and shoot fresh
mass relative to the control. At 20%
concentration of PEG the root and shoot
fresh mass has decreased by 42% and
25% respectively relative to the control.
Again at 30% concentration of PEG the
root and shoot fresh mass has decreased
by 37% and 50% respectively. Tatrai et
al.18 also reported a similar decrease in
shoot fresh mass in Thymus citriodorus
under different concentration of PEG
treatment. Water stress reduces the rate
and changes the pattern of protein
synthesis in Avena coleoptiles as judged
by a double labeling ratio technique 19.
This reduced synthesis of protein under
water stress due to polyethylene glycol
treatment can be attributed to the reduced
fresh mass of rice compared to the
control.
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The catalase activity of the root has
decreased with the increase in the
concentration of PEG with maximum
decrease of 46% at 20% PEG concen-
tration relative to the control (Fig.2.). At
10%, 15%, 25% and 30% PEG
concentration there was 45%, 33%, 41%
and 44% decrease in catalase activity
relative to the control respectively. In case
of shoot with the increase of PEG
concentration there is a decrease in
catalase activity with respect to control.
Murthy et al. 20 also reported a similar
decrease in catalase activity with the
increase in concentration of PEG in
Macrotyloma uniflorum. The data suggest
that there is 30% decrease in catalase
activity at 30% PEG concentration
relative to the control. There is a decrease
of catalase activity by 10%, 11% and 14%
at 15%, 20% and 25% of PEG
concentration relative to the control
respectively (Fig.2.). However at 5% and
10% of PEG concentration there is no
significant change in catalase activity
relative to the control.
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Fig.2. Changes in CAT and GPx activities of root and shoot of growing rice
seedlings subjected to different concentration of PEG (0, 5, 10, 15, 20, 25 and 30%)

As depicted in Fig.2, the GPx activity of
the root also has decreased with rise in
PEG concentration compared to control.
At 20% PEG concentration there is a
maximum decrease in GPx activity of
19% relative to the control of root. The
GPx activity has decreased by 8%, 14%,
16% and 17% at 5%, 10% and 15% and
25% PEG solution respectively. Similarly
in case of shoot the GPx activity has
decreased relative to the control. The
activity of GPx has decreased by 37% and
36% at 15% and 20% strength of PEG
solution. Sharma et al.21 also reported a
decrease in GPx activity at higher level of
drought stress in rice seedlings.
The GR activity of the rice seedlings
treated with 20% PEG has also decreased
in both root and shoot with the increase in
PEG concentration relative to the control.

ECOBIOS, Vol. VIII. (I&II), 2015 79

ECOBIOS, Vol. 8. (1&2), 2015
The GR activity has decreased by 26%
and 41% in root and shoot at 25% PEG
concentration. Again at 30%
concentration of PEG the GR activity has
decreased by 37% and 60% in root and
shoot relative to the control suggesting
Fig.3. Changes in GR activities of root and shoot of growing rice seedlings
subjected to different concentration of PEG (0, 5, 10, 15, 20, 25 and 30%)
CONCLUSION
The present study suggest that water
stress induced by PEG-6000 in rice
significantly reduces the root length,
shoot length, root fresh mass, shoot fresh
mass at 20% and 30% of PEG-6000
treatment. Similarly the antioxidant
enzyme activity viz. catalase, guaiacol
peroxidase (GPx) and glutathione
reductase (GR) also decreases with
increasing concentration of PEG-6000
suggesting the damaging effect of water
ECOBIOS, Vol. VIII. (I&II), 2015
ISSN: 0972-6446
the damaging effect of water stress on
enzyme activity. Fan et al.22 also reported
a similar decrease in GR activity relative
to control in the root tissue of cucumber
seedlings treated with PEG.
stress on growth, antioxidant enzyme
activity. The reduced antioxidant enzyme
activity may have resulted in higher levels
of reactive oxygen species that cause
damage to protein synthesis machinery
mediating reduced level of antioxidant
enzyme level and thus alter plant cell
growth. Further studies on the various
strategies for mitigating water stress in
rice plant can be carried out to improve
rice plant growth and productivity by
enhancing antioxidant function in rice
plants.
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ACKNOWLEDGEMENTS
The author (HU) thankfully acknowledge
the financial support from SERB (Vide
SO No: SB/YS/LS- 44/2013 dated 6th
Sept, 2013), DST, Govt. of India. SS,
MKB, SKP and HU also express their
sincere thanks to the DBT, Govt of India,
for funding DBT RGYI Project (SAN
No.102/IFD/SAN/1716/2013-2014 dated
July15 2013).
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