J Agro Crop Sci (2014) ISSN 0931-2250

DROUGHT STRESS

Interactive Effects of Sudden and Gradual Drought Stress

and Foliar-applied Glycinebetaine on Growth, Water
Relations, Osmolyte Accumulation and Antioxidant Defence
System in Two Maize Cultivars Differing in Drought
Tolerance
L.-X. Zhang1, J.-H. Lai1, Z.-S. Liang1 & M. Ashraf2,3
1 College of Life Sciences, Northwest A&F University, Yangling, China
2 University College of Agriculture, University of Sargodha, Sargodha, Pakistan
3 Department of Botany and Microbiology, King Saud University, Riyadh, Saudi Arabia

Keywords Abstract
antioxidative system; drought stress type;
glycinebetaine; maize cultivar; osmolyte Influence of sudden and gradual drought stress (DS) and foliar-applied glycineb-
accumulation; water status etaine (GB) on growth, water relations, osmolyte accumulation and antioxidant
defence system were investigated in the plants of two maize (Zea mays L.) culti-
Correspondence vars, that is, drought-tolerant Shaandan 9 (S9) and drought-sensitive Shaandan
University College of Agriculture
911 (S911). Sudden DS caused less accumulation of GB and free proline, but a
University of Sargodha
more accumulation of malondialdehyde (MDA), which resulted in a greater
POB 40100, Sargodha
Pakistan reduction in leaf relative water content (RWC) and dry matter (DM) in both cul-
Tel.: +92 48 9230618 tivars compared with the gradual DS. Exogenous GB application caused a rise in
Fax: +92 48 9230837 DM, RWC, contents of GB and free proline as well as the activities of SOD, CAT
Email: ashrafbot@yahoo.com and POD along with a decline in MDA content to various extent in both cultivars
under both types of DS. A more pronounced effectiveness of GB application was
Accepted April 30, 2014
observed in S911 than that in S9 under the same type of DS. It seemed that the
more serious damage of DS was on maize plants, and the better positive role of
doi:10.1111/jac.12081
GB was observed in terms of mitigating the adverse effects of DS. From this
study, it was possible to propose that hardening for drought resistance by gradual
DS treatment and GB application are effective to make plants robust to thrive
under water-deficit conditions.

and physiological processes as a consequence of drought-

Introduction
Drought stress (DS) is one of the main causes of reduced
agricultural productivity worldwide and can result in con-
siderable yield losses of many crops including maize (Zea
mays L.) (Anjum et al. 2011, Gholipoor et al. 2013). The
DS tolerance of crops is largely dependent on the crop cul-
tivar and DS type. The more sensitive to drought the culti-
var is, the more serious reduction in yield would be
(Chandrasekar et al. 2000, Wang et al. 2002, Zhang et al.
2009, Ashraf 2010, Gholipoor et al. 2013). It is now widely
accepted that sudden DS causes a greater detrimental effect
on crop plants compared with gradual DS (Gao et al. 1999,
Mafakheri et al. 2011, Zhang et al. 2011). Drought stress is
known to cause a wide array of changes in key biochemical
induced osmotic stress, which is considered as the primary
effects of DS, whereas secondary drought-induced effects
such as oxidative damage may also occur in most plants
(Ashraf 2010).
Decline in leaf relative water content (RWC) is consid-
ered as a better indicator of water status in plants under
DS (Zhang et al. 2011). Crucial changes in water homo-
eostasis lead to molecular damage, growth arrest and
even death of most plants. Among the above biochemical
and physiological responses, free radicals and other active
derivatives of oxygen are inevitable by-products of physi-
ological redox reactions (Arora et al. 2002, Ashraf 2009,
2010). Reactive oxygen species (ROS) inactivate enzymes
and damage important cellular components if the extent

© 2014 Blackwell Verlag GmbH, 200 (2014) 425–433 425

Zhang et al.
of their production exceeds the antioxidant defence capa-
bility of cells exposed to various abiotic stresses (Ashraf
2009). Reactive oxygen species can destroy normal
metabolism through oxidative-induced lipid peroxidation
(Mafakheri et al. 2011, Zhang et al. 2011). To reduce the
deleterious impacts of DS on normal metabolism and
ensure normal growth and development of crops, plants
have evolved various strategies to counteract this prob-
lem (Ashraf 2010). One of the most metabolic measures
is the synthesis and accumulation of intracellular com-
patible solutes such as endogenous glycinebetaine (GB)
and free proline so as to enhance osmotic adjustment to
counter cell dehydration as cell growth is known as pri-
marily turgor-driven (Bolen 2004, Ashraf and Foolad
2007, Ashraf et al. 2011). On the other hand, plants are
able to synthesise/over-accumulate a variety of antioxi-
dants such as superoxide dismutase (SOD: EC 1.15.1.1),
peroxidase (POD: EC 1.11.1.7) and catalase (CAT: EC
1.11.1.6) that are usually sufficient to protect plants from
oxidative damage (Ashraf 2009). The cascade of studies
on physiological adjustment of crops revealed that
changes in RWC, osmolyte accumulation, activities of
SOD, POD and CAT as well as MDA content were asso-
ciated with crop cultivar and/or DS extent (Hamidou
et al. 2007, Zhang et al. 2009, 2011, 2013).
GB is an efficient osmoprotectant that plays a significant
role in protecting cellular structures and key metabolites in
plants exposed to DS (Ashraf and Foolad 2007). A sensitive
crop cultivar always accumulates less GB than a tolerant
one in response to environmental stress, so that the natural
accumulation of GB is significantly lower to alleviate the
adverse effects of environmental stresses such as DS on
plants (Subbarao et al. 2001, Yang et al. 2002, 2003, Ashraf
and Foolad 2007, Miri and Mohammad 2013). For crops
unable to synthesise sufficient amount of GB, exogenous
application of GB has been suggested as a possible
approach to overcome the environmental limitations of
crop production under DS (Ashraf and Foolad 2007, Zhang
et al. 2009, 2011, 2013).
In view of our present knowledge, there is little informa-
tion available in the literature on the effects of different
modes of DS and GB application on osmolyte accumula-
tion and triggering of antioxidant defence system in maize
cultivars differing in drought tolerance. Keeping in view
the above facts, we hypothesise that higher constitutive or
induced osmolyte accumulation and activities of antioxi-
dant enzymes in the leaves of maize plants are the premier
components of differential tolerance mechanism in two
cultivars differing in drought tolerance, under DS imposed
through two different modes, that is, a sudden and a grad-
ual DS induced by PEG as well as exogenous GB applica-
tion. With this aim in mind, we designed solution culture
experiments to uncover the responses of two maize culti-
426
vars with respect to plant growth, water status, osmolyte
accumulation and antioxidative defence system under DS
imposed in two different ways and exogenous application
of GB.
Materials and Methods
Experiment location and plant material
Solution culture experiments under a sudden and a gradual
DS were performed at Northwest A&F University (Yan-
gling, China). Two maize cultivars, that is, drought-toler-
ant Shaandan 9 (S9) and drought-sensitive Shaandan 911
(S911) were used for the present experiments and their seed
obtained from the Agronomy College of the same
university (Zhang et al. 2009).
Experiment design and sampling
Solution culture experiments were carried out in a con-
trolled growth chamber. The seeds of S9 and S911 were sur-
face-sterilised in 1 % (w/v) sodium hypochlorite solution
on a magnetic stirrer for 20 min and thoroughly rinsed
with sterile deionised water. Seeds of each cultivar were
then transferred to two sheets of sterile filter paper moist-
ened with deionised water after swelling them in deionised
water at 28 °C for 6 h. The seeds were placed in plastic
trays for germination at 28 °C for 72 h in the dark and
then were sown into holes of Styrofoam boards in deion-
ised water in plastic containers (26 9 18 9 12 cm) grown
hydroponically in the growth chamber. The temperature of
the growth chamber was maintained at 27  2 °C, and the
light intensity was 350 lmol m 2 s 1. Daytime humidity
was between 60 % and 70 %. The containers were covered
with black plastic to protect roots from light.
Five and ten days after placement of the seedlings in
deionised water, the deionised water was replaced by half-
strength and full-strength nutrient solution (Hoagland and
Arnon 1938), respectively, which contained all essential
minerals for plant growth. The pH of the nutrient solution
was adjusted to 5.6–5.7 by adding HCl or NaOH. Three
seedlings at three-leaf stage were grown in plastic pots
(3.8 l) in the growth room. Each pot was filled with acid-
washed and sterilised quartz sand up to 20 cm and sup-
plied with distilled water. The seedlings in each pot were
thinned to one plant per pot and then DS treatments initi-
ated. Sudden DS treatment was created by adding 15 %
(w/v) polyethylene glycol (PEG-6000, provided by Xilong
Chemical Factory, Shantou, China) to each of the pots
under this group, whereas for gradual DS, 7.5 % of PEG-
6000 was added after every 24 h. PEG-6000 was dissolved
in full-strength nutrient solution to achieve final osmotic
potentials (ws) of 0.72 MPa determined by a vapour
© 2014 Blackwell Verlag GmbH, 200 (2014) 425–433

pressure Wescor 5500 osmometer (Wang and Li 2002).
Full-strength nutrient solution without PEG-6000 served as
the control group. For each group, each seedling was
sprayed with 20 ml of each of [0 (T0) and 50 (T) mg l 1]
GB in 0.10 % Tween-20 solution (produced in Shiying
Chemical Plant, Changping, Beijing, China) each time. The
spray was applied twice per day on the 1st, 3rd and 6th day
after the initiation of PEG treatment, respectively. There
were six treatments: sudden DS (T0), sudden DS (T); grad-
ual DS (T0), gradual DS (T); control (T0), control (T).
Water lost by evapotranspiration was compensated for by
daily addition of deionised water and aerated the solution
once every day. A backpack sprayer was used to spray the
nutrient solution or water. The foliar application wet the
whole leaf surface. All treatments were replicated four times
in a randomised block design. Desired PEG concentrations
were maintained by irrigating sufficiently with fresh solu-
tion. The solution was aerated 12 h a day. The whole
experiment was carried out twice independently under the
same environmental conditions. Data presented here are
means of four replicates of the two experiments (n = 8).
The maize plants were harvested on the 12th day after
the onset of drought or foliar GB treatments. Shoots were
sampled to determine dry weight. The second or third leaf
from the top of each plant was used for determining RWC,
activities of SOD, POD and CAT, and MDA content. The
samples for examining the activities of antioxidant enzymes
and levels of MDA were stored at 40 °C prior to assay.
Determinations of dry matter (DM) and leaf relative water
content (RWC)
Dry matter of shoots was measured after drying the sam-
ples in a forced-ventilation oven at 65 °C until constant
dry weight. Leaf RWC was estimated by recording the tur-
gid weight of 0.5 g fresh leaf samples by keeping them in
water for 4 h, followed by drying in a hot air oven till
constant weight.
Osmolyte content measurement
Free proline content in leaves was assayed using 5 ml of
3 % sulfosalycylic acid (SSA) following the method of Bates
et al. (1973). Glycinebetaine content was measured follow-
ing Grieve and Grattan (1983) with some modifications.
Dried and finely powdered samples (0.5 g) were shaken
with 20 ml of deionised water for 24 h at 25 °C. The
extracts were diluted 1 : 1 with 2 N H2SO4. Aliquots of
0.5 ml were put in test tubes, mixed with a cold KI-I2
reagent (200 ll) and cooled in ice water for 1 h. The tubes
were stored at 0–4 °C for 16 h and then centrifuged at
12 000 g for 15 min at 4 °C. The supernatant was aspi-
rated. The periodite crystals were dissolved in 5 ml of 1,2-
© 2014 Blackwell Verlag GmbH, 200 (2014) 425–433
Drought Stress and Foliar-Applied Glycinebetaine
dichloroethane. After 2–2.5 h, the absorbance was read at
365 nm with a UV–visible spectrophotometer. Reference
standards of GB were used for calibration and estimation
of GB concentration in the unknown samples.
Enzyme extractions and lipid peroxidation assay
The leaf samples (1.0 g each) were homogenised in a pre-
chilled mortar using ice cold 4 ml of 50 mM phosphate
buffer (pH 7.8) containing 1 % PVP (V/V) and a little
quartz sand. The homogenate was transferred to centrifuge
tubes and centrifuged at 4 °C for 20 min at 10 000 g. The
supernatant was used as an enzyme extract to measure anti-
oxidant enzyme activities.
SOD (EC 1.15.1.1) activity was determined spectropho-
tometrically by measuring its ability to inhibit the photo-
chemical reduction in superoxide-nitro blue tetrazolium as
described by Dhindsa et al. (1981). POD (EC 1.11.1.7)
activity was assayed specifically by measuring the rate of
guaiacol, which was oxidised in 3 min following Putter
(1974). CAT (EC 1.11.1.6) was determined by measuring
the rate of disappearance of H2O2 by Tris-HCl reagent
using the method of Taranishi et al. (1974). The activity of
each enzyme was expressed on protein basis (U mg 1 pro-
tein). Protein concentration of the crude extract was mea-
sured following the method of Gao (2000). The level of
lipid peroxidation in leaf samples was determined in terms
of malondialdehyde (MDA) content. Malondialdehyde was
extracted with 10 % thiobarbituric acid (TBA) and deter-
mined at 450, 532 and 600 nm with 0.6 % TBA as
described by Gao (2000).
Statistical analysis of data
The mean values of all parameters were taken from the
measurements of eight replicates and the standard errors of
the means (S.E.) calculated. One-way ANOVA was applied to
determine the significance of the results between different
treatments, and then, least significant differences (LSD) at
5 % probability level were performed. Duncan’s multiple
range test was used for comparing treatments within two
or three factor combinations. All the statistical analyses
were performed using the SAS software package (SAS
Institute Inc. 1996).
Results
Plant growth performance
Plant growth was appraised by measuring DM of shoots.
The DM of the two maize cultivars was significantly inhib-
ited by DS imposed as a sudden shock or gradually by add-
ing PEG-6000 to the growth medium (Fig. 1). Under
427
Zhang et al.

(a) 12 Foliar-applied GB was found to be effective in increasing

a
10 b b T0 T leaf RWC of both cultivars under both modes of DS
a
(Fig. 1). The greater increases in RWC were obtained
DM (g plant–1)

8 c under sudden DS (by 18 %) than those under gradual DS

d e e
6 fg
f g particularly in S911 due to GB application. However, S9
h
responded almost uniformly to GB application under both
4
modes of DS (Fig. 1).
2

0 Osmolyte accumulation
(b) 110
Accumulation of compatible osmolytes such as GB and free
100 a a a a proline in leaves suggests that osmotic regulation may
90 b b
c d c
80 d improve drought tolerance of maize (Fig. 2). Accumulation
e
RWC (%)

70 f of GB and free proline was more pronounced in S9 than in

60
50 S911 under both modes of DS application. Contents of GB
40 and free proline were greatly enhanced under gradual DS
30 than sudden DS in each cultivar. Leaf RWC of both culti-
20
10 vars declined more under sudden DS (by 7–16 % in S9 and
0 by 14–28 % in S911) than that under gradual DS. Exoge-
Control Gradual DS Sudden DS Control Gradual DS Sudden DS
nous GB treatment induced significant accumulation of GB
S9 S911 and free proline in the maize leaves, which was dependent
on the type of cultivar or DS (Fig. 2). The greater effects of
Fig. 1 Differential effects of two types of drought stress and exogenous
glycinebetaine application on dry matter (DM) (g per plant) (a) and leaf
exogenous GB on raising the endogenous contents of GB
relative water content (RWC) (%) (b) in two maize cultivars. DS repre- and free proline were more apparent in S911 than those in
sents drought stress induced by PEG-6000; S9 and S911 represent culti-
vars Shaandan 9 and Shaandan 911, respectively; T0 and T represent
without and with GB application, respectively. At the top of each col- (a) 4.50
umn, different letters indicate significant differences between twelve a
4.00 b T0 T
Glycinebetaine content

c
treatments (P < 0.05).
3.50 d e
(μmol g–1 DW)

3.00 f g
2.50 h
2.00
gradual DS conditions, DM of S9 and S911 decreased signifi-
1.50 j i ij
cantly by about 23–30 % and 35–41 %, respectively, k
1.00
whereas under the sudden shock of PEG-6000, the decrease 0.50
in shoot DM was more pronounced being 44–55 % 0.00
decrease in S911 and 33–42 % decrease in S9. Overall, S9
(b) 50
maintained higher values of DM than S911 under both 45 a
b
modes of DS application (Fig. 1).
Free proline content

40 c
(μmol g–1 DW)

Exogenous GB application improved plant growth per- 35 d

30 f e
formance of both cultivars under DS in terms of DM accu- g
25 h
mulation (Fig. 1). The greater effectiveness was obtained in 20
drought-sensitive S911 (10–25 % increase) than tolerant S9 15 i i
j j
(only 8–17 % increase), being more under the sudden 10
5
shock than that under gradual DS (Fig. 1).
0
Control Gradual DS Sudden DS Control Gradual DS Sudden DS
S9 S911
Plant water status
Relative water content in leaf tissues is known as a good Fig. 2 Differential effects of two types of drought stress and exogenous
glycinebetaine application on endogenous glycinebetaine (a) and free
indicator for plant water status. Leaf RWC of both cultivars
proline (lmol g 1 DW) (b) in two maize cultivars. DS represents drought
declined more under sudden DS (by 7–16 % in S9 and by stress induced by PEG-6000; S9 and S911 represent cultivars Shaandan
14–28 % in S911) than that under gradual DS (by 5–13 % 9 and Shaandan 911, respectively; T0 and T represent without and with
in S9 and by 11–24 % in S911) compared with the controls GB application, respectively. At the top of each column, different letters
(Fig. 1). indicate significant differences between twelve treatments (P < 0.05).

428 © 2014 Blackwell Verlag GmbH, 200 (2014) 425–433

 J Agro Crop Sci (2014) ISSN 0931-2250

DROUGHT STRESS

Interactive Effects of Sudden and Gradual Drought Stress

and Foliar-applied Glycinebetaine on Growth, Water
Relations, Osmolyte Accumulation and Antioxidant Defence
System in Two Maize Cultivars Differing in Drought
Tolerance
L.-X. Zhang1, J.-H. Lai1, Z.-S. Liang1 & M. Ashraf2,3
1 College of Life Sciences, Northwest A&F University, Yangling, China
2 University College of Agriculture, University of Sargodha, Sargodha, Pakistan
3 Department of Botany and Microbiology, King Saud University, Riyadh, Saudi Arabia

Keywords Abstract
antioxidative system; drought stress type;
glycinebetaine; maize cultivar; osmolyte Influence of sudden and gradual drought stress (DS) and foliar-applied glycineb-
accumulation; water status etaine (GB) on growth, water relations, osmolyte accumulation and antioxidant
defence system were investigated in the plants of two maize (Zea mays L.) culti-
Correspondence vars, that is, drought-tolerant Shaandan 9 (S9) and drought-sensitive Shaandan
University College of Agriculture
911 (S911). Sudden DS caused less accumulation of GB and free proline, but a
University of Sargodha
more accumulation of malondialdehyde (MDA), which resulted in a greater
POB 40100, Sargodha
Pakistan reduction in leaf relative water content (RWC) and dry matter (DM) in both cul-
Tel.: +92 48 9230618 tivars compared with the gradual DS. Exogenous GB application caused a rise in
Fax: +92 48 9230837 DM, RWC, contents of GB and free proline as well as the activities of SOD, CAT
Email: ashrafbot@yahoo.com and POD along with a decline in MDA content to various extent in both cultivars
under both types of DS. A more pronounced effectiveness of GB application was
Accepted April 30, 2014
observed in S911 than that in S9 under the same type of DS. It seemed that the
more serious damage of DS was on maize plants, and the better positive role of
doi:10.1111/jac.12081
GB was observed in terms of mitigating the adverse effects of DS. From this
study, it was possible to propose that hardening for drought resistance by gradual
DS treatment and GB application are effective to make plants robust to thrive
under water-deficit conditions.

and physiological processes as a consequence of drought-

Introduction
Drought stress (DS) is one of the main causes of reduced
agricultural productivity worldwide and can result in con-
siderable yield losses of many crops including maize (Zea
mays L.) (Anjum et al. 2011, Gholipoor et al. 2013). The
DS tolerance of crops is largely dependent on the crop cul-
tivar and DS type. The more sensitive to drought the culti-
var is, the more serious reduction in yield would be
(Chandrasekar et al. 2000, Wang et al. 2002, Zhang et al.
2009, Ashraf 2010, Gholipoor et al. 2013). It is now widely
accepted that sudden DS causes a greater detrimental effect
on crop plants compared with gradual DS (Gao et al. 1999,
Mafakheri et al. 2011, Zhang et al. 2011). Drought stress is
known to cause a wide array of changes in key biochemical
induced osmotic stress, which is considered as the primary
effects of DS, whereas secondary drought-induced effects
such as oxidative damage may also occur in most plants
(Ashraf 2010).
Decline in leaf relative water content (RWC) is consid-
ered as a better indicator of water status in plants under
DS (Zhang et al. 2011). Crucial changes in water homo-
eostasis lead to molecular damage, growth arrest and
even death of most plants. Among the above biochemical
and physiological responses, free radicals and other active
derivatives of oxygen are inevitable by-products of physi-
ological redox reactions (Arora et al. 2002, Ashraf 2009,
2010). Reactive oxygen species (ROS) inactivate enzymes
and damage important cellular components if the extent

© 2014 Blackwell Verlag GmbH, 200 (2014) 425–433 425

Zhang et al.

responses to DS in terms of leaf RWC and DM due to their

Lipid peroxidation
The MDA content, an efficient indicator for lipid peroxida-
tion, greatly increased under sudden DS than that under
gradual DS with the same cultivar. Cultivar S911 had more
MDA accumulation than S9 under the same DS type
(Fig. 3d).
Exogenous GB application significantly decreased MDA
accumulation in both cultivars under two types of DS
and thus alleviated the DS effects by reducing lipid per-
oxidation. The alleviation of lipid peroxidation by exoge-
nous GB was greater in S911 than in S9 under each DS
type (Fig. 3d).
The analysis of variance showed that the effects of differ-
ent treatments, that is, cultivar (C), water regime (W) and
exogenous GB (T), were significant with respect to DM,
contents of GB and free proline, activities of SOD, POD
and CAT, and MDA content (Table 1). Two-way and
three-way interactions were also significant for most of
parameters except for C 9 T and C 9 W 9 T for DM,
GB content and CAT activity (Table 1).
Discussion
Drought stress can be considered as one of the most com-
mon and frequently occurring abiotic stresses worldwide
(Anjum et al. 2011, Zhang et al. 2011, 2013). Growth and
water status of crops are variedly affected by different types
of DS (Zhang et al. 2009, 2013, Gholipoor et al. 2013, Sun
et al. 2014). However, DM production and tissue RWC are
known as two potential measures of growth performance
and water status of plants exposed to DS (Flower and Lud-
low 1986, Harb et al. 2010, Zhang et al. 2011). Decrease in
RWC indicates a loss of turgor that results in limited water
availability for the cell extension processes in crop plants
(Zhang et al. 2011). Crucial changes in water status lead to
molecular damage, growth inhibition and even death of tis-
sues/organs (Harb et al. 2010, Anjum et al. 2011). It is
already known that different crop cultivars show different
differential drought resistance and their differential
responses to different types of DS (Gao et al. 1999,
Chandrasekar et al. 2000, Wang et al. 2002, Zhang et al.
2009, Ashraf 2010). The present study elucidated that grad-
ual DS or sudden DS created more marked reduction in
RWC and DM production in the drought-sensitive cultivar
(S911) than in the drought-resistant one (S9), which is anal-
ogous to what has been already observed in wheat by
Chandrasekar et al. (2000) and in maize by Zhang et al.
(2009). Obviously, higher RWC and DM were obtained in
the drought-tolerant cultivar (S9) compared with that in
the drought-sensitive one (S911) under both types of DS.
Furthermore, the greater per cent reduction in RWC and
DM production occurred in each cultivar due to sudden
DS than that due to gradual DS (Fig. 1).
The tolerance mechanism in plants under water-deficit
may be associated with the accumulation of osmoprotec-
tants such as endogenous-free proline and GB, which
respond actively to DS to mitigate the adverse effects of
water deficit on crop production (Bolen 2004, Ashraf and
Foolad 2007, Ashraf et al. 2011, Miri and Mohammad
2013). Osmolyte accumulation causes a decrease in osmotic
potential which helps the maintenance of adequate water
absorption and an increase in cell turgor pressure to
improve the physiological activity of the plant during DS
(Harb et al. 2010, Anjum et al. 2011). Accumulation of
osmolytes (free proline and endogenous GB) in response to
DS is variable with different cultivars and types of DS (Ash-
raf and Foolad 2007, Anjum et al. 2011). The results of our
study show that free proline and endogenous GB contents
increased due to DS (Fig. 2). In plants subjected to DS, the
contents of free proline and endogenous GB were greater
for the drought-tolerant cultivar (S9) than that for the
drought-sensitive cultivar (S911), especially under gradual
DS (Fig. 2). DS induced accumulation of osmolytes to
decrease the cellular osmotic potential so as to stabilise
membranes and/or macromolecular structures (Harb et al.
2010, Anjum et al. 2011).

Table 1 F values from analysis of variance of two maize cultivars (S9 and S911) with known difference in drought tolerance tested for all parameters
measured at three water regimes (gradual drought stress, sudden drought stress and control) and two levels of GB concentration

Source of variance Cultivar (C) Water Regime (W) Exogenous GB(T) C9W C9T W9T C9W9T

DM 28.49*** 705.56*** 30.91*** 18.73*** 0.67 8.67** 0.07

RWC 1147.82*** 2223.72*** 867.45*** 185.07*** 49.40*** 293.11*** 19.92***
GB content 1759.63*** 6428.13*** 290.92*** 6428.13*** 0.76 33.91*** 0.19
FP content 14221.6*** 27366.3*** 697.59*** 2439.63*** 9.78** 182.33*** 4.95*
SOD activity 6652.93*** 1897.45*** 2385.52*** 1544.71*** 18.31*** 599.17*** 7.12**
POD activity 9331.01*** 428.80*** 2928.80*** 2009.63*** 24.22*** 529.68*** 6.98**
CAT activity 4399.14*** 1611.64*** 647.55*** 553.74*** 4.19 110.25*** 0.37
MDA content 648.05*** 4178.60*** 1542.72*** 155.37*** 81.59*** 428.86*** 24.27***

*Significant at the P = 0.05, **Significant at the P = 0.01, ***Significant at the P = 0.001.

430 © 2014 Blackwell Verlag GmbH, 200 (2014) 425–433


It is now well established that the capacity of the antioxi-
dative defence system and level of lipid peroxidation are
important in limiting oxidative damage and counteracting
excessive active oxygen species (AOS) for normal metabo-
lism under water-deficit conditions (Arora et al. 2002, Ash-
raf 2009). Hence, the activities of antioxidant enzymes and
MDA content in plant tissues determine the degree of
impairment of growth in water-stressed crops (Mafakheri
et al. 2011, Zhang et al. 2011). The production of antioxi-
dants is a widespread response, which results in reducing
membrane lipid peroxidation and maintaining macromo-
lecular structures or functions. It is also well known that
the changes in the activities of SOD, POD and CAT as well
as MDA content occur under DS depending on drought
resistance of crop cultivars and DS types (Ramachandra
et al. 2004, Ge et al. 2006, Ma et al. 2006, Hamidou et al.
2007, Zhang et al. 2009, 2011, 2013, Fig. 1). In the present
work, the comparison of lipid peroxidation and antioxi-
dant enzymes in the two maize cultivars differing in
drought tolerance and exposed to two types of DS may be
helpful in developing a better understanding of tolerance
mechanisms to DS (Harb et al. 2010, Anjum et al. 2011,
Fig. 3). In general, the oxidative enzymes in sensitive crop
plants usually decline at higher rates or increase at low rates
than those in drought-tolerant crop plants under the same
intensity of DS (Zhang and Kirkham 1994, Arora et al.
2002, Ge et al. 2006). It is well evident that the drought-
sensitive cultivar (S911) showed lower activities of antioxi-
dant enzymes and higher MDA content compared with the
drought-tolerant one (S911). Thus, the stress-induced
increased activities of antioxidative enzymes can protect
cell membranes, proteins and metabolic machinery, which
would preserve subcellular structures from drought-
induced damage (Harb et al. 2010, Fig. 3). Thus, a
drought-tolerant cultivar such as S9 can possess a stronger
ability to eliminate active oxygen species and lipid antiper-
oxidation (Ge et al. 2006, Zhang et al. 2009), which in turn
ultimately result in higher crop production. These findings
have been reported in an earlier study under additional GB
supply under DS conditions under long-term mild DS
(Zhang et al. 2011). Overall, gradual DS showed higher
activities of all antioxidative enzymes and lower MDA con-
tent than under sudden DS (Fig. 3). It seemed that gradual
DS could create less detrimental effects on plants than sud-
den DS because of its inferior ability to produce MDA,
which could maintain reversible membrane damage and
enable plants to endure severe DS (Harb et al. 2010, Anjum
et al. 2011, Fig. 3).
Crop losses caused by DS could be alleviated by the
application of osmoprotectants such as GB to crop cano-
pies (M€akela et al. 1996, Agboma et al. 1997, Yang and Lu
2005, Ashraf and Foolad 2007, Zhang et al. 2013). Glycin-
ebetaine may play an osmoprotective role in preventing cell
© 2014 Blackwell Verlag GmbH, 200 (2014) 425–433
Drought Stress and Foliar-Applied Glycinebetaine
injury from stress-induced dehydration (Ashraf and Foolad
2007). There are many reports demonstrating positive
effects of exogenous application of GB on water relations,
plant growth and final crop yield under DS: examples
include those in tobacco, wheat, barley, sorghum, soybean,
common beans and maize (Agboma et al. 1997, Ashraf and
Foolad 2007, Fig. 1). However, differential effects of GB
application on modulation of crop production and water
relations were closely associated with drought resistance of
the cultivar and types of DS (Zhang et al. 2009, 2011,
2013). Whether gradual DS or sudden DS, foliar-applied
GB produced a significant increase in DM and leaf RWC in
the two maize cultivars (Ashraf and Foolad 2007, Fig. 1).
The sensitive cultivar (S911) was more responsive than the
drought-tolerant S9 to GB application in terms of DM
production and RWC (Fig. 1).
Exogenous GB application significantly affects osmolyte
accumulation in crops under DS (Ashraf and Foolad 2007).
With decreasing soil moisture, foliar spray of GB signifi-
cantly increased leaf-free proline content in wheat (Sun
et al. 2001) and endogenous GB in soybean [Glycine max
(L.) Merr.] (Agboma et al. 1997). Exogenous GB applica-
tion caused increased accumulation of endogenous osmo-
lytes such as free proline and GB in maize plants under DS
(Ashraf and Foolad 2007, Fig. 2). On the other hand, exog-
enous GB application also increased the activities of antiox-
idative enzymes and alleviated lipid peroxidation, which
indicate that by this means, it is possible to confer drought
tolerance mechanism. The work reported here elucidates
the responses of two maize cultivars differing in degree of
drought tolerance to GB application and DS. The protec-
tive role of GB was more pronounced on the sensitive culti-
var (S911) than on the tolerant one (S9). The higher
increase in the activities of antioxidant enzymes and a
decrease in MDA content were obtained under sudden DS,
but not under gradual DS. Thus, it is well established that
type of DS and exogenous GB application may obviously
influence plant growth, water relations, osmolyte accumu-
lation and antioxidant defence system of crops (Ashraf and
Foolad 2007, Dıaz-Zorita et al. 2001, Zhang et al. 2011,
Figs 1–3). Gradual DS could create more accumulation of
endogenous-free proline and GB but less MDA accumula-
tion resulting in less reduction of leaf RWC and DM, which
are improved by GB application, especially for a drought-
sensitive cultivar.
Different crop cultivars maintain different DM produc-
tions, RWC, osmolytes contents, antioxidant enzymes
activities and MDA content, which are significantly affected
by type of DS and foliar-applied GB (Ashraf and Foolad
2007, Zhang et al. 2009, 2011, 2013, Miri and Mohammad
2013). It is, therefore, suggested that the optimal applica-
tion of GB can benefit plant growth, water relations and
antioxidant defence system under DS, but this response is
431
Zhang et al.
cultivar-specific and DS model-specific as is evident from
data reported in the current study (Table 1; Figs 1–3),
spraying dose specific and time specific as is evident from
data reported by Miri and Mohammad (2013).
Conclusion
In summary, we have confirmed the differences in GB-
induced biological and physiological characteristics and
modulating ability in two maize cultivars differing in
drought tolerance under two modes of DS application. The
results obtained in our previous studies and the present
study suggest that sudden DS induces substantial growth
inhibition and perturbance in water status by hastening lipid
peroxidation and imbalancing antioxidant defence system as
compared with those under gradual DS. Significantly higher
values of growth parameters, water status and activities of
antioxidative enzymes as well as lower MDA accumulation
were found to be the major contributors to a drought-toler-
ant cultivar to thrive under water-deficit conditions. In
addition, gradual DS appeared to produce weaker detrimen-
tal effects on plants than did the sudden DS. Furthermore,
plant growth, water status, antioxidant defence system and
lipid peroxidation could be modulated by GB application to
different levels under DS. Better effects in terms of GB appli-
cation might be obtained in a drought-sensitive cultivar
(S911) than a drought-tolerant one (S9). It seemed that more
severe damage by DS, that is, sudden DS is, the better effec-
tiveness of GB application would be. Thus, we propose that
the hardening for drought resistance by gradual DS treat-
ment and exogenous GB should preferably be applied to a
drought-sensitive cultivar under DS to bring out its poten-
tial to grow vigorously under drought-prone environments.
Acknowledgements
This work was jointly supported by the China Postdoctoral
Science Foundation (20070421133) and the Key Project
(30230230) of the National Natural Science Foundation of
China (NSFC) as well as the Foundation of State Key Labo-
ratory of Soil Erosion and Dryland Farming (10501-181).
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