improving water relations and gas exchange with brassinosteroids in rice under drought stress

DROUGHT STRESS

Improving Water Relations and Gas Exchange withBrassinosteroids in Rice under Drought StressM. Farooq1, A. Wahid2, S. M. A. Basra3 & Islam-ud-Din4

1 Department of Agronomy, University of Agriculture, Faisalabad, Pakistan

2 Department of Botany, University of Agriculture, Faisalabad, Pakistan

3 Department of Crop Physiology, University of Agriculture, Faisalabad, Pakistan

4 Department of Mathematics and Statistics, University of Agriculture, Faisalabad, Pakistan

Introduction

Drought stress reduces the dry matter production and

final yield (Tahir and Mehdi 2001, Hussain et al. 2008)

most probably due to diminished carbon assimilation

(Wahid and Rasul 2005) and photosynthetic pigments

(Farooq et al. 2009a). Damaged photosynthetic apparatus

(Fu and Huang 2001, Farooq et al. 2008a, 2009a,b,c) and

decreased activities of Calvin cycle enzymes (Monakhova

and Chernyadev 2002) are important causes of reduced

crop yield. It also distresses the water relations of plants

at cellular, tissue and organ levels (Beck et al. 2007,

Farooq et al. 2008a, 2009a,b,c). Another important reason

in this regard is the loss of balance between the produc-

tion of reactive oxygen species (ROS) and the antioxidant

defence system (Fu and Huang 2001, Reddy et al. 2004),

leading to accumulation of ROS and induction of oxida-

tive damage to cell components (Fu and Huang 2001).

To prevent this, plants have developed a variety of anti-

oxidant enzymes and ROS scavenging molecules, the most

important among those are peroxidase, ascorbate peroxi-

dase (APX), glutathione reductase, superoxide dismutase

(SOD) and catalase (CAT) (Halliwell and Gutteridge

1999, Hasegawa et al. 2000, Fazeli et al. 2007).

Brassinosteroids (BRs) are a group of naturally

occurring steroidal plant hormones that regulate plant

growth and development by producing an array of

physiological changes (Fujioka and Yokota 2003, Sasse

Keywords

antioxidants; brassinosteroid; drought stress;

photosynthesis; water relations

Correspondence

Dr M. Farooq

Department of Agronomy, University of

Agriculture, Faisalabad 38040, Pakistan

Tel.: +92 41 9200161 9/2917

Fax: +92 41 9200605

Email: [email protected]

Accepted March 20, 2009

doi:10.1111/j.1439-037X.2009.00368.x

Abstract

Drought stress is the most pervasive threat to sustainable rice production and

mainly disrupts membrane structure and cell-water relations. Exogenously

applied brassinosteroids (BRs) may produce profound changes that may

improve drought tolerance in rice. In this study, we monitored some physio-

logical basis of the exogenously applied BRs in improving drought tolerance in

fine grain aromatic rice (Oryza sativa L.). Two BRs i.e. 28-homobrassinolide

(HBL) and 24-epibrassinolide (EBL) were used both as seed priming and foliar

spray. To prime, the seeds were soaked in 0.01 lm aerated solution each of

HBL and EBL for 48 h and dried back to original weight. Treated and

untreated seeds were sown in plastic pots with normal irrigation in a phyto-

tron. At four-leaf stage (3 weeks after sowing), plants were subjected to

drought stress at 50 % field capacity by cutting down the water supply. For

foliar spray, 0.01 lm of HBL and EBL solutions were sprayed at five-leaf stage.

Drought stress severely reduced fresh and dry weights, whilst exogenously

applied BRs improved net CO2 assimilation, water use efficiency, leaf water sta-

tus, membrane properties, production of free proline, anthocyanins, soluble

phenolics, but declined the malondialdehyde and H2O2 production. In conclu-

sion, BRs application improved the leaf water economy and CO2 assimilation,

and enabled rice to withstand drought. Moreover, foliar spray had better effect

under drought than seed treatments and of the two BRs, EBL proved more

effective.

J. Agronomy & Crop Science (2009) ISSN 0931-2250

262 ª 2009 Blackwell Verlag, 195 (2009) 262–269

2003, Feldmann 2006). They are also known to amelio-

rate various biotic and abiotic stress effects (Krishna

2003, Ali et al. 2007, Jager et al. 2008). Application of

24-epibrassinolide (EBL) increased the membrane stabil-

ity and proline contents of rice during chilling (Wang

and Zeng 1993). Sairam (1994) reported the involve-

ment of BRs in the maintenance of tissue water status,

photosynthetic rate and biomass yield in wheat under

drought stress. Likewise, application of BRs to radish

(Raphanus sativus) improved cadmium tolerance by

activation of antioxidant enzymes (Anuradha and Rao

2007) and photosynthesis under nickel toxicity (Alam

et al. 2007).

Seed treatment with various priming agents such as

glycinebetaine, CaCl2, KCl and salicylic acid have been

reported to improve drought tolerance and stand estab-

lishment in rice (Farooq et al. 2006b,c, 2008a), and chill-

ing tolerance in maize (Farooq et al. 2008b,c,d,e) and

wheat (Farooq et al. 2008f). Likewise, seed treatment with

brassinolide significantly increased the dry mass accumu-

lation and activities of antioxidant enzymes in lucerne

(Medicago sativa) under salinity (Zhang et al. 2007) and

improved the survival and growth of Robinia pseudoacacia

under drought (Li et al. 2007).

Although some reports have provided evidence that

BRs can improve drought tolerance in moderate drought-

tolerant plants, these roles are mainly related to the allevi-

ation of oxidative damage (Sairam 1994, Li et al. 2007).

Despite that, exploring novel roles of BRs is the subject of

intensive research. To best of our knowledge, no study

has ever discovered the potential of exogenous BRs appli-

cation to improve drought tolerance in submerged plants

like rice. We hypothesized that BRs can improve leaf

water status and photosynthesis, leading to improved

drought tolerance. This study was undertaken to explore

the possible role of BRs to improve drought tolerance in

rice, based on changes in some growth and physiological

attributes.

Materials and Methods

Experimental details and growth conditions

Fine (aromatic) rice (Oryza sativa L. cv. Super-basmati)

was used in the study as experimental material. The

seeds were surface sterilized with 0.2 % HgCl2 solution

for 5 min and thoroughly rinsed with tap water. Two

BRs, 28-homobrassinolide (HBL) and EBL were used

both for seed priming (SP) and foliar spray. For prim-

ing, rice seeds were soaked in 0.01 lm aerated solution

of both the BRs for 48 h at 28 �C. The BRs were

dissolved initially in small amount of ethanol and final

volume made up with distilled water containing Tween-

20 (0.05 % of the final volume). The seeds were soaked

in this solution (1 : 5 w/v) (Farooq et al. 2006a). After

each treatment, seeds were given three surface washings

with distilled water and dried back closer to original

moisture under forced air at 27 ± 3 �C, sealed in poly-

thene bags and stored in a refrigerator at 5 �C until use

(Lee and Kim 2000).

Treated and untreated seeds were grown in plastic

pots (20 cm in diameter and 18 cm in height) contain-

ing loam soil. The pots were kept in a phytotron with a

photosynthetically active photon flux density of

300 mmol m)2 s)1, 27 �C, 70–80 % relative humidity

and a photoperiod of 14/10 h light/dark. Experimental

design was completely randomized with five replications.

All pots were well watered (maintained at 100 % field

capacity) up to four leaf stage (3 weeks after sowing)

and then subjected to drought stress. Drought was main-

tained at 50 % field capacity by applying water on alter-

nate days or whenever needed. For foliar application

(FA), 0.01 lm solution each of HBL and EBL was

mechanically sprayed on leaves at five leaf stage (4 weeks

after sowing). For comparison, two controls were main-

tained; both receiving no BRs as FA or seed treatments,

one under drought conditions and the other under well-

watered conditions. Hoagland nutrient solution (300 ml)

was applied with irrigation water once in a week

(Hoagland and Arnon 1950).

All the observations, except seedling fresh and dry

weight, were taken 1 week after FA of BRs. The experi-

ment was terminated after 3 weeks (stem elongation

stage; Zadokos et al. 1974) when about 50 % leaves of

drought stressed plants showed sings of wilting. At har-

vest, the seedlings were tested for vigour after carefully

removing from the soil. Seedling fresh weight was deter-

mined immediately after harvest while dry weight was

taken after drying at 70 �C for 7 days.

Leaf gas-exchange measurements

Net rate of photosynthesis (A), intercellular CO2 concen-

tration (Ci), transpiration rate (E) and stomatal conduc-

tance (gs) in the penultimate fully expanded leaves was

measured using a portable infrared gas analyser based

photosynthesis system (LI-6400, LiCor, Lincoln, Nebraska,

USA). Data were recorded at 09:00 to 10:00 am 1 week

after foliar BRs spray. At the time of data collection, air

relative humidity was about 75 % and the ambient CO2

concentration 450 lmol CO2 mol)1. Water use efficiency

(WUE) was calculated as ratio between net photosynthesis

and transpiration rate. Limitation to CO2 uptake by sto-

mata, a measure of carboxylation efficiency of Rubisco,

was computed as ratio between net photosynthesis and

sub-stomatal CO2 concentration.

Inducing Drought Tolerance in Rice with Brassinosteroids

ª 2009 Blackwell Verlag, 195 (2009) 262–269 263

Plant water relations

Leaf water potential (ww) was determined with pressure

chamber (Soil Moisture Equipment Corp., Santa Barbara,

CA, USA) from penultimate leaf. Frozen leaf tissues were

thawed, sap expressed, centrifuged at 5000 g and osmotic

potential (ws) was determined with an osmometer (Digi-

tal Osmometer, Wescor, Logan, UT, USA). Leaf pressure

potential (wp) was computed as a difference of ww and

ws. To determine relative water contents (RWC), fresh

leaves (0.5 g) were weighed to get fresh weight (Wf).

Later, these leaves were floated on water for 4 h and satu-

rated weight (WS) was determined. These leaves were

dried for 24 h at 85 �C to determine dry weigh (Wd).

RWC (%) were calculated as:

RWC ¼ ðWf �WdÞ=ðWs �WdÞ � 100 %

Membrane permeability

To determine membrane permeability, electrolyte leakage

from leaves was measured following the protocol of Blum

and Ebercon (1981). Six leaf segments of similar size were

briefly washed with distilled water and engrossed in a test

tube having 6 ml distilled water for 12 h at room temper-

ature. Then electrical conductivity (EC1) of solution was

measured with a conductivity meter (Model DDS-11A,

Shanghai Leici Instrument Inc., Shanghai, China). Sam-

ples were then heated in boiling water for 20 min and

cooled to room temperature. The conductivity of killed

tissues (EC2) was again measured. Electrolyte leakage was

measured as the ratio between EC1 and EC2 and

expressed in percentage.

Metabolite levels

For free proline (Pro) estimation following the method

of Bates et al. (1973), 0.5 g of fresh leaf material was

homogenized in 10 ml of 3 % aqueous sulphosalicyclic

acid and filtered through Whatman No. 2 filter paper.

Two ml of filtrate was mixed with 2 ml each of acid-

ninhydrin and glacial acetic acid in a test tube. The

mixture was placed in a water bath for 1 h at 100 �C.

The reaction mixture was extracted with 4 ml toluene

and the chromophore containing toluene was aspirated,

cooled to room temperature and absorbance was mea-

sured at 520 nm with a spectrophotometer (Shimadzu

UV 1601, Shimadzu Corporation, Kyoto, Japan). Tannic

acid-equivalent soluble phenolics were determined with

spectrophotometer from 80 % acetone extracts of leaves

as described by Julkenen-Titto (1985). Anthocyanins

were determined after extraction of leaves in acidified

methanol (1 % HCl v/v), vacuum filtered and quantified

using spectrophotometer at 535 nm according to the

method described by Stark and Wray (1989).

Lipid peroxidation was measured in terms of malondi-

aldehyde (MDA) content following the method of Heath

and Packer (1968). Leaf sample (1 g) was homogenized in

10 ml 0.1 % trichloroacetic acid. The homogenate was

centrifuged at 15 000 g for 5 min and 4 ml of 0.5 % thio-

barbituric acid in 20 % trichloroacetic acid was added to a

1 ml aliquot of the supernatant. The mixture was heated

at 95 �C for 30 min and quickly cooled on ice. After cen-

trifugation at 10 000 g for 10 min, the absorbance was

recorded at 532 nm. The value for non-specific absorption

at 600 nm was subtracted. The MDA content was calcu-

lated using its absorption coefficient of 155 mmol)1 cm)1.

Leaf hydrogen peroxide was extracted as described by

Prasad et al. (1994) and estimated using titanium reagent

following the method of Teranishi et al. (1974).

Antioxidants

Total extractable SOD activity was determined following

the method of McCord and Fridovitch (1969). Inhibition

of colour formation (measured at 560 nm) was deter-

mined with the addition of 0–50 ll of the extract to a

reaction mixture containing 50 mm HEPES/KOH buffer

(pH 7.8), 0.05 units xanthine oxidase, 0.5 mm nitroblue

tetrazolium and 4 mm xanthine. One unit of SOD activity

equalled the volume of extract needed to cause 50 %

inhibition of the colour reaction. CAT activity was mea-

sured following the modified method of Luck (1974).

Enzyme extract (50 ll) was added to 3 ml of H2O2-phos-

phate buffer (pH 7.0). Time required for decrease in the

absorbance from 0.45 to 0.40 was noted. Enzyme solution

containing H2O2-free phosphate buffer was used as

control. Enzyme activity was expressed in mmol H2O2

consumed min)1 mg)1 chl. APX activity was estimated

according to the method of Nakano and Asada (1987).

Ascorbate oxidation to dehydroascorbate was followed at

265 nm in 1 ml reaction mixture containing 50 mm

HEPES/KOH (pH 7.6), 0.1 mm EDTA, 0.05 mm

ascorbate, 10 ll extract and 0.1 mm H2O2.

Statistical analysis

The data were subjected to analysis of variance using

COSTAT computer software (CoHort Software, Berkeley,

CA, USA). Least significant difference test was applied to

compare the treatment means. Correlations of growth

and photosynthetic attributes were established with water

relations, metabolites, membrane characteristics and

antioxidants levels in the rice leaves. Correlations of

membrane characteristics were also established with water

relations, metabolites and antioxidants.

Farooq et al.


Results

Rice growth was severely hampered under drought stress,

while exogenous application of BRs alleviated this adverse

effect. Maximum plant height and seedling fresh and dry

weight were observed from rice plants raised under well-

watered conditions (Table 1). Of the two BRs, maximum

plant height and seedling fresh and dry weight were

recorded from the plants treated with EBL-FA. However,

EBL-FA performed similar to EBL-SP in case of plant

height (Table 1).

The highest leaf water (ww), osmotic (ws) and pressure

(wp) potentials and RWC were observed in well-watered

plants, although stress drought drastically reduced these

characteristics. However, exogenous BRs application sub-

stantially improved the leaf water relation attributes

under drought. Maximum ww, ws, wp and RWC were

recorded from EBL-FA under drought (Table 2). Simi-

larly, maximum A, Ci, gs, E and A/Ci were evident in

well-watered plants, while drought stress significantly

reduced these attributes. Application of BRs notably

improved A, Ci and A/Ci but decreased gs and E under

drought. Maximum A, Ci and A/Ci were measured from

EBL-FA, which was at par with EBL-SP in case of A and

carboxylation efficiency of Rubisco and HBL-FA for Ci;

however, minimum gs was recorded from HBL-FA under

drought stress. Lowest E was measured from HBL-SP

followed by EBL-SP and HBL-FA (Table 3). Likewise,

maximum WUE (1.52) was recorded from EBL-FA that

was at par with well-watered control (1.48) and EBL-FA

(1.42), while it was minimum (0.99) in drought stresses

plants without BRs applied (Table 3).

Minimum phenolics, anthocyanins and free proline

(Pro) contents were measured from plants raised under

well-watered conditions; while these metabolites levels

were higher under drought and the highest under drought

with BRs application. Maximum phenolics and anthocya-

nins were recorded from EBL-FA, while maximum Pro

was noted from HBL-FA (Table 4). Leaf H2O2, MDA

contents and membrane permeability were minimal under

well-watered conditions, which increased significantly

under drought. However, BRs application significantly

lessened the values of these attributes under drought

stress. Minimum leaf H2O2, MDA and membrane perme-

ability under drought stress was observed from EBL-FA,

which was at par with ELB-SP in case of MAD and all

BRs treatments for membrane permeability (Table 4).

Although SOD contents were decreased by drought

stress, BRs application significantly improved it (Table 5).

Maximum SOD was recorded from EBL-FA treatment

while maximum CAT and APX activities noted in well-

watered rice plants, which were decreased significantly

upon exposure to drought stress. Nevertheless, BRs

application improved the CAT and APX under stress

conditions; EBL-FA was the most promising (Table 5).

To validate the above findings, fresh and dry weight,

net photosynthesis and WUE were correlated with water

relations, metabolites levels, membrane characteristics and

antioxidant activities (Table 6). Fresh and dry weight, A,

Table 1 Influence of brassinosteroid application on the seedling vig-

our under drought stress in rice

Treatment

Seedling fresh

weight (g plant)1)

Seedling dry

weight (g plant)1) Plant height (cm)

CK1 53.11 ± 1.44 a1 15.01 ± 0.44 a 57.11 ± 1.41 a

CK2 30.21 ± 1.21 e 07.41 ± 0.34 d 39.17 ± 2.11 e

HBL-SP 34.13 ± 1.61 d 09.23 ± 0.21 c 44.33 ± 1.01 cd

EBL-SP 38.14 ± 1.37 c 10.17 ± 0.51 c 47.61 ± 1.01 bc

HBL-FA 35.17 ± 1.22 d 09.13 ± 0.41 c 45.27 ± 1.23 c

EBL-FA 42.33 ± 1.17 b 12.27 ± 0.13 b 49.41 ± 2.22 b

1Means sharing the same letters in a column do not differ significantly

at P £ 0.05 according to least significant difference test. Each value

indicates treatment mean ± S.E.

CK1, well watered, no brassinosteroid application; CK2, drought

stress, no brassinosteroid application; SP, seed priming; FA, foliar

application; HBL, 28-homobrassinolide; EBL, 24-epibrassinolide.

Table 2 Effects of brassinosteroid application

on the plant water relations under drought

stress in rice Treatment

Water potential

(ww, )MPa)

Osmotic potential

(ws, )MPa)

Pressure potential

(wp, MPa)

Relative water

contents (%)

CK1 0.41 ± 0.031 e1 0.96 ± 0.042 e 0.55 ± 0.031 a 84.71 ± 5.53 a

CK2 1.16 ± 0.021 a 1.32 ± 0.062 a 0.16 ± 0.026 e 42.37 ± 6.33 e

HBL-SP 1.05 ± 0.032 b 1.24 ± 0.053 b 0.19 ± 0.031 d 46.62 ± 3.45 d

EBL-SP 0.90 ± 0.023 c 1.21 ± 0.037 b 0.31 ± 0.023 b 52.54 ± 2.66 c

HBL-FA 0.92 ± 0.051 c 1.15 ± 0.022 c 0.23 ± 0.044 c 47.81 ± 4.61 d

EBL-FA 0.73 ± 0.053 d 1.04 ± 0.021 d 0.31 ± 0.031 b 55.15 ± 2.34 b

1Means sharing the same letters in a column do not differ significantly at P £ 0.05 according to

least significant difference test. Each value indicates treatment mean ± S.E.

CK1, well watered, no brassinosteroid application; CK2, drought stress, no brassinosteroid appli-

cation; SP, seed priming; FA, foliar application; HBL, 28-homobrassinolide; EBL, 24-epibrassino-

lide.



Ci and ratio of A/Ci were correlated negatively with water

and osmotic potentials but positively with leaf turgor and

RWC except Ci, which had no correlation with leaf turgor

and relative water. Hydrogen peroxide was negatively

related to fresh and dry weight, A, Ci, WUE and ratio of

A/Ci, while there was negative correlation of MDA with

fresh and dry weight, A, Ci and A/Ci ratio. Membrane

permeability was related negatively to fresh and dry

weight, Ci and WUE. Soluble phenolics were negatively

correlated with stomatal conductance and transpiration

rate, while anthocyanins were negatively related with sto-

matal conductance only. Activities of CAT and APX were

positively related to seedling fresh and dry weight, A, Ci,

WUE and ratio of A/Ci, while SOD has positive correla-

tion only with WUE (Table 6).

Discussion

Drought stress impaired the rice growth in terms of plant

length, fresh and dry mass (Table 1) mainly by disrupting

water relations (Table 2) and leaf gas exchange properties

(Table 3). However, use of BRs (EBL and HBL) as seed

and foliar treatments improved these attributes compared

with the untreated-stressed plants (Table 1). BRs can

improve carbon-assimilation by Rubisco and WUE of

Table 4 Effects of brassinosteroid application on the metabolites in rice under drought stress

Treatment

Soluble phenolics

(lg g)1 FW)

Anthocyanins

(lg g)1 FW)

Leaf-free proline

content (lmol g)1 DW)

Leaf H2O2

(lmol g)1 FW)

MAD

(lmol g)1 FW)

Membrane

permeability (%)

CK1 39.33 ± 1.11 f1 22.43 ± 1.41 d 6.64 ± 0.43 c 9.33 ± 0.74 e 12.41 ± 1.05 d 10.13 ± 1.15 c

CK2 43.41 ± 1.23 e 25.33 ± 1.32 c 8.57 ± 0.45 b 18.21 ± 0.91 a 25.32 ± 1.31 a 23.65 ± 1.29 a

HBL-SP 46.67 ± 0.79 d 28.37 ± 1.16 b 10.41 ± 0.63 a 15.23 ± 0.73 b 21.41 ± 1.51 b 14.12 ± 1.83 b

EBL-SP 50.34 ± 1.05 c 29.42 ± 1.34 b 11.24 ± 0.71 a 13.27 ± 1.21 c 19.41 ± 1.31 c 13.15 ± 1.43 b

HBL-FA 52.27 ± 0.68 b 28.31 ± 1.27 b 10.72 ± 0.61 a 12.21 ± 0.53 d 20.22 ± 1.41 b 14.31 ± 1.26 b

EBL-FA 54.37 ± 1.12 a 31.44 ± 1.21 a 10.46 ± 0.72 a 12.31 ± 1.10 d 19.31 ± 1.23 c 12.24 ± 1.27 b

1Means sharing the same letters in a column do not differ significantly at P £ 0.05 according to least significant difference test. Each value


CK1, well watered, no brassinosteroid application; CK2, drought stress, no brassinosteroid application; SP, seed priming; FA, foliar application;

HBL, 28-homobrassinolide; EBL, 24-epibrassinolide.

Table 3 Effects of brassinosteroid application on the leaf CO2 net assimilation rate (A), intercellular CO2 concentration (Ci), stomatal conductance

(gs), transpiration rate (E), water use efficiency and stomatal limitations to CO2 uptake under drought stress in rice

Treatment

Leaf CO2 net

assimilation rate (A)

(lmol m)2 s)1)

Intercellular CO2

concentration (Ci)

(lmol mol)1)

Stomatal

conductance (gs)

(mol m)2 s)1)

Transpiration

rate (E)

(lmol m)2 s)1)

Water use

efficiency (A/E)

Stomatal limitations

to CO2 uptake (A/Ci)

CK1 16.67 ± 1.23 a1 291 ± 4.23 a 0.471 ± 0.011 a 11.23 ± 1.13 a 1.48 ± 0.31 a 0.057 ± 0.0012 a

CK2 09.31 ± 1.45 d 257 ± 3.22 d 0.391 ± 0.018 b 09.41 ± 1.41 b 0.99 ± 0.42 d 0.036 ± 0.0017 d

HBL-SP 11.13 ± 1.33 c 278 ± 3.83 c 0.331 ± 0.027 c 08.13 ± 1.17 c 1.37 ± 0.37 bc 0.040 ± 0.0018 c

EBL-SP 12.07 ± 1.27 bc 276 ± 4.11 c 0.332 ± 0.037 c 08.47 ± 1.23 c 1.42 ± 0.43 ab 0.044 ± 0.0013 b

HBL-FA 11.13 ± 1.67 c 281 ± 2.96 b 0.321 ± 0.036 c 08.32 ± 1.41 c 1.34 ± 0.19 c 0.040 ± 0.0015 c

EBL-FA 13.03 ± 1.88 b 283 ± 3.39 b 0.341 ± 0.027 c 08.57 ± 1.36 c 1.52 ± 0.36 a 0.046 ± 0.0011 b

1Means sharing the same letters in a column do not differ significantly at P £ 0.05 according to least significant difference test. Each value


CK1, well watered, no brassinosteroid application; CK2, drought stress, no brassinosteroid application; SP, seed priming; FA, foliar application;

HBL, 28-homobrassinolide; EBL, 24-epibrassinolide.

Table 5 Effects of brassinosteroid application on the antioxidants in

under drought stress in rice

Treatment

SOD

(unit g)1 protein)1CAT (lmol min)1

g)1 protein)

APX (lmol min)1

g)1 protein)

CK1 13.47 ± 0.21 b2 13.61 ± 1.41 a 12.53 ± 0.43 a

CK2 10.87 ± 0.41 c 8.47 ± 0.45 d 9.35 ± 0.35 d

HBL-SP 13.49 ± 0.51 b 10.59 ± 0.69 c 10.54 ± 0.61 c

EBL-SP 15.59 ± 0.37 a 10.53 ± 0.51 c 10.69 ± 0.65 c

HBL-FA 14.39 ± 0.34 b 10.44 ± 0.67 c 10.67 ± 0.53 c

EBL-FA 16.45 ± 0.21 a 12.52 ± 0.41 b 11.71 ± 0.56 b

1One unit of SOD activity is equivalent to the volume of extract

needed to cause 50 % inhibition of the colour reaction.2Means sharing the same letters in a column do not differ significantly

at P £ 0.05 according to least significant difference test. Each value


CK1, well watered, no brassinosteroid application; CK2, drought

stress, no brassinosteroid application; SP, seed priming; FA, foliar

application; HBL, 28-homobrassinolide; EBL, 24-epibrassinolide; SOD,

superoxide dismutase; CAT, catalase; APX, ascorbate peroxidase.

Farooq et al.


leaves as a result of their involvement in the plasma

membrane permeability under stressful conditions

(Hamada 1986, Table 4). In our study, improved A/Ci

and A/E ratios with BRs application substantiated that

improved water economy during photosynthesis is impor-

tant to rice under drought stress.

Although drought stress hampered the plant water rela-

tions, the exogenous application of BRs maintained the

tissue water status (Table 2), possibly by stimulating the

proton pumping (Khripach et al. 2003), activating nucleic

acid and protein synthesis (Bajguz 2000) and regulation of

genes expression (Felner 2003). In this study, BRs stimu-

lated the biosynthesis of free proline, soluble phenolics

and anthocyanins (Table 4), which is important manifesta-

tion of drought tolerance in many plant species ((Wahid

and Ghazanfar 2006, Anuradha and Rao 2007, Wahid

2007). Both anthocyanins and soluble phenolics have

aromatic ring in their structure, which act as membrane

stabilizer during episodes of abiotic stress (Olga et al.

2003, Taiz and Zeiger 2006) and induce ROS scavenging

cascades in the cells (Takahama and Oniki 1997). BRs sig-

nificantly enhanced the activity of SOD, CAT and APX

(Table 5), which protected the leaves from oxidative dam-

age. From the synthesis of metabolites and induction of

antioxidants, it is clear that BRs induce multiple pathways

of protection from oxidative damage in rice.

Contents of H2O2 and MDA and membrane permeabil-

ity were significantly increased (Table 4). There is a close

association of these parameters in producing oxidative

damage under stressful condition (Feng et al. 2003,

Munne-Bosch and Penuelas 2003, and the present find-

ings). Such changes quite often arise because of the gener-

ation of ROS, mainly H2O2, which is a relatively long-

lived molecule (Apel and Hirt 2004). The ROS react with

proteins, lipids and DNA, and impair the normal cellular

functions (Foyer and Fletcher 2001). As noted in this

study, correlations of water relations parameters, activities

of antioxidants and H2O2, MDA and membrane perme-

ability were closer with the gas exchange attributes, while

soluble phenolics and anthocyanins were rarely associated

(Table 6). This indicated that soluble phenolics and an-

thocyanins had little roles in improving the leaf water sta-

tus, rather their main function remained protection of

non-photosynthetic membranes from oxidative damage in

rice, which is contrary to earlier finding in various plant

species (Olga et al. 2003, Wahid 2007). However, from

tight correlations of CAT and APX activities (involved in

elimination of H2O2) with CO2 assimilation and mainte-

nance of leaf water status (Table 6), it was revealed that

BRs play a crucial role in improved leaf water status,

integrity of chloroplastic membranes and Rubisco activity

under drought.

The above effects of BRs appear to be related to

changes in their structure during abiotic stress tolerance.

EBL is with predominant lactone structure, while HBL

has 6-ketone (Zullo and Adam 2002). Moving from the

lactone to the 6-ketone, brassinolide activity decreases

substantially (Takatsuto et al. 1983, Zullo and Adam

2002). Thompson et al. (1982) reported that EBL is about

three times more active than HBL. In this study, EBL was

more effective in improving drought tolerance of rice,

irrespective of the application method. This seems the

reason that EBL performed better than HBL to induce

drought tolerance.

In sum, in addition to the scavenging of ROS with the

enhanced expression of antioxidants twined with the

synthesis of phenolics and anthocyanins, improved leaf

water status and carboxylation efficiency of Rubisco are

Table 6 Correlation coefficients of some growth and gas exchange attributes of rice with water relations, metabolites and activity of antioxidant

Characteristics

Fresh

weight

Dry

weight

Intercellular CO2

concentration

Net

photosynthesis

Stomatal

conductance

Transpiration

rate

Water use

efficiency A/Ci

Water potential )0.990*** )0.983*** )0.843* )0.983*** )0.617 ns )0.680 ns )0.725 ns )0.979***

Osmotic potential )0.934*** )0.945*** )0.884* )0.921*** )0.471 ns )0.536 ns )0.779 ns )0.902*

Turgor potential 0.982*** 0.952*** 0.749 ns 0.979*** 0.716 ns 0.773 ns 0.628 ns 0.989***

Relative water content 0.972*** 0.940*** 0.730 ns 0.973*** 0.792 ns 0.834* 0.562 ns 0.980***

Free proline )0.561 ns )0.500 ns )0.124 ns )0.543 ns )0.979*** )0.958*** 0.151 ns )0.567 ns

Hydrogen peroxide )0.881* )0.875* )0.946*** )0.890* )0.327 ns )0.416 ns )0.838* )0.867*

MDA )0.965*** )0.946*** )0.890* )0.980*** )0.568 ns )0.631 ns )0.759 ns )0.971***

Membrane permeability )0.992*** )0.832* )0.967*** )0.802 ns )0.077 ns )0.141 ns )0.976*** )0.769 ns

Soluble phenolics )0.347 ns )0.278 ns 0.071 ns )0.379 ns )0.859* )0.869* 0.307 ns )0.361 ns

Anthocyanins )0.341 ns 0.240 ns )0.01 ns )0.331 ns )0.885* )0.809 ns 0.316 ns )0.404 ns

Superoxide dismutase 0.354 ns 0.424 ns 0.624 ns 0.353 ns )0.441 ns )0.368 ns 0.847* )0.742 ns

Catalase 0.944*** 0.973*** 0.917*** 0.946*** 0.439 ns 0.485 ns 0.859* 0.925***

Ascorbate peroxidase 0.957*** 0.977*** 0.928*** 0.960*** 0.452 ns 0.507 ns 0.857* 0.940***

Significant at *P < 0.05; **P < 0.01; ***P < 0.01; ns, non-significant.

MDA, malondialdehyde.



important findings of this study. Such mechanisms are

important for sustained rice production in relatively water

scarce areas and at critical stages of rice growth. Of the

two methods of BRs application, foliar spray was more

effective in improving rice growth under drought, while

of the BRs, EBL was more effective.

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