J.

Agronomy & Crop Science (2009) ISSN 0931-2250

DROUGHT STRESS

Improving the Drought Tolerance in Rice (Oryza sativa L.)

by Exogenous Application of Salicylic Acid
M. Farooq1, S. M. A. Basra2, A. Wahid3, N. Ahmad2 & B. A. Saleem4
1 Department of Agronomy, University of Agriculture, Faisalabad, Pakistan
2 Department of Crop Physiology, University of Agriculture, Faisalabad, Pakistan
3 Department of Botany, University of Agriculture, Faisalabad, Pakistan
4 Institute of Horticultural Sciences, University of Agriculture, Faisalabad, Pakistan

Keywords Abstract
antioxidant system; drought stress;
photosynthesis; rice; ROS; salicylic acid; seed Drought stress encumbers the rice growth predominantly by oxidative damage
priming; water relations to biological membranes and disturbed tissue water status. In this study, the
role of salicylic acid (SA) to induce drought tolerance in aromatic fine grain
Correspondence rice cultivar Basmati 2000 was evaluated. SA was applied as seed and foliar
Dr M. Farooq
treatments. For seed treatment, rice seeds were soaked in 50, 100 and
Department of Agronomy, University of
Agriculture, Faisalabad-38040, Pakistan
150 mg l)1 aerated solution of SA for 48 h and then dried back. Treated and
Tel.: +92 41 9200161 extn. 9/2936 untreated seeds were sown in plastic pots in a phytotron. At four leaf stage,
Fax: +92 41 9200605 one set of plants was subjected to drought stress, while the other remained well
Email: [email protected] watered. Drought was maintained at 50 % of field capacity by watering every
alternate day. For exogenous application, SA was applied 50, 100 and
Accepted February 19, 2009 150 mg l)1 at five leaf stage. In the control, SA was neither applied exogenously
nor as seed treatment. Drought stress severely affected the seedling fresh and
doi:10.1111/j.1439-037X.2009.00365.x
dry weight, photosynthesis, stomatal conductance, plant water relations and
starch metabolism; however, SA application improved the performance of rice
under both normal and stress conditions. Drought tolerance in rice was well
associated with the accumulation of compatible solutes, maintenance of tissue
water potential and enhanced potency of antioxidant system, which improved
the integrity of cellular membranes and facilitated the rice plant to sustain
photosynthesis and general metabolism. Foliar treatments were more effective
than the seed treatments. Foliar application with 100 mg l)1 (FA 100) was
the best treatment to induce the drought tolerance and improve the performance
under normal and stress conditions compared with the control or other treat-
ments used in this study.

components (Loggini et al. 1999), damages photosyn-

Introduction
thetic apparatus (Fu and Huang 2001) and decreases the
Salicylic acid (SA) is a naturally existing phenolic com- activities of Calvin cycle enzymes (Monakhova and
pound. As it can function as a growth regulator, many Chernyadév 2004). Moreover, exposure of plants to
researchers have included it in the group of plant growth drought quite often leads to the generation of reactive
regulators (Raskin 1992). oxygen species (ROS) (Munne-Bosch and Penuelas 2003,
Exposure of plants to drought stress substantially El-Tayeb and Ahmed 2007, Farooq et al. 2009a). Being
decrease leaf water potential (ww), relative water contents highly reactive, ROS can seriously damage plants by lipid
(RWC) and transpiration rate with associated increase in peroxidation, protein degradation, DNA fragmentation
leaf temperature (Halder and Burrage 2003). Drought and ultimately cell death (Beligni and Lamattina 1999,
stress produces changes in photosynthetic pigments and Kratsch and Wise 2000, Foyer and Fletcher 2001).

ª 2009 Blackwell Verlag, 195 (2009) 237–246 237

Farooq et al.

However, activation of antioxidant system better ensures Materials and Methods

the tolerance ability of plants in stress prone environ-
Experimental details
ments. This system consisting of several antioxidant
enzymes, such as superoxide dismutase (SOD), ascorbate- Fine rice (Oryza sativa L.) cultivar Basmati 2000 was used
peroxidase (APX), glutathione reductase (GR) and cata- in the study as experimental material. The seeds were sur-
lase (CAT) protects membranes from the injurious effects face sterilized with 0.2 % HgCl2 solution for 5 min and
of ROS (Hasegawa et al. 2000, Fazeli et al. 2007). thoroughly rinsed with tap water. SA was applied exoge-
There are several reports available highlighting the role nously as seed treatment and foliar application (FA).
of SA to induce stress tolerance in plants (Fletcher and For seed priming treatments, seeds were soaked in 5,
Hofstra 1988, Dat et al. 1998a, Senaratna et al. 2000). SA 10 and 15 mg l)1 aerated solution of SA solution for 48 h
has been found to induce heat tolerance in mustard (Dat at 28 ± 2 C. The ratio of seed weight to solution volume
et al. 1998a,b), chilling tolerance in maize (Janda et al. was 1 : 5 (w/v) (Farooq et al. 2006a). After each treat-
1999, Farooq et al. 2008c) and wheat (Taşgn et al. 2003), ment, seeds were given three surface washings with dis-
heavy metal stress tolerance in barley (Metwally et al. tilled water and dried back close to original moisture
2003) and drought tolerance in wheat plants (Singh and level under forced air at 27 C ± 3, sealed in polythene
Usha 2003, Waseem et al. 2006, Horváth et al. 2007). Kho- bags and stored in a refrigerator at 5 C until use (Lee
dary (2004) reported increased chlorophyll and carotenoids and Kim 2000).
contents in maize plants by SA application. Senaratna et al. Treated and untreated seeds were grown in plastic pots
(2000) found that SA is one of the important signal mole- (20 cm in diameter and 18 cm in height) in a phytotron
cules, which modulate plant responses to environmental with a photosynthetically active photon flux density
stress. Burketová et al. (2003) reported that in leaf tissues, (PPFD) of 350 mmol m)2 s)1, 27 C, 70–80 % relative
Benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester humidity and a photoperiod of 14/10 h (light/dark).
(BTH) and SA application induced proteins more effec- Experimental design was completely randomized with five
tively than glycinebetaine but the effect of glycinebetaine replications. Till four leaf stage, all the pots were irrigated
(GB) in roots was as efficient as BTH and SA. daily to keep them well watered (maintained at 100 %
Salicylic acid caused activation of SOD and peroxidase, field capacity). Afterwards one set of plants was subjected
which resulted in reduced injurious effects of salinity and to drought stress, while the other remained well watered.
water deficit on plants. SA treatment also reduced the Drought was maintained at 50 % of field capacity by
level of lipid peroxidation and electrolyte leakage (Shakir- watering every alternate day or whenever needed.
ova 2007). Nonetheless, SA application is reported to Hoagland nutrient solution (300 ml) was applied with
reduce the CAT activity and enhance H2O2 level (Janda irrigation water once in a week. For exogenous applica-
et al. 2003). This increased H2O2 acts as signalling mole- tion, SA was applied in 5, 10 and 15 mg l)1 at five leaf
cule to activate antioxidant enzymes (Klessig et al. 2000). stage. In the control, SA was neither applied exogenously
In seed priming, seeds are partially hydrated to a point nor as seed treatment.
where germination processes begin but radicle emergence All the observations, except seedling fresh and dry
does not occur (Bradford 1986, Farooq et al. 2006a,b,c, weight, were taken 1 week after FA of SA. The experiment
2008b). Recently, seed priming with GB (Farooq et al. was terminated (3 weeks after the induction of drought
2008a), SA (Farooq et al. 2008c), KCl (Farooq et al. stress) when 50 % of stressed plants were wilted. At
2008d) and CaCl2 (Farooq et al. 2008e) have been found harvest, the seedlings were tested for vigour after carefully
to induce chilling tolerance in hybrid maize mainly by removing from the soil. Seedling fresh weight was
the activation of antioxidant system. determined immediately after harvest while dry weight
In view of gradually depleting irrigation water resources was taken after drying at 70 C for 7 days.
throughout the world, it is highly imperative to investi-
gate the effects and mechanisms of drought on sub-
Leaf gas-exchange measurements
merged food crops like rice. Although some studies
report the general effects of drought stress on rice growth Leaf gas-exchange attributes of the penultimate fully
and yield, the basic effects at the biochemical and molec- expanded leaves were recorded using a portable infrared
ular levels have not been investigated in depth. It is gas analyzer based photosynthesis system (LI-6400; LiCor,
hypothesized that exogenous SA application may alleviate Inc., Lincoln, NE, USA). Observations were recorded at
the oxidative damage with the enhanced activities of 09 : 00 to 10 : 00 am 1 week after foliar SA application.
enzyme antioxidants and promote seedling growth. This During recording of data, air relative humidity was about
study therefore was carried out to investigate the possible 75 % and the ambient CO2 concentration was 500 lmol
role of SA to induce drought tolerance in rice. CO2 mol)1.

238 ª 2009 Blackwell Verlag, 195 (2009) 237–246

 Drought Tolerance in Rice by Salicylic Acid

Plant water relations Lipid peroxidation was estimated in terms of malondi-

aldehyde (MDA) content according to the method of
Leaf water potential (ww) was estimated with pressure Heath and Packer (1968). Leaf samples (1 g) were
chamber (Soil Moisture Equipment Corp., Santa Barbara, homogenized in 10 ml of trichloroacetic acid. The
CA, USA). Frozen leaf tissues were thawed, sap expressed, homogenate was centrifuged at 15 000 g for 5 min. Four
centrifuged at 5000 g and leaf osmotic potential (ws) was millilitre (0.5 %) of thiobarbituric acid in 20 % trichloro-
determined with an osmometer (Digital Osmometer, acetic acid was added to 1 ml aliquot of the supernatant.
Wescor, Logan, UT, USA), while leaf pressure potential Mixture was heated at 95 C for 30 min and then cooled
(wp) was computed as a difference of ww and ws. For, rapidly in an ice bath. After centrifugation at 10 000 g for
RWC, fresh leaves (0.5 g) (Wf) were rinsed in water until 10 min, the absorbance was recorded at 532 nm. The
constant weight. Saturated leaves were weighed (WS) and value for non-specific absorption at 600 nm was sub-
then dried for 24 h at 85 C to determine dry weigh (Wd). tracted. The MDA content was calculated using its
RWC were calculated according to following formula: absorption coefficient of 155 mmol)1 cm)1 and expressed
as lmol MDA g)1 fresh weight.
RWC ¼ ðWf  Wd Þ=ðWS  Wd Þ  100 %:
Salicylic acid
The extraction and determination of SA was performed
Membrane permeability
following the method of Malamy and Klessig (1992). Leaf
Membrane permeability was estimated by measuring the samples (0.5 g) (taken from the second top leaves of the
leaf electrolyte leakage following the protocol of Blum rice seedlings) were ground with 1.5 ml of 90 % methanol
and Ebercon (1981). Six leaf segments of same size were and then centrifuged at 10 000 g for 15 min. The pellets
washed thoroughly with distilled water and immersed in were re-extracted with 1.5 ml of 100 % methanol and
a test tube having 6 ml distilled water for 12 h. Then centrifuged again as mentioned above. The later
electrical conductivity (EC1) of solution was measured supernatants were mixed with those of the first extraction
with a conductivity metre (Model DDS-11A; Shanghai and evaporated in vacuo at 35 C until the methanol totally
Leici Instrument Inc., Shanghai, China). Samples were disappeared. Distilled water (2.5 ml) was added to the
then heated in boiling water for 20 min and cooled to aqueous phases and dissolved at 80 C in a water bath for
room temperature. The conductivity of killed tissues 2 min, which were then incubated with 2.5 ml of sodium
(EC2) was again measured. Membrane permeability was acetate buffer (pH 4.5) at 37 C for 10 min, extracted with
calculated as the ratio between EC1 and EC2. 10 ml of ethyl acetate/cyclopentane/2-propanol.

Starch metabolism Osmoprotectants

To estimate a-amylase activity, ground leaf sample (1 g) Leaf GB contents were extracted and contents estimated
was mixed with 10 ml phosphate buffer (pH 7.0) and left by the method of Grieve and Grattan (1983). Leaf extracts
for 24 h at 4 C. The enzyme activity was determined from were prepared by vigorous shaking in 2 m H2SO4, cooled
the supernatant by the dinitrosalicyclic acid (DNS) method and mixed with equal volume of periodide, vortexed and
(Bernfeld 1955). To determine total soluble sugars, ground kept at 0–4 C for 16 h. The mixture was centrifuged at
leaf sample (1 g) was mixed with 10 ml distilled water and 10 000 g at 4 C for 15 min and the supernatant was
left for 24 h at 25 C (Lee and Kim 2000). Mixture was fil- aspirated while cool. The resultant crystals were dissolved
tered (with Whatman No. 42; Whatman plc, Kent, UK) in 1, 2-dichloroethane and the absorbance was taken at
and the final volume was made to 10 ml with distilled 365 nm.
water. Total soluble sugars were determined by the phenol For free proline estimation, method of Bates et al.
sulphuric method (Dubois et al. 1956). (1973) was followed. Fresh leaf material (0.5 g) from the
mid-section of each plant was homogenized in 10 ml of
3 % aqueous sulfosalicyclic acid and filtered through
Hydrogen peroxide and lipid peroxidation
Whatman No. 2 filter paper. Two millilitre of filtrate was
Hydrogen peroxide in leaf tissues was determined using mixed with 2 ml acid-ninhydrin and 2 ml of glacial acetic
titanium reagent according to the method of Teranishi acid in a test tube. The mixture was placed in a water
et al. (1974). Sample preparation and hydrogen peroxide bath for 1 h at 100 C. The reaction mixture was
estimation were performed as described previously by extracted with 4 ml toluene and the chromophore
Prasad et al. (1994). containing toluene was aspirated cooled to room

ª 2009 Blackwell Verlag, 195 (2009) 237–246 239

Farooq et al.

temperature, and the absorbance was measured (a)

Well watered

Seedling fresh weight (g)

at 520 nm with a Shimadzu UV 1601 Spectrometer 60 Drought
(Shimadzu Corp., Kyoto, Japan). 50
40
30
Antioxidants
20
Total extractable SOD activity was determined following 10
the method of McCord and Fridovitch (1969). Inhibition 0
of colour formation (measured at 560 nm) was deter- Control SP50 SP100 SP150 FA50 FA100 FA150
mined by addition of 0–50 ll of the extract to a reaction (b)
25

Seedling dry weight (g)

mixture containing 50 mm HEPES/KOH buffer (pH 7.8), Well watered
20 Drought
0.05 unit xanthine oxidase, 0.5 mm nitroblue tetrazolium
and 4 mm xanthine. One unit of SOD activity equalled 15
the volume of extract needed to cause 50 % inhibition 10
of the colour reaction. CAT activity was measured follow-
5
ing the modified method of Luck (1974). Enzyme extract
(50 ll) was added to 3 ml of H2O2-phosphate buffer (pH 0
Control SP50 SP100 SP150 FA50 FA100 FA150
7.0). The time required for decrease in the absorbance Salicylic acid treatments
from 0.45 to 0.40 was noted. Enzyme solution containing
H2O2-free phosphate buffer was used as a control. Fig. 1 Influence of salicylic acid treatments on the (a) seedling fresh
and (b) dry weight in rice under well-watered and drought condi-
Enzyme activity was expressed in mmol of H2O2 con-
tions ± S.E. (the results are the average of five replications repeated
sumed min)1 mg)1 chl. APX activity was estimated twice). SP 50 = seed priming with 50 ppm SA, SP 100 = seed priming
according to the method of Nakano and Asada (1987) with 100 ppm SA, SP 150 = seed priming with 150 ppm SA, FA
with slight modification. Ascorbate oxidation to dehydro- 50 = foliar application of 50 ppm SA, FA 100 = foliar application of
ascorbate was followed at 265 nm in 1 ml reaction 100 ppm SA, FA 150 = foliar application of 150 ppm SA.
mixture containing 50 mm HEPES/KOH (pH 7.6),
0.1 mm EDTA, 0.05 mm ascorbate, 10 ll extract and (a) Well watered
0.1 mm H2O2.
Leaf CO2 net assimilation

20 Drought
rate (µmol m–2 s–1)

16

Statistical analysis 12

8
The data were subjected to statistical analysis using
costat computer software (CoHort Computer Software, 4
Berkeley, CA, USA). Least significant difference (LSD) test 0
was applied to compare the treatment means. Graphical Control SP50 SP100 SP150 FA50 FA100 FA150

presentation of data was carried out using microsoft (b) Well watered
excel program (Microsoft Corporation, Los Angeles, CA, 0.6 Drought
Stomatal conductance

USA). Parallels were drawn between GB, free proline and 0.5
(mol m–2 s–1)

SA, and leaf ww, wp and RWC, and between antioxidants 0.4
and membrane permeability, MDA and H2O2. 0.3
0.2
0.1
Results
0
Control SP50 SP100 SP150 FA50 FA100 FA150
The performance of rice was considerably affected under Salicylic acid treatments
drought conditions. Drought stress resulted in declined
seedling fresh and dry weight (Fig. 1), net photosynthesis Fig. 2 Influence of salicylic acid treatments on the (a) leaf CO2 net
(Pn), stomatal conductance (gs; Fig. 2), ww, ws and wp assimilation rate (b) Stomatal conductance in rice under well-watered
(Fig. 3), RWC (Fig. 4a), a-amylase activity, total soluble and drought conditions ± S.E. (the results are the average of five
replications repeated twice). SP = seed priming; FA 100 = foliar
sugars (Fig. 5) and antioxidant activities (SOD, CAT, APX;
application; 50,100, 150 = 50, 100 and 150 ppm SA, respectively.
Fig. 8) in rice compared with well-watered conditions.
Nonetheless, membrane permeability (Fig. 4b), H2O2, All the SA treatments improved all the attributes
MDA (Fig. 6), leaf GB, free proline and SA contents studied under stress conditions (Figs 1–8). Nonetheless
(Fig. 7) were considerably increased by drought stress. under well-watered conditions seedling fresh and dry

240 ª 2009 Blackwell Verlag, 195 (2009) 237–246

 Drought Tolerance in Rice by Salicylic Acid

(a) Well watered (a)

1.4 100 Well watered
Drought
Water potential (-MPa)

1.2 Drought
80
1

RWC (%)
0.8 60
0.6 40
0.4
20
0.2
0
0 Control SP50 SP100 SP150 FA50 FA100 FA150
Control SP50 SP100 SP150 FA50 FA100 FA150

Well watered
(b) Well watered
(b) 25
Drought Drought

Electrolyte leakage (%)

1.5
Osmotic potential (-MPa)

20
1.2
15
0.9
10
0.6
5
0.3
0
0 Control SP50 SP100 SP150 FA50 FA100 FA150
Control SP50 SP100 SP150 FA50 FA100 FA150
Salicylic acid treatments
(c)
Pressure potential (-MPa)

0.7 Well watered Fig. 4 Influence of salicylic acid treatments on the (a) relative water
0.6 Drought
content (RWC) and (b) electrolyte leakage in rice under well-watered
0.5
and drought conditions ± S.E. (the results are the average of five
0.4
replications repeated twice). SP = seed priming; FA 100 = foliar
0.3
application; 50,100, 150 = 50, 100 and 150 ppm SA, respectively.
0.2
0.1
0
(a)
a-amylase activity (unit)*

Control SP50 SP100 SP150 FA50 FA100 FA150 10 Well watered

Salicylic acid treatments Drought
8

Fig. 3 Influence of salicylic acid treatments on the (a) water potential, 6

(b) osmotic potential and (c) pressure potential in rice under well- 4
watered and drought conditions ± S.E. (the results are the average of
2
five replications repeated twice). SP = seed priming; FA 100 = foliar
application; 50,100, 150 = 50, 100 and 150 ppm SA, respectively. 0
Control SP50 SP100 SP150 FA50 FA100 FA150

(b)
weight (Fig. 1), ww, ws and wp (Fig. 3), RWC (Fig. 4a), 18 Well watered
(mg g–1 fresh weight)

Drought
a-amylase activity, total soluble sugars (Fig. 5), leaf GB, 15
Soluble sugars

free proline and SA contents (Fig. 7) and antioxidant 12

activities were improved, other attributes remain 9
unaffected. However, foliar treatments were more effective 6
than the seed treatments. FA with 100 ppm (FA 100) was 3
the best treatment that gave maximum seedling fresh and 0
Control SP50 SP100 SP150 FA50 FA100 FA150
dry weight, net photosynthesis (Fig. 2b), leaf water poten- Salicylic acid treatments
tial, osmotic potential and pressure potential (Fig. 3),
RWC (Fig. 4a), a-amylase activity, total soluble sugars Fig. 5 Influence of salicylic acid treatments on the (a) a-amylase activ-
(Fig. 5), leaf GB and proline (Fig. 7), and antioxidant ity and (b) soluble sugars in rice under well-watered and drought con-
activities (Fig. 8). The same treatment resulted in mini- ditions ± S.E. (the results are the average of five replications repeated
twice). *One unit of the enzyme’s activity is the amount of enzyme
mum leaf electrolyte leakage (Fig. 4b) and leaf GB and
which released 1 lmol of maltose by 1 ml original enzyme solution in
proline contents (Fig. 7) under both well-watered and 1 min. SP = seed priming; FA 100 = foliar application; 50,100,
stress conditions and hydrogen peroxide and lipid peroxi- 150 = 50, 100 and 150 ppm SA, respectively.
dation (Fig. 6) under drought stress.
Correlation drawn between ww, wp and RWC of leaves proline, while SA was strongly correlated with ww under
and GB, free proline and SA contents indicated that ww, both well-watered and drought-stress conditions
wp and RWC were strongly correlated with GB and free (Table 1). Positive correlation between SA and RWC was

ª 2009 Blackwell Verlag, 195 (2009) 237–246 241

Farooq et al.

(a) Well watered (a) 21

Drought Well watered

SOD (Unit g–1 protein)*

15
18 Drought
Leaf H2O2 content

12
(µmol g–1 FW)

15
9 12
6 9
6
3
3
0
Control SP50 SP100 SP150 FA50 FA100 FA150 0
Control SP50 SP100 SP150 FA50 FA100 FA150

(b) Well watered (b)

15 Well watered

CAT (µmol min–1 g–1

25 Drought
Drought
Leaf MDA content

12
20
(µmol g–1 FW)

protein)
9
15
6
10
3
5
0
0 Control SP50 SP100 SP150 FA50 FA100 FA150
Control SP50 SP100 SP150 FA50 FA100 FA150
Salicylic acid treatments (c) 12
Well watered

APX (µmol min–1 g–1

10 Drought
Fig. 6 Influence of salicylic acid treatments on the (a) leaf hydrogen
peroxide (H2O2) (b) leaf malondialdehyde (MDA) content in rice under 8

protein)
well-watered and drought conditions ± S.E. (the results are the average 6
of five replications repeated twice). SP = seed priming; FA 100 = foliar 4
application; 50,100, 150 = 50, 100 and 150 ppm SA, respectively.
2
0
(a) Control SP50 SP100 SP150 FA50 FA100 FA150
18 Well watered Salicylic acid treatments
Leaf GB content
(µmol g–1 DW)

15 Drought
12 Fig. 8 Influence of salicylic acid treatments on the (a) superoxide
9 dismutase (SOD), (b) catalase (CAT) and (c) ascorbate peroxidase (APX)
6 in rice under well-watered and drought conditions ± S.E. (the results
3
are the average of five replications repeated twice). *One unit of SOD
0
Control SP50 SP100 SP150 FA50 FA100 FA150 activity is equivalent to the volume of extract needed to cause 50%
inhibition of the colour reaction. SP = seed priming; FA 100 = foliar
(b)
15 application; 50,100, 150 = 50, 100 and 150 ppm SA, respectively.
Leaf proline content

Well watered
Drought
(µmol g–1 DW)

12
9 also found under drought conditions but there was no
6 correlation between SA and wp under both drought-stress
3 and well-watered conditions and between SA and RWC
0 under well-watered conditions (Table 1). Membrane
Control SP50 SP100 SP150 FA50 FA100 FA150 electrolyte leakage (membrane permeability), H2O2 and
(c) MDA were negatively correlated with the antioxidant sys-
6
Well watered
tem under drought stress. Under well-watered condition,
Leaf SA content

5 Drought
(µg g–1 FW)

4 there was negative correlation between all antioxidants

3 (studied) and EC, and between CAT and MDA; and APX
2 and MDA (Table 2).
1
0
Control SP50 SP100 SP150 FA50 FA100 FA150
Discussion
Salicylic acid treatments
Drought stress drastically reduced the seedling fresh and
Fig. 7 Influence of salicylic acid treatments on the (a) leaf GB
dry weight (Fig. 1), which seems to be direct result of
content, (b) leaf proline content and (c) leaf salicylic acid content in
rice under well-watered and drought conditions ± S.E. (the results are
diminished photosynthesis (Fig. 2a). Under drought
the average of five replications repeated twice). SP = seed priming; FA stress, photosynthesis is hampered mainly due to
100 = foliar application; 50,100, 150 = 50, 100 and 150 ppm SA, reduced stomatal conductance (Fig. 2b; Farooq et al.
respectively. 2008f, 2009b,c), changes in photosynthetic pigments

242 ª 2009 Blackwell Verlag, 195 (2009) 237–246

 Drought Tolerance in Rice by Salicylic Acid

Table 1 Correlation coefficients (r) of GB, free proline and SA with changes in leaf water potential (ww) and relative leaf water content (RWC) of
rice under well-watered and drought conditions

Water potential Pressure potential Relative water contents

Osmoticum/SA WW DS WW DS WW DS

GB 0.93*** 0.81* 0.73* 0.80* 0.84** 0.85**

Free proline 0.74* 0.96*** 0.83** 0.94*** 0.86** 0.94***
SA 0.81* 0.73* 0.53 ns 0.63 ns 0.70 ns 0.83**

*P < 0.05, **P < 0.01, ***P < 0.001; ns, non significant; WW, well watered; DS, drought stress.

Table 2 Correlation coefficients (r) of antioxidants with changes in membrane thermostability (EC), hydrogen peroxide (H2O2) and malondialde-
hyde (MDA) of rice under well-watered and drought conditions

EC H2O2 MDA

Antioxidants WW DS WW DS WW DS

SOD )0.80* )0.91** )0.56 ns )0.90** )0.64 ns )0.97***

CAT )0.86** )0.84** )0.35 ns )0.89** )0.84** )0.94***
APX )0.86** )0.89** )0.63 ns )0.90** )0.73 * )0.93***

*P < 0.05, **P < 0.01, ***P < 0.001, ns, non-significant; WW, well watered; DS, drought stress.

(Loggini et al. 1999) and decreased activities of Calvin increased the leaf H2O2 and MDA contents (Fig. 6).
cycle enzymes (Monakhova and Chernyadév 2004). However, SA application reduced the membrane electro-
Moreover, ROS production (Ma et al. 2006) and imbal- lyte leakage significantly (Fig. 4b) and leaf H2O2 and
ance between ROS and antioxidants (Hasegawa et al. MDA contents (Fig. 6). Enhanced electrolyte leakage, leaf
2000, Fazeli et al. 2007) under drought stress also dis- H2O2 and MDA contents are considered to be a symptom
turb the photosynthetic apparatus and system (Munne- of stress-induced damage and deterioration (Feng et al.
Bosch and Penuelas 2003, Farooq et al. 2008b). 2003). Exposure of plants to certain environmental stres-
However, studies with different applications of SA have ses quite often leads to the generation of ROS (Munne-
revealed a positive effect on photosynthesis and plant Bosch and Penuelas 2003), which may react with proteins,
growth under drought stress (Rajasekaran and Blum lipids and DNA causing oxidative damage and impairing
1999, Singh and Usha 2003). For example, SA applica- the normal cellular functions (Foyer and Fletcher 2001).
tion in drought stressed wheat increased the photosyn- ROS in plants are scavenged by a variety of antioxidant
thetic pigments and carboxylase activity of Rubisco enzymes and/or lipid-soluble and water-soluble molecules
(Singh and Usha 2003). (Foyer and Fletcher 2001). Of these, antioxidant enzymes
Plant water relations in rice were also disturbed under are the most effective against oxidative damage (Halliwell
drought stress (Fig. 3, 4a); however, SA application and Gutteridge 1999). The study suggested that enzymatic
improved these relations. The strong correlation between antioxidant activities of rice seedlings were substantially
plant water relation components and the accumulation of induced by SA application. Although the previous studies
compatible solutes (GB and free proline) under drought report the increase in SOD and peroxidases and not the
(Table 1) indicated the involvement of compatible solutes CAT by SA treatments ((Janda et al. 2003, Shakirova
with the maintenance of ww, wp and improved leaf water 2007), CAT activity was also increased in this study. Even
status under drought. High levels of compatible solutes if SA has proved capable of binding directly to CAT
(Fig. 7) enable a plant to maintain low water potentials. enzyme, isolated from tobacco, inhibiting its activity
By lowering water potentials, the accumulation of com- (Chen et al. 1993, Conrath et al. 1995), the implication of
patible osmolytes involved in osmoregulation allows addi- CAT inhibition by SA can not be validated in all plant
tional water to be taken up from the environment, thus species. For example, in tobacco all the CAT isoenzymes
buffering the immediate effect of water shortages within are inhibited by SA (Durner and Klessig 1996) but not in
the organism (Kumar et al. 2003, Farooq et al. 2008f, rice (Sánchez-Casas and Klessig 1994). In rice, SA inhib-
2009a,b,c). ited the activity of the CATb isoenzyme, but not that of
Membrane permeability was significantly increased as CATa (Chen et al. 1997).
indicated by increased level of electrolyte leakage under This increased antioxidant production lowered the
drought stress (Fig. 4b). Moreover, drought stress ROS-based damages in the plant system as is evident

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Farooq et al.

from the negative correlation between membrane perme- El-Tayeb, M. A., and M. K. Ahmed, 2007: Apoplastic protein
ability, H2O2 and MDA and antioxidant system (Table 2). pattern, hydrolases and peroxidase activity of Vicia faba
In conclusion, drought stress severely hampers the rice cultivars as influenced by drought. Int. J. Agric. Biol. 9,
performance. Augmented synthesis of compatible solutes 226–230.
and activation of antioxidant system by SA application Farooq, M., S. M. A. Basra, and K. Hafeez, 2006a: Seed invigo-
improved the integrity of cellular membranes and enabled ration by osmohardening in coarse and fine rice. Seed Sci.
the rice plant to maintain tissue water status and as a Technol. 34, 181–187.
result, photosynthesis and general metabolism. Results Farooq, M., S. M. A. Basra, M. Khalid, R. Tabassum, and
T. Mehmood, 2006b: Nutrient homeostasis, reserves
further suggest that FA of SA is better than seed
metabolism and seedling vigor as affected by seed priming
treatment.
in coarse rice. Can. J. Bot. 84, 1196–1202.
Farooq, M., S. M. A. Basra, R. Tabassum, and I. Afzal, 2006c:
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