J.

Agronomy & Crop Science (2008) ISSN 0931-2250

CHILLING/FREEZING STRESS

Exploring the Role of Calcium to Improve Chilling Tolerance

in Hybrid Maize
M. Farooq1, T. Aziz1, S. M. A. Basra2, A. Wahid3, A. Khaliq1 & M. A. Cheema1
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

Keywords
antioxidants; Ca2+; chilling stress; maize;
seedling growth
Correspondence
Dr M. Farooq
Department of Agronomy, University of
Agriculture, Faisalabad 38040, Pakistan
Tel.: +92 41 9200161-9/2917
Fax: +92 41 9200605
Email: farooqcp@gmail.com
Accepted June 4, 2008
doi:10.1111/j.1439-037X.2008.00322.x

Abstract
Abiotic stresses, including chilling, impede the plant growth and development
mainly by oxidative damage. In this study, seed priming with CaCl2 was
employed to reduce the damage caused by chilling stress in hybrid maize.
Maize hybrid (Hycorn 8288) seeds were soaked in 50, 100 and 150 mg l)1
(ppm) aerated solution of CaCl2 for 24 h and dried. Treated and untreated
seeds were sown at 27 C (optimal temperature) and 15 C (chilling stress)
under controlled conditions. Seed priming with CaCl2 significantly reduced the
chilling damage and improved the germination rate, root and shoot length,
and seedling fresh and dry weights. Activities of antioxidants, including catalase, superoxide dismutase and ascorbate peroxidase, were also improved. Soluble sugars and a-amylase concentrations determined as general metabolic
indicators of stress were also increased by seed priming with CaCl2. Priming
also improved the performance of maize at optimal temperature. Maintenance
of tissue water contents, reduction in membrane leakage and increase in
antioxidant activities, and carbohydrate metabolism seemed to induce chilling
tolerance by CaCl2. Seed priming with 100 mg l)1 CaCl2 was the optimal concentration in improving the performance of hybrid maize both under optimal
and stress conditions.

Introduction
Domino consequence of the most of abiotic stresses,
including temperature extremes, drought, submergence,
salinity and mineral toxicities, is the production of reactive oxygen species (ROS) at higher levels (Hodgson and
Raison 1991, Bartosz 1997, Shalata and Tal 1998, Kratsch
and Wise 2000, Foyer and Fletcher 2001, Neill et al. 2002,
Farooq et al. 2008c). Excessive ROS generation damages
macromolecules including proteins, nucleic acids and lipids (Kratsch and Wise 2000, Xing and Rajashekar 2001).
To prevent this, plants have developed variety of antioxidant enzymes and scavenging molecules (Hasegawa et al.
2000, Farooq et al. 2008c). The most important among
them are peroxidase (POD), ascorbate peroxidase (APX),
glutathione reductase, superoxide dismutase (SOD) and
catalase (CAT), which play important role in protecting
350

cells from oxidative damage (Halliwell and Gutteridge

1999, Hasegawa et al. 2000, Fazeli et al. 2007, Farooq
et al. 2008c). Chilling reduced leaf dry weight and carbohydrates by 34 % and 59 % respectively (Keller and
Steffen 1995), while in another study, total protein contents were also reduced but proline, H2O2 and relative
electrical conductivity (EC) were increased by chilling
stress (Feng et al. 2003).
Calcium (Ca2+) has been reported to improve cold
acclimation in cold-resistant plants (Monroy and Dhindsa
1995, Agarwal et al. 2005). It acts as a second messenger
in many plant-signalling processes (Gilroy et al. 1990,
Bush 1995), and also plays an important role as the
primary physiological transducer of different stresses
(Rincon and Hanson 1986). Minorsky (1989) concluded
that change in calcium levels is a necessary step in a
temperature-sensing mechanism that enables the plant to
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Chilling Tolerance in Maize

withstand future cold stress better. In another study,

Knight et al. (1991) reported that chilling stress signalling
in tobacco was accompanied by a large transient rise in
cytosolic Ca2+. Recently, Agarwal et al. (2005) suggested
that abiotic stress signal is transduced via ABA, Ca2+ and
H2O2, which might be responsible for the activation of
some common transcription factors associated with SOD,
APX and CAT.
Various seed priming strategies have been reported to
improve the germination and seedling establishment at
suboptimal temperatures. For example, soaking rice seed
in various concentrations of proline, betaine, putrescine,
spermidine and spermine increased low-temperature tolerance (Naidu and Williams 2004). Priming with CaCl2
has been found to be the most effective technique in
improving the growth of rice nursery seedling (Farooq
et al. 2007), and stand establishment in coarse and fine
rice (Farooq et al. 2006ac). Zheng et al. (2002) also
reported significantly improved chilling tolerance in rice
by priming with CaCl2. In another study, Farooq et al.
(2008a) reported improved chilling tolerance in late-sown
wheat by seed priming with CaCl2.
In Pakistan, maize is normally sown during the
month of February, when the temperature is quite low
(17/10 3 C, day/night) for maize seed germination,
and therefore crop stand is hampered. This warrant
finding strategies to accomplish optimal stand and seed
priming with CaCl2 might be a pragmatic approach.
No information is available reporting the leeway to
induce chilling tolerance in hybrid maize by CaCl2 seed
treatments. It is hypothesized that maize seed priming
with CaCl2 solution may alleviate the oxidative damage
with the enhanced activities of enzyme antioxidants and
promote seedling emergence and subsequent growth.
This study therefore was carried out to let slip the role
of CaCl2 seed treatments in improved chilling tolerance
in hybrid maize.

Materials and Methods

Source of seed
Seeds of maize hybrid (Hycorn 8288) obtained from ICI
Life Sciences, Sahiwal, Pakistan (8.54 % seed moisture
contents) were used in the study. For all treatments,
selected healthy seeds were used in the same numbers.
Seed priming treatments
For priming, maize seeds were soaked in aerated solution
of CaCl2 [having concentrations 50, 100 and 150 mg l)1
(ppm)] for 24 h at 28 2 C. Seed weight to solution
volume ratio was 1 : 5 (w/v) (Farooq et al. 2006a). After
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each treatment, seeds were rinsed thoroughly with

distilled water and dried back closer to original moisture
level under forced air at 27 3 C, sealed in polythene
bags and stored in a refrigerator at 5 C until use (Lee
and Kim 2000). Untreated dry seeds were taken as
control.
Emergence and seedling vigour evaluation
Treated and control seeds were sown in 10-kg plastic
pots (15 in each) containing moist acid/water washed
sand and placed in a growth room with a photosynthetically active photon flux density of 350 mmol
m)2 s)1 and a photoperiod of 14/10 h light/dark.
Experimental design was completely randomized in
factorial arrangement with four replications. One set of
pots was placed at 27 C (optimal temperature, control), while the other was kept at 15 C (chilling stress)
during the whole period of study. Number of emerged
seeds was recorded daily according to the seedling
evaluation Handbook of Association of Official Seed
Analysis (1990). Time taken to 50 % emergence of
seedlings (E50) was calculated according to the formulae
of Coolbear et al. (1984), modified by Farooq et al.
(2005). Mean emergence time (MET) was calculated
using the formulae of Ellis and Roberts (1981). Energy
of emergence (EE) was determined according to Farooq
et al. (2008b). Coefficient of uniformity of emergence
(CUE) was calculated using the formulae of Bewley and
Black (1985). On 15th day after emergence, samples
(second top leaf) for biochemical analysis were collected
from plants raised under optimal and stress conditions.
Thereafter, the seedlings were tested for vigour after
carefully removing from the sand. Number of secondary
roots and leaves were taken as root and leaf scores
respectively. Seedling fresh weight was determined
immediately after harvest while dry weight was taken
after drying at 70 C for 7 days.
Membrane permeability
Membrane permeability was determined in terms of
electrolyte leakage according to the method of Blum
and Ebercon (1981). Six leaf segments of about equal
size were rinsed with distilled water and immersed in
6 ml distilled water for 12 h followed by EC1 of
solution measured with a conductivity meter (Model
DDS-11A; Shanghai Leici Instrument Inc., Shanghai,
China). Samples 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 calculated as the ratio between EC1
and EC2.
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Farooq et al.

Relative water content

Antioxidants activities

Fresh leaves (0.5 g; Wf) were washed in water until constant weight of the leaves was attained. The saturated
leaves were weighed (WS) and then dried for 24 h at
80 C for determinations of the dry weigh (Wd). Relative
water content (RWC) was calculated by the following
formula:

Superoxide dismutase activity was estimated according to

the method of McCord and Fridovitch (1969). Inhibition
of colour formation (measured at 560 nm) was determined by the addition of 050 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 measured following the modified method of Luck (1974).
Enzyme extract (50 ll) was added to 3 ml of hydrogenperoxide-phosphate buffer (pH 7.0). The 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 of H2O2 consumed min)1 mg)1 chl. APX activity was estimated as described by Nakano and Asada
(1987) with slight modification. 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.

RWC Wf  Wd =WS  Wd  100%:

Sugar metabolism
To determine a-amylase activity, 1 g ground leaf sample
was mixed with 10 ml phosphate buffer (pH 7.0) and left
for 24 h at 4 C. The enzyme activity was determined
from the supernatant using the dinitrosalicyclic acid
method (Bernfeld 1955). To determine total soluble sugars, 1 g ground leaf sample was mixed with 10 ml distilled water and left for 24 h at 25 C (Lee and Kim
2000). The mixture was filtered (with Whatman No. 42)
and the final volume made to 10 ml with distilled water.
Total soluble sugars were determined using the phenol
sulphuric acid method (Dubois et al. 1956).

Fig. 1 Influence of pre-sowing CaCl2 seed

treatments on the (a) time to 50 % emergence and (b) mean emergence time in maize
at optimal and low temperatures S.E.

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Chilling Tolerance in Maize

Statistical analysis
The experiment was laid out in a completely randomized
design in factorial arrangement with four replications. The
experiments were conducted twice; data were pooled, and
subjected to statistical analysis using costat computer
package (CoHort Software, Berkeley, CA, USA). Least significant difference test was applied to compare the treatment means. Graphical presentation of data was carried
out using Microsoft Excel program (Microsoft Corporation, Los Angeles, CA, USA). For comparison of treatment
means, standard errors were computed using Microsoft
Excel program. Parallels were drawn between antioxidants
and membrane electrolyte leakage and RWC.
Results
Chilling stress increased time to 50 % emergence (E50)
and MET (Fig. 1), whereas EE, final emergence percentage (FEP) (Fig. 2), CUE (Fig. 3), shoot and root length
(Fig. 4), seedling fresh and dry weights (Fig. 5), and leaf
and root scores (Fig. 6) were decreased under stress conditions than at optimal temperature. Likewise, RWC
(Fig. 7a) and starch metabolism (Fig. 8) were also
decreased by chilling stress. However, electrolyte leakage
(Fig. 7b), and SOD, CAT and APX activities (Fig. 9) were
increased by chilling stress.

Seed priming treatments with CaCl2 reduced the E50

and MET, and increased the EE, FEP and CUE at both
the temperatures (Figs 13). Under both optimal and
stress conditions, seed priming with 100 and 150 ppm
CaCl2 were the best treatments to reduce E50 and MET,
respectively (Fig. 1), whereas maximum EE and FEP were
observed from seed treatment with 100 ppm CaCl2
(Fig. 2). While maximum CUE was recorded from seed
treatment with 150 ppm CaCl2 (Fig. 3). Although all seed
treatments improved the performance at both temperatures, FEP from 50 and 150 ppm treatments was similar
to that of control at optimal temperature (Fig. 2b).
All the CaCl2 treatments improved the shoot and root
lengths (Fig. 4), and seedling fresh and dry weights at
both temperatures, while leaf and root scores were
improved only at optimal temperature (Fig. 6). Maximum
shoot and root length (Fig. 4), and seedling dry weight
(Fig. 5b) under both optimal and stress conditions, and
seedling fresh weight (Fig. 5b) and leaf score (Fig. 6a)
under optimal conditions were recorded from seed priming with 100 ppm CaCl2. Maximum seedling fresh weight
(Fig. 5) under stress conditions, and root score (Fig. 6b)
at optimal conditions were recorded from seed treatment
with 150 ppm CaCl2.
None of the priming techniques improved the RWC at
optimal temperature (Fig. 7a). However, all the CaCl2

Fig. 2 Influence of pre-sowing CaCl2 seed

treatments on the (a) energy of emergence
and (b) final emergence percentage in maize
at optimal and low temperatures S.E.
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Farooq et al.

Fig. 3 Influence pre-sowing CaCl2 seed

treatments on the on the coefficient of
uniformity of emergence in maize at optimal
and low temperatures S.E.

Fig. 4 Influence of pre-sowing CaCl2 seed

treatments on the (a) shoot and (b) root
lengths in maize at optimal and low
temperatures S.E.

treatments improved the RWC under chilling (Fig. 7a),

and reduced the electrolyte leakage under optimal and
chilling conditions (Fig. 7b). Maximum RWC under
stress condition and minimum electrolyte leakage under
both stress and optimal conditions were recorded from
seed priming with 100 ppm CaCl2 (Fig. 7b).
At both temperatures, all CaCl2 treatments improved
the a-amylase activity and soluble sugars content when
compared with control (Fig. 8a,b). However, maximum
sugars, and a-amylase were recorded from 100 ppm CaCl2
treatment under both conditions (Fig. 8). At optimal and
354

low temperatures, CaCl2 seed treatments improved the

SOD (Fig. 9a), CAT (Fig. 9b) and APX (Fig. 9c) compared
with control. However, maximum SOD and APX activities
under both conditions, and CAT activity under optimal
conditions were recorded from seed priming with 100
ppm CaCl2, whereas maximum CAT activity under stress
conditions was observed from seed priming with 150 ppm
CaCl2 (Fig. 9).
Parallels between antioxidants and RWC showed a
positive correlation between RWC and CAT, and RWC
and APX under both stress and optimal conditions,
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Chilling Tolerance in Maize

Fig. 5 Influence of pre-sowing CaCl2 seed

treatments on the (a) seedling fresh and
(b) dry weights in maize at optimal and low
temperatures S.E.

Fig. 6 pre-sowing CaCl2 seed treatments on

the (a) leaf and (b) root scores in maize at
optimal and low temperatures S.E.
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Farooq et al.

Fig. 7 Influence of pre-sowing CaCl2 seed

treatments on the (a) relative water content
(RWC) and (b) electrolyte leakage in maize at
optimal and low temperatures S.E.

Fig. 8 Influence of pre-sowing CaCl2 seed

treatments on the (a) a-amylase activity and
(b) soluble sugars in maize at optimal and low
temperatures S.E. *One unit of the enzymes
activity is the amount of enzyme that released
1 lmol of maltose by 1 ml original enzyme
solution in 1 min.

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Chilling Tolerance in Maize

Fig. 9 Influence of pre-sowing CaCl2 seed

treatments on the (a) superoxide dismutase
(SOD), (b) catalase (CAT) and (c) ascorbate
peroxidase (APX) in maize at optimal and low
temperatures S.E. *One unit of SOD activity
is equivalent to the volume of extract needed
to cause 50% inhibition of the colour
reaction.

whereas, there was positive correlation between RWC and

SOD under stress and not under optimal conditions
(Table 1). Nevertheless, parallels between antioxidants

and membrane electrolyte leakage showed a strong negative correlation under both stress and optimal conditions.
Discussion

Table 1 Correlations coefficients (r) of antioxidants with changes in

membrane electrolyte leakage and relative leaf water content of
maize under normal and chilling stress conditions
Electrolyte leakage

Relative water contents

Antioxidants

Optimal

Chilling stress

Optimal

Chilling stress

SOD
CAT
APX

)0.88*
)0.96**
)0.91*

)0.90*
)0.90*
)0.94**

0.76 ns
0.83*
0.83*

0.87*
0.98***
0.84*

*P < 0.05; **P < 0.01; ***P < 0.001; ns, non-significant (n = 4).
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Chilling stress significantly reduced the germination and

early seedling growth in hybrid maize; however, seed
priming with CaCl2 successfully induced the chilling
tolerance. Moreover, seed priming with CaCl2 improved
the performance under both the optimal and stress conditions. Under both optimal and stress conditions, seed
priming with 100 ppm CaCl2 was the most suitable treatment to significantly improve the emergence (Figs 13),
early seedling growth (Figs 4 and 5), antioxidant activities
(Fig. 9) and general metabolism (Fig. 8) in hybrid maize.
Seed priming with CaCl2 treatments not only improved
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Farooq et al.

the germination rate (Figs 13) but also enhanced the

seedling vigour as indicated by higher root and shoot
lengths, and seedling fresh and dry weights (Figs 4 and 5).
Membrane permeability was significantly reduced as
indicated by the increased level of electrolyte leakage
under chilling stress (Fig. 7b). Enhanced electrolyte leakage was considered to be a symptom of stress-induced
membrane damage and deterioration (Feng et al. 2003).
However, seed priming decreased the membrane leakage
significantly (Fig. 7b) possibly by increased antioxidant
activities (Fig. 9). This increased antioxidant production
reduced the ROS-based damages in the plant system as
evident from negative correlation between membrane
electrolyte leakage and antioxidants (Table 1). Calcium
is well known for its role in maintaining membrane
structure and function (Paliyath et al. 1984) possibly by
retaining inter- and intramolecular linkages (Clarkson
and Hanson 1980).
Under low-temperature stress, RWC was also decreased
significantly (Fig. 7a) possibly due to decreased metabolites and osmotica available to hold the water within the
cells. Chilling stress signalling was reported to be accompanied by a large transient rise in cytosolic calcium concentration (Knight et al. 1991), an indicator of the role of
calcium in chilling tolerance. Increased calcium contents
in chilling-tolerant plant species can be correlated with
osmoregulation-based maintenance of tissue water status
(Minorsky 1989).
Exposure of plants to certain environmental stresses
often leads to the generation of ROS (Munne-Bosch and
Penuelas 2003), which may react with proteins, lipids and
DNA causing oxidative damage and impairing the optimal cellular functions (Foyer and Fletcher 2001).
Increased expression of antioxidants occurs in several
plants under chilling stress (Herouart et al. 1991, Farooq
et al. 2008b,d). ROS in plants are scavenged by a variety
of antioxidant enzymes and/or lipid- and water-soluble
molecules (Foyer et al. 1994). Of these, antioxidant
enzymes are the most effective against oxidative damage
(Halliwell and Gutteridge 1999). Agarwal et al. (2005)
suggested that abiotic stress signal is transduced via ABA,
Ca2+ and H2O2, which might be responsible for the activation of some common transcription factors associated
with SOD, APOX and CAT. The present study also suggest that enzymatic antioxidant activities of maize seedlings were substantially induced during chilling stress
compared with those grown at optimal temperature. This
implied that maize hybrid Hycorn 8288 has the ability to
activate its antioxidant system upon exposure to chilling
and possibly under other stresses.
In conclusion, seed priming with CaCl2 can improve
the low-temperature tolerance in hybrid maize mainly by
the maintenance of high tissue water contents, reduced
358

electrolyte leakage and improved antioxidant enzymes

activities, indicating it as a major metabolic event during
tolerance to low temperature. Performance of hybrid
maize was also improved at optimal temperature seed
priming with CaCl2, 100 ppm being the most effective
under both conditions.
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