DROUGHT STRESS

Seed Priming with Ascorbic Acid Improves DroughtResistance of WheatM. Farooq1,2, M. Irfan1, T. Aziz1, I. Ahmad1 & S. A. Cheema1

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

2 The UWA Institute of Agriculture, The University of Western Australia, Crawley, WA, Australia

Introduction

Wheat (Triticum aestivum L.), the most important winter

cereal, is the staple for millions around the world.

Drought is the major factor limiting crop growth and

productivity in many regions of the world, the loss of

which is more than any other single environmental factor

(Farooq et al. 2009a,b). However, changing global climate

is making the situation more serious (IPCC 2007).

Water deficit during initial stage of crop results in

delayed and erratic seedling emergence and stand establish-

ment (Almansouri et al. 2001, Kaya et al. 2006), and in

severe cases, complete inhibition of seedling emergence

may also result (Kaya et al. 2006). Decrease in water uptake

during imbibition phase of germination is the primary rea-

son for this decline in stand establishment (Muril-

lo-Amador et al. 2002). Drought also disturbs the plant

growth owing to loss of turgor (Farooq et al. 2009a, Taiz

Keywords

ascorbate; drought; osmotic adjustment; seed

priming; water relations

Correspondence

M. Farooq

Department of Agronomy

University of Agriculture

Faisalabad-38040

Pakistan

Tel.: +92 41 9200161 9/2931

Fax: +92 41 9200605

Email: farooqcp@gmail.com;

mfarooq@uaf.edu.pk

Accepted April 20, 2012

doi:10.1111/j.1439-037X.2012.00521.x

Abstract

The study, consisting of two independent experiments, was conducted to evalu-

ate the role of seed priming with ascorbic acid (AsA) in drought resistance of

wheat. In the first experiment, seeds of wheat cultivars Mairaj-2008 and Lasani-

2008 were either soaked in aerated water (hydropriming) for 10 h or not

soaked (control). In the second experiment, seeds of same wheat cultivars were

soaked in aerated (2 mm) AsA solution (osmopriming) or water (hydropri-

ming) for 10 h. In both experiments, seeds were sown in plastic pots (10 kg)

maintained at 70 % and 35 % of water-holding capacity designated as well

watered and drought stressed, respectively. Both experiments were laid out in a

completely randomized design with six replications. Drought caused delayed

and erratic emergence and disturbed the plant water relations, chlorophyll con-

tents and membranes because of oxidative damage; however, root length in

cultivar Lasani-2008 was increased under drought. Hydropriming significantly

improved the seedling emergence and early growth under drought and well-

watered conditions; however, improvement was substantially higher from

osmopriming with AsA. Similarly, osmopriming with AsA significantly

improved the leaf emergence and elongation, leaf area, specific leaf area, chlo-

rophyll contents, root length and seedling dry weight. Owing to increase in

proline accumulation, phenolics and AsA, by seed priming with AsA, plant

water status was improved with simultaneous decrease in oxidative damages.

These improved the leaf emergence and elongation, and shoot and root growth

under drought. However, there was no difference between the cultivars in this

regard. In conclusion, osmopriming with AsA improved the drought resistance

of wheat owing to proline accumulation and antioxidant action of AsA and

phenolics, leading to tissue water maintenance, membrane stability, and better

and uniform seedling stand and growth.

J. Agronomy & Crop Science (2013) ISSN 0931-2250

12 ª 2012 Blackwell Verlag GmbH, 199 (2013) 12–22

and Zeiger 2010), as water supply from the xylem to the

surrounding elongating cells is interrupted (Nonami 1998).

Production and accumulation of osmoprotectants, for

example, proline, glycinebetaine in plant tissues during

drought, the osmotic adjustment, is an adaptive response

(Vendruscolo et al. 2007, Cattivelli et al. 2008, Farooq

et al. 2008a, Izanloo et al. 2008, Hussain et al. 2009).

Osmotic adjustment helps in improving water uptake

(Tangpremsri et al. 1991, Chimenti et al. 2006, Farooq

et al. 2009a) and enables leaf turgor maintenance for the

same leaf water potential, thus supporting stomatal con-

ductance and carbon assimilation under drought (Ali

et al. 1999, Farooq et al. 2009a).

Over-production of reactive oxygen species (ROS) than

their dousing is one of the key responses of plants to

environmental stresses (Smirnoff 1998, Farooq et al.

2009a, 2011). The ROS, thus generated, deteriorate the

cellular membranes and several other vital substances and

may even lead to cell death (Beligni and Lamattina 1999,

Kratsch and Wise 2000). Malondialdehyde (MDA) pro-

duction is taken as an index of ROS-induced oxidative

damage (Teisseire and Guy 2000, Zhang et al. 2007).

However, plants have evolved several antioxidative

defence mechanisms, including the production of enzy-

matic and non-enzymatic antioxidants to reduce ROS-

induced oxidative damages (Posmyk et al. 2009).

Seed priming, a controlled hydration technique that

allows the pre-germination metabolisms without actual

germination (Bradford 1986, Farooq et al. 2009c), is one

of the most pragmatic and short-term approaches to

combat the effects of drought (Kaya et al. 2006, Farooq

et al. 2010) and other environmental stresses (Farooq

et al. 2008b,c, 2010, Jafar et al. 2012) on seedling emer-

gence and stand establishment. Primed seeds usually have

higher and synchronized germination (Brocklehurst et al.

1984, Kaya et al. 2006, Farooq et al. 2009c) owing to sim-

ply a reduction in the lag time of imbibitions (Brockle-

hurst and Dearman 2008), build-up of germination-

enhancing metabolites (Farooq et al. 2006a), metabolic

repair during imbibition (Burgass and Powell 1984, Bray

et al. 1989) and osmotic adjustment (Bradford 1986).

Ascorbic acid (AsA) is one of the important metabo-

lites involved in cell division, osmotic adjustment

(De-Gara et al. 2003) and also plays vital role during the

initial stages of germination (Arrigoni et al. 1997). Ascor-

bic acid also possesses strong antioxidant potential and

helps in balancing the production and scavenging of ROS

(Muller-Moule et al. 2003, 2004); however; high endoge-

nous AsA level is required to maintain the balance. Inter-

estingly, exogenous application of AsA can increase the

endogenous AsA level (Chen and Gallie 2004).

Application of AsA through seed priming may thus be

helpful in improving the stand establishment and allome-

try of wheat under drought. This study was conducted to

evaluate the potential of AsA in improving the drought

resistance in wheat. It was hypothesized that seed priming

with AsA improves the drought resistance of wheat

through increase in endogenous AsA contents,

antioxidant potential and osmotic adjustment.

Materials and Methods

Plant material

Seeds of wheat cultivars ‘Lasani-2008’ and ‘Mairaj-2008’,

used in this study, were obtained from Wheat Research

Institute, Faisalabad, Pakistan and Regional Agriculture

Research Institute, Bahawalpur, Pakistan, respectively. Ini-

tial moisture contents and germination percentage were

9.14 %, 9.04 % and 95.5 %, 96.25 % in cultivars Mairaj-

2008 and Lasani-2008, respectively.

Experimental details

The study consisted of two independent experiments. In

the first experiment, seeds of both wheat cultivars were

either soaked in aerated water (hydropriming) for 10 h or

not soaked (control). In the second experiment, wheat

seeds of both cultivars were soaked in aerated 2 mm solu-

tion of ascorbic acid (AsA; osmopriming) or distilled

water (hydropriming) for 10 h, keeping seed to solution

ratio of 1 : 5 (w/v) (Farooq et al. 2006b). Seeds were then

removed, rinsed thoroughly with distilled water and

re-dried near to their original weight with forced air at

27 �C ± 2 under shade.

In both experiments, seeds were sown (eight in each

pot) in soil-filled (10 kg) plastic pots maintained at 70 %

and 35 % of water-holding capacity designated as well

watered and drought stressed, respectively. Plants were

thinned to four plants per pot after achieving the con-

stant count. Soil moisture was monitored and maintained

every alternate day. Experimental soil was sandy loam

having ECe 1.65 dS m)1 and pH 7.8. Both experiments

were laid out in a completely randomized design in a

factorial arrangement with six replicates per treatment.

Stand establishment

Seedling emergence was observed daily according to the

Association of Official Seed Analysts (AOSA) (1990) until

a constant count was achieved. The time to 50 % emer-

gence (E50) was calculated following the method of Farooq

et al. (2005). Mean emergence time (MET) was calculated

according to the equation of Ellis and Roberts (1981).

Coefficient of uniformity of emergence (CUE) was calcu-

lated using the formulae of Bewley and Black (1994).

Ascorbic Acid Improves Drought Resistance of Wheat

ª 2012 Blackwell Verlag GmbH, 199 (2013) 12–22 13

Allometry

Number of leaves was counted daily, starting from the

seedling emergence; length of each individual leaf was

measured daily to derive the leaf elongation rates. Four

weeks after the emergence, plants were harvested to

record root and shoot lengths and seedling dry weight.

Leaf area was measured manually with a ruler while spe-

cific leaf area (SLA) was calculated as the ratio of leaf area

to leaf weight.

Plant water relations

Leaf water potential (ww) of penultimate leaves was mea-

sured by pressure chamber (Model 3005; Soil Moisture

Equipment Corp., Santa Barbara, CA, USA) between

6 : 00 and 9 : 00 am 1 day before the final harvest. The

same leaves were put in a glass vial and stored at )20 �C

for 24 h. After thawing at room temperature (for

15 min), cell sap was extracted, and the osmotic potential

(ws) was measured using vapour pressure osmometer

(Model 5500; Wescor Inc., Logan, UT, USA). The relative

leaf water contents (RWC) were measured following the

technique of Barrs and Weatherly (1962).

Photosynthetic pigments

Photosynthetic pigments, chlorophyll-a and -b, were

determined following Arnon (1949) using 500 mg fresh

leaf extracted overnight with 80 % acetone and centri-

fuged at 14 000 g for 5 min.

Membrane stability index

Membrane stability was estimated by measuring the con-

ductivity of leachates owing to damaged plasma mem-

brane following the method of Shanahan et al. (1990).

One gram of leaf material (10 · 10 mm pieces) was taken

in 10 ml distilled water in glass vials and kept at 10 �C

for 24 h with shaking. The initial conductivity (C1) was

recorded after bringing sample to 25 �C with conductivity

meter. The samples were then autoclaved for 10 min,

cooled to 25 �C, and final conductivity (C2) was

recorded. Membrane stability index (MSI) was calculated

as MSI = [1 ) (C1/C2)] · 100.

Metabolite determination

Oxidative damage to the membrane lipids was estimated

by analysing the content of total thiobarbituric acid–reac-

tive substances (TBARS), expressed as equivalents of mal-

ondialdehyde (MDA). The amount of MDA was

determined following Hichem et al. (2009). For the

estimation of total phenolics, leaf samples were extracted

with 95 % methanol, and the phenolics were estimated

using Folin–Ciocalteu method (Ainsworth and Gillespie

2007). To determine the amount of free proline, fresh leaf

material was homogenized in 3 % aqueous sulfosalicylic

acid, and free leaf proline was estimated following the

method of Bates et al. (1973). To determine ascorbic acid,

leaves were homogenized in 6 % trichloroacetic acid and

ascorbic acid was estimated according to the procedure of

Mukherjee and Choudhuri (1983).

Statistical analysis

Data collected were subjected to statistical analysis by

analysis of variance using the computer software costat

(Cohort Software, Berkeley, CA, USA). The mean values

were compared with the least significance difference test

following the procedure of Snedecor and Cochran (1980).

Microsoft Excel was used for the graphical presentation

and calculation of correlation coefficients.

Results

Experiment 1

Drought significantly delayed the MET and E50; however,

hydropriming significantly decreased the MET and E50

under drought and well-watered conditions (Table 1).

Likewise, drought decreased the CUE and seedling dry

weight in both cultivars, hydropriming but improved

CUE and seedling dry weight under drought and well-

watered conditions (Table 1).

Experiment 2

In both wheat cultivars, MET, E50 and CUE were signifi-

cantly affected by drought and seed priming (Table 2).

Drought increased the MET and E50 in wheat cultivars;

however, osmopriming with AsA significantly decreased

the MET and E50 both under drought and well-watered

conditions than hydropriming (Table 2). Likewise

drought decreased the CUE; nonetheless, osmopriming

with AsA substantially improved that under control and

drought conditions than hydropriming (Table 2).

Drought stress significantly decreased the seedling dry

weight (Table 1), shoot length, leaf area and specific leaf

area (SLA) (Table 3) in both wheat cultivars. However,

osmopriming with AsA significantly improved seedling

dry weight (Table 3), shoot length, leaf area and SLA in

wheat cultivars Lasani-2008 and Mairaj-2008 (Table 3).

Although root length was increased under drought in cul-

tivar Lasani-2008, there was no difference for root length

in cultivar Mairaj-2008 under drought and well-watered

Farooq et al.

14 ª 2012 Blackwell Verlag GmbH, 199 (2013) 12–22

conditions (Table 3). However, in all cases, osmopriming

with AsA substantially increased the root length (Table 3).

Maximum root length was recorded from cultivar Lasani-

2008 under drought raised from seeds osmoprimed with

AsA followed by cultivar Mairaj-2008 under same

conditions (Table 3).

Drought substantially delayed the leaf emergence and

decreased the number of leaves (Fig. 1) and elongation of

1st (Fig. 2a), 2nd (Fig. 2b), 3rd (Fig. 2c) and 4th

(Fig. 2d) leaf in both wheat cultivars. Nonetheless, seed

priming with AsA substantially improved the leaf emer-

gence (Fig. 1) and elongation of 2nd (Fig. 2b), 3rd

(Fig. 2c) and 4th (Fig. 2d) leaf under well-watered and

drought conditions in both wheat cultivars. However, the

elongation of 1st leaf was improved by seed priming with

AsA under well-watered conditions in both cultivars,

whereas under drought, seed priming with AsA improved

the elongation of 1st leaf in cultivar Mairaj-2008 only

(Fig. 2a).

Drought significantly decreased the leaf water potential,

osmotic potential and relative leaf water contents in both

cultivars (Table 4). Although, there was no difference in

leaf water potential from hydropriming and osmopriming

with AsA under well-watered conditions in cultivar

Lasani-2008 and under both well-watered and drought

conditions in cultivar Mairaj-2008, leaf water potential

was higher in cultivar Lasani-2008 osmoprimed with AsA

under drought than hydroprimed seeds (Table 4). There

was no statistical difference between hydropriming and

osmopriming with AsA under well-watered conditions in

cultivar Lasani-2008 for osmotic potential; however, in

rest of the cases, osmotic potential was higher from

Table 1 Influence of hydropriming on stand establishment and seedling dry weight in wheat cultivars under well-watered and drought conditions

Treatments

MET (days) E50 (days) CUE Seedling dry weight (g)

Lasani-2008 Mairaj-2008 Lasani-2008 Mairaj-2008 Lasani-2008 Mairaj-2008 Lasani-2008 Mairaj-2008

WW DS WW DS WW DS WW DS WW DS WW DS WW DS WW DS

Control 8.44c 9.65a 8.23d 9.09b 6.33e 8.18a 6.52d 8.00b 0.45c 0.29e 0.42c 0.28e 0.13b 0.09d 0.14b 0.09d

Hydropriming 7.41e 8.33c,d 7.49e 8.43c 5.71f 7.19c 5.87f 7.11c 0.71a 0.37d 0.68b 0.36d 0.17a 0.11c 0.18a 0.12b,c

Means sharing the same letter for a single parameter do not differ significantly at P < 0.05.

WW, well-watered; DS, drought stress; MET, mean emergence time; E50, time to 50 % emergence; CUE, coefficient of uniformity of emergence.

Table 2 Influence of seed priming with ascorbic acid on stand establishment and seedling dry weight in wheat cultivars under well-watered and

drought conditions

Treatments

MET (days) E50 (days) CUE Seedling dry weight (g)

Lasani-2008 Mairaj-2008 Lasani-2008 Mairaj-2008 Lasani-2008 Mairaj-2008 Lasani-2008 Mairaj-2008

WW DS WW DS WW DS WW DS WW DS WW DS WW DS WW DS

Hydropriming 7.29b 8.09a 7.23b 8.09a 5.66e 7.08a 5.92d,e 7.00a,b 0.67b 0.33d 0.62b 0.31d 0.151d 0.113g 0.161c 0.117f

Osmopriming 6.47c 7.42b 6.29c 7.25b 4.80f 6.20c,d 5.04f 6.53b,c 0.82a 0.41c 0.78a 0.43c 0.169b 0.123e 0.178a 0.126e

Means sharing the same letter for a single parameter do not differ significantly at P < 0.05.

WW, well-watered; DS, drought stress; MET, mean emergence time; E50, time to 50 % emergence; CUE, coefficient of uniformity of emergence.

Table 3 Influence of seed priming with ascorbic acid on root and shoot lengths, leaf area and specific leaf area in wheat cultivars under well-

watered and drought conditions

Treatments

Root length (cm) Shoot length (cm) Leaf area (cm2) Specific leaf area (cm2 g)1)

Lasani-2008 Mairaj-2008 Lasani-2008 Mairaj-2008 Lasani-2008 Mairaj-2008 Lasani-2008 Mairaj-2008

WW DS WW DS WW DS WW DS WW DS WW DS WW DS WW DS

Hydropriming 8.82e 9.79b,c 9.63c,d 9.13d,e 5.29b,c 3.67e 5.39b 3.92e 15.77d 13.80e 17.95c 15.65d 257.22b 253.71d 256.76b 253.89d

Osmopriming 9.79b,c 10.55a 10.07a,b,c 10.20a,b 5.47a,b 4.50d 5.90a 4.92cd 19.87b 17.47c 21.37a 20.33b 259.16a 255.31c 259.78a 255.55c

Means sharing the same letter for a single parameter do not differ significantly at P < 0.05.

WW, well-watered; DS, drought stress.

Ascorbic Acid Improves Drought Resistance of Wheat

ª 2012 Blackwell Verlag GmbH, 199 (2013) 12–22 15

osmopriming than hydropriming (Table 4). Osmopriming

with AsA significantly improved the relative leaf water

contents in both cultivars under both well-watered and

drought conditions (Table 4).

Drought stress significantly decreased the chlorophyll

contents in both wheat cultivars. In both cultivars, chlo-

rophyll-a contents were substantially improved by osmo-

priming with AsA than hydropriming under both

drought and well-watered conditions. However, chloro-

phyll-b contents were improved by osmopriming with

AsA only under well-watered conditions but not under

drought (Table 5).

Drought significantly disturbed the membrane stability

in both cultivars (Table 5). Although there was no differ-

ence for membrane stability between hydropriming and

osmopriming with AsA under well-watered conditions in

both wheat cultivars, under drought, osmopriming with

AsA substantially increased the membrane stability in

both wheat cultivars (Table 5).

Malondialdehyde contents, an index of oxidative stress,

were substantially increased under drought in both wheat

cultivars (Table 6); however, osmopriming with AsA sub-

stantially decreased the MDA contents in both wheat cul-

tivars (Table 6). Likewise, soluble phenolics, leaf proline

contents and ascorbic acid contents were also increased

under drought in both cultivars (Table 6). Osmopriming

with AsA significantly increased soluble phenolics, leaf

proline contents and ascorbic acid contents in both culti-

vars under drought and well-watered conditions

(Table 6). There was no difference between both cultivars

for MDA, soluble phenolics and leaf proline contents

under drought and well-watered condition; however,

under well-watered conditions, ascorbic acid contents

were higher in cultivar Lasani-2008 while under drought,

ascorbic acid contents were higher in cultivar Mairaj-2008

(Table 6).

Under well-watered conditions, CUE was positively

correlated with SLA, chlorophyll-a and proline contents;

Num

ber o

f lea

ves p

er p

lant

(a)

0

1

2

3

4

5

10 12 14 16 18 20 22 24 26 11 13 15 17 19 21 23 25

OsmoprimingHydropriming

(b)

0

1

2

3

4

5

10 12 14 16 18 20 22 24 26 11 13 15 17 19 21 23 25

OsmoprimingHydropriming

DroughtWell-wateredDays after sowing

Fig. 1 Influence of seed priming with ascor-

bic acid on leaf emergence in wheat cultivar

(a) Lasani-2008 and (b) Mairaj-2008 under

well-watered and drought conditions ±S.E.

Farooq et al.

16 ª 2012 Blackwell Verlag GmbH, 199 (2013) 12–22

seedling dry weight had positive correlation with leaf area,

SLA, chlorophyll-a, membrane stability, phenolics and

proline contents, and leaf area was positively correlated

with seedling dry weight, SLA, chlorophyll-a, membrane

stability, phenolics and proline contents (Table 7). While

SLA and chlorophyll-a had positive correlation with each

other and with CUE, seedling dry weight, leaf area, RWC,

membrane stability, phenolics and proline contents, RWC

Lea

f len

gth

(cm

)

(a)

0

2

4

6

8

11 13 15 17 19 21 11 13 15 17 19 21

Osmopriming

Hydropriming

(a)

0

2

4

6

8

11 13 15 17 19 21 11 13 15 17 19 21

Osmopriming

Hydropriming

(b)

0

2

4

6

8

10

12

14 16 18 20 22 24 26 28 15 17 19 21 23 25 27

Osmopriming

Hydropriming

(b)

0

2

4

6

8

10

12

14 16 18 20 22 24 26 28 15 17 19 21 23 25 27

Osmopriming

Hydropriming

(c)

0

2

4

6

8

10

12

14

15 17 19 21 23 25 27 15 17 19 21 23 25 27

OsmoprimingHydropriming

(c)

0

2

4

6

8

10

12

14

15 17 19 21 23 25 27 16 18 20 22 24 26 28

OsmoprimingHydropriming

(d)

0

2

4

6

8

20 21 22 23 24 25 26 27 28 22 23 24 25 26 27 28

OsmoprimingHydropriming

(d)

0

2

4

6

8

20 21 22 23 24 25 26 27 28 22 23 24 25 26 27 28

OsmoprimingHydropriming

Well-watered Drought Well-watered DroughtMairaj-2008Lasani-2008

Days after sowing

Fig. 2 Influence of seed priming with ascorbic acid on leaf elongation of (a) first, (b) second, (c) third and (d) fourth leaf in wheat cultivars Lasan-

i-2008 and Mairaj-2008 under well-watered and drought conditions ±S.E.

Ascorbic Acid Improves Drought Resistance of Wheat

ª 2012 Blackwell Verlag GmbH, 199 (2013) 12–22 17

was positively correlated with membrane stability, pheno-

lics and proline contents (Table 7). Membrane stability

index was positively correlated with phenolics and proline

contents; whereas, phenolics were positively correlated

with proline contents (Table 7). Under both well-watered

and drought conditions, MDA had negative correlation

with CUE, seedling dry weight, leaf area, SLA, chl-a,

RWC, membrane stability, phenolics and proline contents

(Tables 7 and 8).

Under drought, CUE had positive correlation with

seedling dry weight, leaf area, SLA, chlorophyll-a, RWC,

phenolics and proline contents; seedling dry weight was

positively correlated with leaf area, SLA, chlorophyll-a,

membrane stability, AsA, phenolics and proline contents

(Table 8). Leaf area was positively correlated with SLA,

chl-a, AsA, membrane stability, phenolics and proline

contents whereas SLA had positive correlation with chlo-

rophyll-a, RWC, membrane stability, AsA, phenolics and

proline contents and chlorophyll-a was positively corre-

lated with RWC, membrane stability, phenolics and pro-

line contents (Table 8). RWC had positive correlation

with phenolics and proline contents whereas membrane

stability index was positively correlated with AsA, pheno-

lics and proline contents and AsA and phenolics were

positively correlated with proline contents (Table 8).

Discussion

This study investigated whether seed priming with AsA

can improve drought resistance in wheat. Drought caused

Table 4 Effect of seed priming with ascorbic acid on plant water relations in wheat cultivars under well-watered and drought conditions

Treatments

Water potential ()MPa) Osmotic potential ()MPa) Relative leaf water contents (%)

Lasani-2008 Mairaj-2008 Lasani-2008 Mairaj-2008 Lasani-2008 Mairaj-2008

WW DS WW DS WW DS WW DS WW DS WW DS

Hydropriming 0.146c 0.180a 0.113d 0.143c 1.68c,d 1.84a 1.70c 1.83a 90.55b,c 86.86e,f 89.95c 86.62f

Osmopriming 0.134c 0.162b 0.110d 0.134c 1.66d 1.79b 1.65d 1.76b 91.41a 87.99d 91.95a 87.52d,e

Means sharing the same letter for a single parameter do not differ significantly at P < 0.05.

WW, well-watered; DS, drought stress.

Table 5 Effect of seed priming with ascorbic acid on chlorophyll contents and membrane stability index in wheat cultivars under well-watered

and drought conditions

Treatments

Chl-a (mg g)1 FW) Chl-b (mg g)1 FW) Membrane stability index (%)

Lasani-2008 Mairaj-2008 Lasani-2008 Mairaj-2008 Lasani-2008 Mairaj-2008

WW DS WW DS WW DS WW DS WW DS WW DS

Hydropriming 6.76b 3.95d 6.56b 4.00d 2.82c 2.08d 3.21b 2.22d 43.88a,b 33.61d 45.34a 37.28c,d

Osmopriming 7.88a 5.31c 8.17a 5.27c 3.87a 2.25d 3.67a 2.33d 46.50a 38.74b,c 48.89a 39.93b,c

Means sharing the same letter for a single parameter do not differ significantly at P < 0.05.

WW, well-watered; DS, drought stress.

Table 6 Effect of seed priming with ascorbic acid on chlorophyll contents, soluble phenolics and membrane stability index in wheat cultivars

under well-watered and drought conditions

Treatments

Leaf MDA content

(lmol g)1FW) Soluble phenolics (mg g)1 FW)

Leaf free proline contents

(lmol g)1 FW) Ascorbic acid (mg g)1 FW)

Lasani-2008 Mairaj-2008 Lasani-2008 Mairaj-2008 Lasani-2008 Mairaj-2008 Lasani-2008 Mairaj-2008

WW DS WW DS WW DS WW DS WW DS WW DS WW DS WW DS

Hydropriming 15.56c 21.29a 15.37c 22.03a 33.74e 43.76b 34.94e 44.31b 7.87e 12.37c 8.23e 14.45c 16.75e 27.05c 14.21f 29.87b

Osmopriming 13.35d 17.56b 13.67d 17.24b 36.95d 45.89a 38.48c 46.52a 9.81d 17.56a 9.75d 18.25a 18.23d 29.33b 16.44e 31.17a

Means sharing the same letter for a single parameter do not differ significantly at P < 0.05.

WW, well-watered; DS, drought stress.

Farooq et al.

18 ª 2012 Blackwell Verlag GmbH, 199 (2013) 12–22

delayed and erratic stand establishment (Table 2),

decreased the seedling dry weight (Table 2), shoot length,

leaf area, specific leaf area (Table 3), leaf emergence

(Fig. 1) and elongation (Fig. 2) in tested wheat cultivars.

Limited water availability during the imbibition phase of

germination is the primary reason for delayed and erratic

stand establishment (Murillo-Amador et al. 2002). How-

ever, the results of this study indicated improvement in

stand establishment (Table 2), seedling dry weight

(Table 2), shoot length, leaf area and specific leaf area by

seed priming with AsA (Table 3). Seed priming with AsA

substantially improved the stand establishment under

drought and well-watered conditions owing to early com-

pletion of pre-germination metabolic activities during

priming. Modulation of hydrolases during lag phase of

germination by AsA helped to build germination metabo-

lites (Farooq et al. 2006a), resulting in earlier and uni-

form stand establishment (Table 2). This better

germination start also helped in improving the leaf emer-

gence (Fig. 1) and elongation (Fig. 2) under both well-

watered and drought conditions. AsA also enhances the

shoot organogenesis, resulting in better leaf expansion

and plant growth (Stasolla and Yeung 2001).

Although drought decreased the plant water relations

(Table 4), chlorophyll contents and membrane stability

(Table 5), in both wheat cultivars, root length (Table 3),

leaf MDA contents, soluble phenolics, leaf free proline

contents and AsA contents were improved under drought

(Table 6). Nonetheless, seed priming with AsA improved

the plant water relations (Table 4), chlorophyll contents,

membrane stability (Table 5), soluble phenolics, leaf free

proline contents and AsA contents (Table 6) with simulta-

neous decrease in MDA under drought and well-watered

conditions. However, for mitigating the drought effects,

seed priming with AsA was seen as an excellent strategy in

maintaining plant water relation attributes (Table 4) by

the accumulation of osmolytes such as proline (Table 6).

In addition to improvement in chlorophyll-a contents

Table 7 Correlation coefficients of important traits in wheat genotypes under well-watered conditions (n = 6)

DW LA SLA Chl-a RWC MSI AsA Phen Pro MDA

CUE 0.71ns 0.73ns 0.94** 0.94** 0.91* 0.67ns 0.73ns 0.79ns 0.97** )0.99**

DW 0.99** 0.87** 0.88* 0.80ns 0.99** 0.44ns 0.99** 0.98** )0.95**

LA 0.87** 0.88* 0.79ns 0.97** 0.24ns 0.99** 0.93** )0.88**

SLA 0.99** 0.99** 0.87** 0.63ns 0.93** 0.94** )0.93**

Chla-a 0.98** 0.87** 0.63ns 0.93** 0.95** )0.95**

RWC 0.82* 0.66ns 0.87* 0.87** )0.87**

MSI 0.16ns 0.86* 0.98** )0.80 ns

AsA 0.32ns 0.56ns )0.65ns

Phen 0.94** 0.90*

Pro 0.99**

CUE, coefficient of uniformity of emergence; DW, seedling dry weight; LA, leaf area; SLA, specific leaf area; Chl-a, chlorophyl-a; RWC, relative

leaf water contents; MSI, membrane stability index; AsA, ascorbic acid; Phen, total soluble phenolics; Pro, leaf free prolines; MDA, malondialde-

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

Table 8 Correlation coefficients of important traits in wheat genotypes under drought conditions (n = 6)

DW LA SLA Chl-a RWC MSI AsA Phen Pro MDA

CUE 0.91** 0.87** 0.98** 0.97** 0.90** 0.75ns 0.56ns 0.95** 0.90* )0.99**

DW 0.98** 0.97** 0.94** 0.77ns 0.96** 0.84** 0.99** 0.99** )0.91**

LA 0.92** 0.86* 0.64ns 0.93** 0.89** 0.97** 0.95** )0.85*

SLA 0.99** 0.89* 0.87* 0.69ns 0.99** 0.97** )0.98**

Chla-a 0.94** 0.83* 0.61ns 0.96** 0.95** )0.99**

RWC 0.63ns 0.32ns 0.82* 0.81* )0.93**

MSI 0.93** 0.92** 0.96** )0.75ns

AsA 0.78ns 0.82* )0.54ns

Phen 0.99** 0.95**

Prol 0.90*

CUE, coefficient of uniformity of emergence; DW, seedling dry weight; LA, leaf area; SLA, specific leaf area; Chl-a, chlorophyl-a; RWC, relative

leaf water contents; MSI, membrane stability index; AsA, ascorbic acid; Phen, total soluble phenolics; Pro, leaf free prolines; MDA, malondialde-

hyde; ns, non-significant.

*Significant at P < 0.05; **Significant at P < 0.01.

Ascorbic Acid Improves Drought Resistance of Wheat

ª 2012 Blackwell Verlag GmbH, 199 (2013) 12–22 19

(Table 5), exogenous application of AsA, through seed

priming, substantially improved its endogenous level

(Table 6), which triggered the accumulation of proline

and phenolics under drought in particular, as is evident

from strong positive correlation of AsA with proline and

phenolics under drought (Table 8).

Proline is one of the most common osmolytes, which

helps in promoting water retention and alleviating the

negative effect of drought on plants (Serraj and Sinclair

2002). There was strong positive correlation of proline

with RWC, seedling dry weight, leaf area, SLA, chloro-

phyll-a and membrane stability under drought (Table 8),

indicating the proline accumulation improved the plant

water status, thus avoiding the oxidative damages

(Table 6) and allowing the leaf expansion (Fig. 2). At cel-

lular level, maintenance of higher water potential means

increasing stomatal conductance under lower water status

(Sellin 2001), which increases root performance for water

uptake (Chimenti et al. 2006) as has been indicated by

the increase in root length by seed priming with AsA

(Table 3). Increased content of intracellular proline thus

increases the plant’s ability to survive under drought

(Taylor 1996). Improved root system plays key role in

plant surviving during drought (Hoogenboom et al.

1987). Improvement in root system with low decrease of

shoot has been regarded as good indicator of drought

resistance (Guoxiong et al. 2002). However, in rest of the

cases, there was no substantial difference between two

wheat cultivars under well-watered and drought condi-

tions (Tables 1–6).

Malondialdehyde production is taken as an index of

ROS-induced oxidative damage (Teisseire and Guy

2000, Zhang et al. 2007). However, plants have evolved

several antioxidative defence mechanisms, including the

production of enzymatic and non-enzymatic antioxi-

dants to reduce ROS-induced oxidative damages (Pos-

myk et al. 2009). Ascorbic acid is one of the most

important ubiquitous non-enzymatic antioxidants pres-

ent in plants (Smirnoff 2000, Smirnoff and Wheeler

2000) with higher concentration in leaves than that in

other plant parts (Smirnoff 2005). AsA detoxifies several

ROS produced during the Mehler reaction (Foyer and

Noctor 2000). Increase in endogenous level of AsA by

seed priming helped in dousing off the ROS levels as

has been indicated by decrease in MDA contents

(Table 6), strong negative correlation between AsA and

MDA and positive correlation between AsA and mem-

brane stability index under drought (Table 8). Phenolics

also possess antioxidant potential in plant cells (Sgherri

et al. 2004, Wahid and Ghazanfar 2006, Wahid 2007).

Seed priming with AsA improved the phenolics

(Table 6), which helped in decreasing the oxidative

damage, as is evident from its positive and negative

correlations with membrane stability and MDA con-

tents, respectively, under well-watered (Table 7) and

drought (Table 8) conditions.

In conclusion, drought has adverse effects on the seed-

ling emergence, stand establishment, allometry and water

relation of plants. However, seed priming with AsA

improves the drought resistance in wheat through

increase in endogenous AsA contents, antioxidant poten-

tial and osmotic adjustment. Manipulation of endogenous

AsA levels through genetic or biotechnological means

may result in the development of drought resistance in

wheat.

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