Plant Stress 2 (2021) 100031

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Plant Stress
journal homepage: www.sciencedirect.com/journal/plant-stress

Differential responses of antioxidants and osmolytes in upland cotton

(Gossypium hirsutum) cultivars contrasting in drought tolerance
C.K. Singh a, *, B.K. Rajkumar b, V. Kumar c
a
ARS, Junagadh Agricultural University (JAU), Junagadh, Gujarat, India
b
ARS, Main Cotton Research Station, NAU, Surat, Gujarat, India
c
Research Scientist (Retd.), Main Cotton Research Station, NAU, Surat, Gujarat, India

A R T I C L E I N F O A B S T R A C T

Keywords: Major abiotic stresses affecting growth and metabolism of plants include heat and drought or water stress. In
Abiotic stress cotton, drought stress leads to limited plant growth and productivity that results in significant yield losses – up to
Osmotic potential 30% worldwide. Therefore, the present study was carried out in cotton to examine the changes in growth,
Proline
physiological, and oxidative stress mechanisms in response to drought stress. Our results revealed a significant
Catalase
Glycine betaine
(P<0.05) decline in growth parameters such as plant height (cm), root length (cm), and relative water content
(RWC) caused by drought stress. However, a significant increase (P<0.05) was observed in osmotic potential
(OP), electrolyte leakage (EL), glycine betaine (GB) content, lipid peroxidation, proline content, and antioxidant
enzyme activity. Two tolerant cultivars, G.Cot.16 x H-1353/10 and H-1353/10 x G.Cot.16, indicated no sig­
nificant decrease in physio-biochemical traits, whereas sensitive genotypes were markedly affected during
drought stress. The activity of antioxidant enzymes, such as catalase and peroxidase, increased markedly among
the tolerant cultivars, but decreased in sensitive cultivars. Thus, it can be inferred that short-term drought stress
severely disrupted cotton growth and metabolism, and that the apparent osmolyte accumulation and enzymatic
mechanism played a critical role in protecting plants against oxidative damage.

1. Introduction 2018). Furthermore, higher transpiration loss also leads to drought

Cotton (Gossypium spp.; Malvaceae family) is the world’s leading
fiber and oilseed crop. Gossypium hirsutum L. is the dominating elite
cotton species that is usually grown in tropical and subtropical areas
with warm climates, more often with limited water availability.
Approximately, one-third of the cultivated area of the world suffers from
chronically inadequate water supply. In all agricultural regions, yields of
rainfed crops are periodically reduced by drought, and the severity of
the problem may increase because of climate change across the globe.
Reproductive growth stages are sensitive to drought or water deficit
stress, which negatively impacts agricultural yield (Selote & Kha­
na-Chopra, 2004). In cotton, the effect of drought stress on reproductive
growth, yield, and fiber quality is well known as revealed in some of the
recent studies (Bozorov et al., 2018; Ayele et al., 2020). The decline in
cottonseed yield caused by drought stress is observed in the form of a
reduction in the number of bolls and boll weight (Basal et al., 2009).
Abiotic stresses involving water deficiencies adversely affect cell
turgidity that ultimately results in reduced crop production (Singh et al.,
conditions. To cope with drought stress, plants undergo morphological,
physiological, and biochemical changes. These changes mainly help in
the retention of water despite the high external osmoticum. Plant root
systems are critical for drought stress adaptation. Root penetration itself
contributes to significant variation in drought stress tolerance in crop
plants such as cotton (Luo et al., 2016). This may be mainly attributed to
genetic variations in root capacity to reach deeper soil layers (Comas
et al., 2013). During drought stress, morphological plasticity of roots
helps them to cope up with the disruption in the physical conditions of
the soil, which in turn helps the plants to adapt to the change in chemical
and physical soil properties (Kashiwagi et al., 2015). The effects of root
size and design on yield are determined by soil moisture distribution and
the degree of competition for water resources within the plant popula­
tion (King et al., 2009).
During drought stress, the leaf osmotic potential (Ψs) decreases, but
cotton can respond osmotically, allows it to maintain a larger leaf turgor
potential (Ψp) and thus limit the effects of drought stress (Nepomuceno
et al., 1998). Drought tolerance in plants is also determined by the

* Corresponding author.
E-mail address: chandrakant.singh07@gmail.com (C.K. Singh).

https://doi.org/10.1016/j.stress.2021.100031
Received 24 December 2020; Received in revised form 24 August 2021; Accepted 24 August 2021
Available online 26 August 2021
2667-064X/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
C.K. Singh et al.
Fig. 1. Average monthly temperature, rainfall and relative humidity in 2012-13.
amount of water in the leaves. Drought-tolerant plants are typically
those with high water content (de Brito et al., 2011). The higher RWC in
the leaves of the tolerant genotypes was due to a decrease in osmotic
ability in their leaves that heads photosynthesis close to normal.
The production of reactive oxygen species (ROS) – such as super­
oxide, hydroxyl radicals, hydrogen peroxide, and singlet oxygen – is one
of the main reactions of plants in response to abiotic stresses (Rejeb
et al., 2014). The ROS create oxidative stress in plants exposed to
different stressful environments that include drought stress. MDA
(malondialdehyde) production and antioxidant enzyme modulation
have both been demonstrated to induce oxidative damage to membrane
lipid structure (Zhang et al., 2006). This accumulation of MDA and
regulation of the activities of antioxidant enzymes under stressful en­
vironments are prospective indices to assess the status of oxidative stress
(Farooq et al., 2010). Amelioration of the adverse effects of such stresses
at the cellular and whole plant levels could be achieved through other
physiological and biochemical mechanisms, including the accumulation
of compatible osmolytes, which is one of the most defensive biochemical
responses to abiotic stress (Gupta et al., 2014). Proline (PRO), glycine
betaine, and many other organic compatible solutes play a crucial role in
plant stress tolerance (Farooq et al., 2010). Therefore, it is necessary to
understand the mechanism of drought tolerance to maintain produc­
tivity in arid environments. Hence, the present study was planned to
examine the effects of drought stress on different physiological (leaf
water relation and osmotic potential, electrolyte leakage), biochemical
(estimation of proline and glycine betaine, estimation of superoxide
dismutase, ascorbate peroxidase and catalase activities, and lipid per­
oxidation), and morphological traits (plant height and root length),
which may be helpful in the screening of cotton germplasm for tolerance
against drought stress.
2. Materials and methods
2.1. Plant material
Four cotton cultivars viz., G.Cot.16, H-1353/10 (tolerant genotypes),
BS-30, and H-1452/10 (sensitive genotypes) were obtained from Main
Cotton Research Station, Navsari Agricultural University, Surat, Gujarat,
India. These four cultivars were crossed in full diallel fashion during
Kharif season 2011–12. Developed hybrids and their respective parents
2
Plant Stress 2 (2021) 100031
were evaluated in Kharif season 2012–13 under irrigated (nonstress) and
rainfed (stress) conditions.
2.2. Drought induction treatment
The experiment was carried out at Main Cotton Research Station
(20◦ -12’ N, 72◦ -52’ E; altitude 11.34 m), Navsari Agricultural Univer­
sity, Surat, Gujarat, India, during the Kharif season 2012–13. The
experiment was conducted in a split-plot design in which the main plot
consisted of irrigated and rainfed or drought stress conditions. Cotton
cultivars were arranged in subplots under three replications for each
treatment. During the vegetative period, the average maximum tem­
perature ranged from 30.0 C to 39.7 C, while the average minimum
◦ ◦
temperature ranged from 14.7 C to 27.0 C (Fig. 1). The soil type was
◦ ◦
drained clay soil, which represents the typical black cotton soil, origi­
nally composed of predominant montmorillonite clay and has medium
fertility. These soils crack vertically upon drying to a depth of 80 to 120
cm. The clay content ranges from 56.4 to 64.9 percent. The plants were
sown at the end of June under irrigated or well-watered condition and
were allowed to grow. Under the rainfed condition, irrigation was
withheld to impose drought stress because of moisture loss. No rainfall
was observed when cotton plants flowered to maturity.
2.3. Morphological traits
Morphological traits viz., plant height and root length were
measured. The plant height of tagged plants was measured periodically
(in cm) from the base of the plant (ground level) to the tip of the main
shoot and the average values were recorded. The root length of the
plants was measured (in cm) from the base of the plant to the tip of the
main root. Root samples of the cotton plants were collected from the soil
by a root drilling method. Roots were rinsed with running water to
remove the adhered soil. The clean root samples were scanned using a
flatbed scanner (EPSON perfection V700) and the images were captured
(in .tif format) on a computer. DART software was used to calculate root
length (Le Bot et al., 2010).
2.4. Leaf water relation and osmotic potential
Leaf relative water content (RWC) was determined using the
C.K. Singh et al. Plant Stress 2 (2021) 100031

following formula proposed by (Barrs and Weatherley, 1962): RWC=

68.89±10.0cd
(FW - DW)/ (TW - DW) x 100, where FW is fresh weight, DW is dry

72.89±2.3abc
79.85±1.0ab

76.45±2.2ab

77.16±1.9ab
67.01±0.8cd
69.85±0.9cd

71.24±1.9bc
70.47±3.0bc
65.46±1.7d

65.05±8.9d

75.48±1.6b
81.25±1.2a

80.41±3.3a

80.85±2.7a
70.15±9.4c
weight, and TW is the turgid weight after 24 h floating in distilled water

Stress (R)
at 4 C. Leaf osmotic potential (Ψs) was measured in accordance with the
◦

method of Scholander (1964) using a pressure chamber (PMS instru­

ment Co., Coravallis, OR USA).

58.32±9.4abcde

58.41±1.9abcde
Ion leakage (%)

63.25±0.9abcd
2.5. Measurement of electrolyte leakage (EL)
53.41±9.6bcde

55.41±9.2bcde

55.64±2.1bcde
65.63±1.4abc
65.21±2.1abc
51.26±8.8cde

51.23±4.3cde
Non-stress (I)

67.36±8.8ab

67.25±1.8ab
49.85±4.2de

48.22±1.0de
73.58±9.9a

47.37±1.9e
Ion leakage (IL) was measured and calculated in accordance with the
method described by Huo et al. (2016). Briefly, the following procedure
was followed: (i) cotton leaves were cut into strips and incubated in
distilled water for 12 h. (ii) The initial conductivity (C1) was measured
with a conductivity meter. (iii) The samples were then boiled for 30 min
1.89±0.17abcd

1.94±0.14abcd

2.31±0.24abcd
1.86±0.11abcd
2.08±0.32abcd

2.18±0.22abcd
2.31±0.50abcd
1.73±0.53bcd
1.76±0.10bcd

2.35±0.41abc
2.45±0.22ab
2.45±0.22ab
1.59±0.22cd

1.55±0.19cd

1.50±0.11d

to result in complete ion leakage (IL). (iv) After cooling down, the
2.69±0.19a
Stress (R)

electrolyte conductivity (C2) was measured and electrolyte leakage (C)

was calculated using the formula:
Osmotic Potential (-bar)

C (%) = C1/C2 × 100.

With the same column values are the means ± SE of three replications. Variants possessing same letters are not statistically significant at 5% probability level. 2.6. Estimation of free proline
1.02±0.06bcdef
0.85±0.10cdefg

0.89±0.08cdefg
1.05±0.07bcde
0.84±0.01defg
1.11±0.16bcd
0.78±0.10efg

0.75±0.05efg

0.75±0.02efg

0.76±0.02efg
Non-stress (I)

1.25±0.16ab
1.15±0.08bc
0.72±0.06fg

0.73±0.02fg
0.68±0.06g

1.49±0.24a

Proline content was assayed using the ninhydrin method described

by (Maghsoudi et al., 2016) as follows: (i) Leaf sample (0.1 g) was ho­
mogenized in 1 ml of 3% sulfosalicylic acid. (ii) After centrifugation at
12000 g for 5 min, 0.5 ml of supernatant was added to 0.5 ml of glacial
acetic acid and 0.5 ml ninhydrin solution. (iii) The mixture was then
76.4±12.5ab

73.6±10.6ab

67.0±10.2ab

62.0±10.6b
71.3±1.0ab
69.6±1.5ab
64.3±1.3ab
63.7±8.5ab
74.9±0.6ab

82.0±0.6ab

75.2±9.4ab

69.8±9.2ab

73.6±4.2ab
72.5±3.4ab
61.3±1.1b
84.5±1.9a

heated at 100◦ C for 1 h, and quickly cooled on ice for 30 min, followed
Stress (R)

by the addition of 1 ml of toluene, and the extracted organic phase was

read at 520 nm. (iv) Proline concentration was determined by the cali­
Effect of water stress on morpho-physio traits of upland cotton cultivars grown under stress (R) and non-stress (I) condition.

bration curve and expressed as µmole proline g− 1 FW.

Non-stress (I)

80.4±11.8ab

2.7. Glycine betaine (GB) content

75.5±2.9ab
73.7±9.1ab
76.2±9.1ab
69.9±9.3ab
79.6±3.6ab

87.4±5.2ab
80.5±3.1ab
82.2±1.8ab

72.9±9.8ab
79.4±0.9ab
70.5±9.4ab
78.5±3.8ab
79.2±2.0ab
69.6±2.3b
89.3±2.8a
RWC (%)

Estimation of endogenous glycine betaine content from fully

expanded leaves was carried out in accordance with the method sug­
gested by Grieve and Grattan (1983). The procedure included the
following steps: (i) Leaf tissue (0.5 g) was chopped in 5 ml of a
22.0±2.0ab
23.0±5.2ab

18.3±3.7ab
22.7±3.0ab
21.0±1.7ab

20.7±3.0ab
19.7±4.7ab

22.0±3.4ab
20.3±4.5ab

19.3±1.1ab
21.0±2.6ab
18.03±.4ab
17.0±6.2b

17.7±5.5b
24.0±2.3a

26.0±1.0a
Stress (R)

toluene-water mixture (0.05% toluene), and the resulting extract was

collected in a 20 ml test tube. (ii) These test tubes were incubated for 24
h at 25◦ C on a shaker. (iii) The samples were filtered and 0.5 ml of
aliquot was mixed with 1 ml of 2 N HCl solution. (iv) This was followed
Root length (cm)

by the addition of 0.1 ml of KI solution mixed in an ice-cold water bath

Non-stress (I)
24.0±3.6ab
25.0±4.3ab
22.0±3.4ab
23.0±5.0ab
25.0±6.9ab
23.0±5.0ab

24.0±1.0ab
23.0±0.0ab

23.0±2.6ab

21.0±1.0ab
23.0±6.0ab
21.0±3.6ab

for 90 min. (v) To the solution obtained in step iv, firstly, 2 ml of ice-cold
25.0±6.5b

20.0±4.0b
26.0±6.5a

28.0±5.2a

water was added and mixed, and then, 10 ml of chilled (-10◦ C) 1,2
dichloroethane was added to this mixture. (vi) To obtain a two-layer
separation, a continuous stream of air was passed from the above solu­
tion for 1-2 min. (vii) From this two-layer separation, the upper aqueous
57.3±5.0bcd

56.3±1.5bcd
58.0±1.0bcd

55.3±0.5bcd

layer was discarded and the lower organic layer was collected; the op­
54.0±2.0cd

54.0±2.0cd
63.7±4.0bc

64.7±2.0bc

52.0±2.0d

51.3±2.0d
65.0±4.3b

66.0±1.7b
82.3±3.0a
84.0±4.3a

84.0±1.7a

89.0±9.5a
Stress (R)

tical density of this was recorded at 365 nm.

2.8. Lipid peroxidation

Plant height (cm)

Lipid peroxidation in leaf tissues was determined as 2-thiobarbituric

76.0±16.0abcde
78.7±3.5abcde
85.0±3.0abcd

70.3±7.5bcde

acid (TBA) reactive metabolites mainly composed of malondialdehyde

Non-stress (I)
87.0±4.3abc

68.0±9.6cde
89.0±8.7ab

65.3±3.0de
67.0±2.6de
94.0±4.3a
65.0±3.0e
67.0±4.5e

63.7±3.0e
63.0±4.0e
61.7±7.0e
62.3±4.0e

(MDA) (Heath and Packer, 1968). Leaves (0.2 g) were extracted in 2 ml

of 0.25% TBA prepared in 10% TCA. The extract was heated at 95◦ C for
30 min, and then, quickly cooled on ice. The collected extract was
centrifuged at 10,000 g for 10 min and absorbance was measured at 532
nm. The content of malondialdehyde was expressed as nmol of MDA
H-1353/10 X H-1452/10

using an extinction coefficient of 155 mM cm− 1.

G.Cot.16 X H-1452/10
G.Cot.16 X H-1353/10

H-1353/10 X G.Cot.16

H-1452 X H-1353/10
BS-30 X H-1353/10
BS-30 X H-1452/10
H-1353/10 X BS-30

H-1452 X G.Cot.16
BS-30 X G.Cot.16
G.Cot.16 X BS-30

H-1452 X BS-30

2.9. Preparation of enzyme extract and estimation of enzyme activity

H-1353/10

H-1452/10
Cultivars

G.Cot.16

Approximatley 0.5 g of leaf tissue was ground to fine powder in

Table 1

BS-30

liquid N2, and then, homogenized in 2 ml of ice-cold 100 mM potassium

phosphate buffer (pH 7.0) [1 M methylene diamine tetraacetic acid

3
C.K. Singh et al.
Fig. 2. Effect of water stress on (a) Proline, (b) Glycine betaine and (c) MDA content in leaves of G. hirsutum cultivars. Data represent the means ± SD of three
separate measurements. Different letters on the top of the error bars indicate statistically different means at P≤ 0.05.
(EDTA) and 2% (w/v) polyvinylpyrrolidone (PVP)] using a chilled pestle
and mortar. The homogenate was centrifuged at 10,000 g for 10 min at
4◦ C and the supernatant was collected and used for the assays of (a)
superoxide dismutase (SOD), (b) ascorbate peroxidase (APX), and (c)
catalase (CAT).
a) SOD activity: SOD (EC1.15.1.1) activity was assayed using the
photochemical NBT method (Beyer et al, 1991). The assay mix (1 ml)
contained 50 mM phosphate buffer (pH 7.8), 9.9 mM methionine, 57
mM NBT, 0.025% Triton X-100, and 0.0044% riboflavin. The
photoreduction of NBT was measured at 560 nm. One unit of SOD
was defined as the volume of extract that causes inhibition of the
photoreduction of NBT by 50%.
b) APX activity: APX (EC 1.11.1.11) activity was measured immediately
in fresh extract and was assayed as described by Nakano and Asada
(1981). For this purpose, 0.1 ml of fresh extract (mentioned above)
was added to a mixture containing 50 mM sodium phosphate buffer
of pH 7.0, 0.1 mM H2O2, 0.5 mM ascorbate, and 0.1 mM EDTA to
make up the final volume to 3 ml. This resulting decrease in the
absorbance at 290 nm (ε=2.8 mM− 1 cm− 1) was measured as the
indicator of APX activity caused by hydrogen peroxide oxidation.
c) Catalase activity (EC 1.11.1.6): Catalase activity was determined in
the homogenates by measuring the decrease in absorption at 240 nm
as H2O2 (ε = 39.4 mM− 1 cm− 1) in accordance with the method of
Aebi (1984) and enzyme activity expressed as µmol H2O2 oxidized
min− 1 g− 1 protein. In this case, 50 µl enzyme extract (prepared
above) was added to a mixture that contained 50 mM sodium
phosphate buffer (pH 7.0) and 10mM H2O2 to make the volume to 3
4
Plant Stress 2 (2021) 100031
ml. Catalase activity was calculated by using extinction coefficient
39.4 mM− 1 cm− 1.
2.10. Statistical analysis
The data were analyzed using statistical software StatView (SAS,
1999). Significant differences between treatment means were evaluated
using Duncan’s multiple range tests (p < 0.05). Correlation coefficients
were also determined on the selected data using StatView.
3. Results and discussion
3.1. Morphological traits
Morphological characters are of immense importance in screening a
large number of genotypes to drought stress tolerance owing to their
simplicity. In the present study, plant height showed a decreasing trend
at all the stages of drought stress compared to nonstress (irrigated)
conditions. Among the different cultivars, G.Cot.16 x H-1353/10 and its
reciprocal cross H-1353/10 x G.Cot.16, showed significantly higher
(P<0.05) plant height under both the nonstress and stress conditions.
However, marginal reduction in plant height was observed during
drought stress conditions when compared to irrigated conditions. (irri­
gated) (Table 1). The genotypes, BS-30 x H-1452/10 and H-1452/10 x
BS-30, recorded significantly lower plant height. The results demon­
strated wide variations in cultivars, which were corroborated by Hasan
et al. (2018). This decrease in plant height under drought stress might be
attributed to the suppression of cell expansion and cell growth or
because of low turgor pressure. Less reduction in plant height in the case
C.K. Singh et al.
Fig. 3. Effect of water stress on antioxidant enzyme (a) SOD, (b) APX and (c) Catalase activities in leaves of G. hirsutum cultivars. Data represents the means ± SD of
three separate measurements. Different letters on the top of the error bars indicate statistically different means at P≤ 0.05.
of tolerant parents and their hybrids was perhaps because of the
reduction in the number of nodes and/or internodal length—which is in
agreement with the results of Pettigrew (2004), where the plants that
received more water produced more nodes, resulting in increased plant
height. Therefore, the plants with less water will certainly produce fewer
nodes, which lead to reduced height as observed in the present study.
Deep rooting accounts for the difference in drought tolerance be­
tween species as the deep root system provides better tolerance by
providing water under extreme conditions through deep absorption. The
tolerant genotypes (G. Cot.16 x H-1353/10 and H-1353/10 x G. Cot.16)
showed higher root length in comparison to sensitive cultivars under
drought stress conditions in the present study, which is in agreement
with an earlier study (Abdelmoghny et al., 2020). Furthermore, root
length also significantly (P<0.05) differed among the parents and hy­
brids. This may suggest genetic differences in root length among tolerant
and sensitive cultivars as reported earlier (Luo et al., 2014), where
variations in root length were reported in cotton cultivars because of
drought stress.
3.2. Physiological traits
RWC is an important physiological trait that describes the water
status of plants. It is the most important index for drought tolerance, as it
is a measure of plant water status that reflects the metabolic activity of
tissues (Abdelmoghny et al., 2020). It is reported that high RWC is a
drought tolerance mechanism and that high RWC is the result of more
osmotic regulation or less elasticity of the cell wall (Erice et al., 2010).
Among the different genotypes examined for RWC in the present study,
G.Cot.16 x H-1353/10 and H-1353/10 x G.Cot.16, registered high RWC
and minimal reduction than other genotypes grown under stress
5
Plant Stress 2 (2021) 100031
conditions, which is mainly because of the maintenance of osmotic
regulation (Table 1). The significant variation in RWC observed across
different genotypes in our study could be attributed to their genetic
background and response to drought stress (Zahid et al., 2021). Ideally,
leaf osmotic potential is reduced under drought stress conditions (Zhang
et al., 2021); however, cotton can osmotically adjust and maintain a
higher leaf osmotic potential under stress conditions (Nepomuceno
et al., 1998) (Table 1).
In our study, osmotic potential increased significantly under stress
conditions in all the cultivars. However, among the different cultivars,
G.Cot.16 x H-1353/10 and H-1353/10 x G.Cot.16, showed low value
with a higher percentage increase in osmotic potential, under stress. The
reduction in osmotic potential is a consequence of the net accumulation
of solutes in the symplast (Fang & Xiong, 2015). The tolerant cultivars
showed low osmotic potential compared to sensitive cultivars. This
perhaps helps the tolerant cultivars to maintain the water availability
under stress conditions. The decrease in osmotic potential in the tolerant
genotypes is also reflected in the higher RWC of their leaves, which
consequently maintains photosynthesis near to normal (Nepomuceno
et al., 1998). Overall, our findings reveal osmotic adjustment to be a
primary acclimatization response. Osmotic adjustment helps to increase
the concentration of cellular solutes, which helps in maintaining the
required osmotic potential that ensures continued uptake of water
during the stress period (Blum, 2017).
Ion leakage caused by the disruption of cell membranes is regarded
as one of the consequences of oxidative stress that leads to lipid perox­
idation, membrane permeabilization, and cell death (Iqbal et al., 2020).
Ion leakage showed a significant increase under stress conditions and
increased progressively with decreasing RWC (Table 1). Among the
different cultivars, G.Cot.16 x H-1353/10 and H-1353/10 x G.Cot.16,
C.K. Singh et al. Plant Stress 2 (2021) 100031

Fig. 4. Relationship between yield and (a) Proline, (b) Glycine betaine and (c) MDA content of upland cotton cultivars under environment of non-stress (I) and stress
(R) condition.

showed less ion leakage compared to sensitive cultivars under stress
which is in agreement with an earlier study (De Brito et al., 2011). An
increase in ion leakage in sensitive cultivars is perhaps caused by the
negative effects of water deficit. This might lead to the damage of cell
membranes, followed by the release of ions into the intercellular space
that result in higher ion leakage. Our findings indicate the role of ion
leakage as a measure of membrane stability that could be assessed by
determining the leakage of ions from leaf discs exposed to heat and
dehydration stress. In the present study, the membrane of
drought-stressed plants was less stable than unstressed plants.

6
C.K. Singh et al. Plant Stress 2 (2021) 100031

Fig. 5. Relationship between yield and (a) Root length, (b) RWC and (c) Osmotic potential (OP) of upland cotton cultivars under environment of non-stress (I) and
stress (R) condition.

3.3. Proline, MDA, and glycine betaine
Tolerant cultivars showed an increase in proline content as compared
to sensitive cultivars. Drought stress-tolerant cultivars had the highest
rate of proline accumulation (8.89 and 8.61 mg g− 1 FW) during stress.
This is perhaps because the proline acts as an osmolyte and maintains
the osmotic potential after the alleviation of stress (Kumar et al., 2011).
Proline also contributes to stabilizing the subcellular structures (e.g.,
membranes and proteins), scavenges free radicals, and buffers cellular
redox potential under stress conditions (Zahid et al., 2021). Among the

7
C.K. Singh et al.
different cultivars, G.Cot.16 x H-1353/10 and H-1353/10 x G.Cot.16,
showed significantly higher proline accumulation under stress over
nonstress (irrigated) conditions (Fig. 2a). A similar trend of increase in
free proline content with a decrease in water content along with dif­
ferences in the proline level among the cultivars was reported by Pawar
et al. (2010).
Glycine betaine helps to balance the osmotic pressure of cells to
cope-up with osmotic stress and maintain turgidity ((Ashraf and Foolad,
2007). Our results indicated that glycine betaine content increased
significantly under stress over nonstress (irrigated) conditions. The
tolerant cultivars such as G.Cot.16 x H-1353/10 and H-1353/10 x G.
Cot.16, showed a higher increase in glycine betaine compared to sen­
sitive cultivars under stress conditions; this was also further supported
by a positive correlation of glycine betaine levels with tolerance under
drought stress (Fig. 2b). Glycine betaine helps to protect the integrity of
the cell membrane and osmotic adjustment during drought stress (OA)
in cotton plants (Lv et al., 2007, Marimuthu & Murali, 2018). Our results
and also earlier reports (Pawar et al., 2010) reveal that glycine betaine
content could be used as one of the biochemical markers to screen
tolerant and sensitive plants—as tolerant cultivars show more accu­
mulation of glycine betaine compared to sensitive cultivars under
well-watered and water-limited regimes.
Malondialdehyde content (MDA) increased significantly under stress
conditions. High MDA content is a characteristic feature of oxidative
stress-induced structural membrane damage that has been reported as a
common response to environmental constraints, including drought
(Castillo, 2008). In the present study, sensitive cultivars showed
significantly higher MDA content compared to tolerant cultivars under
stress conditions. The situation was reversed in the case of irrigated
conditions, where the tolerant cultivars were characterized by a lower
MDA content than the sensitive cultivars. Thus, the results of the present
study indicated that the cultivars, H-1353/10 x G.Cot.16 and G.Cot.16 x
H-1353/10, showed higher tolerance caused by the low accumulation of
MDA (Fig. 2c). Water limitation resulted in an increase of MDA in both
cultivars. However, drought-subjected tolerant cotton plants maintained
much lower MDA levels than the sensitive plants, as also revealed earlier
(Deeba et al., 2012; Zhang et al., 2015).
3.4. Antioxidant enzymes
Antioxidant enzymes such as SOD, APX, and CAT that were esti­
mated in the present study enable detoxification of excess ROS, and
thereby, maintain the balance in the formation and removal of reactive
oxygen species under stress conditions (Alvarez et al., 2009); Hasa­
nuzzaman et al., 2020). Our results indicated that the activity of all the
three antioxidant enzymes (SOD, APX, and CAT) increased significantly
under drought stress over irrigated condition (Fig. 3 a, b, and c).
Furthermore, this increase in activity was more conspicuous and sig­
nificant in the tolerant cultivars, G.Cot.16 x H-1353/10 and H-1353/10
x G.Cot.16, when compared to sensitive cultivars under stress conditions
which is in agreement with an earlier study (Hasan et al., 2018). SODs
are metalloenzymes located in various cell compartments that catalyze
the disproportionation of O− 2 to O2 and H2O2 (Deeba et al., 2012). In the
same study, it was also observed that APX activity generally increased as
the drought season progressed—with evidence of higher activity in the
two water deficit treatments compared to full-irrigation treatment.
Differences observed in the activity of antioxidant enzymes across ge­
notypes could be related to different genetic behavior for tolerance to
drought stress conditions.
4. Correlation analysis
Yield is a highly complex trait that involves the interactions of many
component factors, known as contributing components. Thus, under­
standing the interaction of characters among themselves and with the
environment is of practical importance in plant breeding. In the present
8
Plant Stress 2 (2021) 100031
study, different biochemical traits were also associated with yield and
these traits could be useful in the selection of cultivars. Biochemical
characters such as glycine betaine and proline showed a significant
positive correlation with seed cotton yield under both nonstress (irri­
gated) and stress (rainfed) conditions (Fig. 4 a and b). Moreover, higher
yield correlated with high glycine betaine may be attributed to
improved photosynthesis and protein synthesis under drought stress
(Zhao et al., 2007) leading to higher yield. Cotton is one of those species
that accumulate a higher amount of glycine betaine than others (Blun­
den et al., 2001). On the contrary, MDA content is negatively correlated
with the yield which is quite obvious because of the osmotic adjustment
that a plant needs to make under stress to tolerate (Fig. 4c).
Morphophysiological traits, along with RWC and OP, also showed
association with yield such as root length. While root length and RWC
showed a significantly positive correlation with seed cotton yield under
both environments (Fig. 5 a and b), OP showed a negative correlation
(Fig. 5c). RWC and root length contribute toward the survival of plants
under drought stress, and therefore, can be used as an early-stage
screening technique in breeding for drought resistance. In the present
study, the decrease in osmotic potential in the tolerant genotypes was
responsible for the higher RWC in their leaves that consequently
maintained photosynthesis near to normal. This was also in agreement
with earlier studies (Nepomuceno et al., 1998; Zahid et al., 2021) where
drought-tolerant cotton genotypes showed a decrease in leaf osmotic
potential at full turgor compared to the sensitive genotypes.
5. Conclusion
Our results revealed that drought stress induces an alteration in
growth with changes in metabolic activities in cotton. The alteration in
the levels of proline, MDA, and glycine betaine contents during stress
conditions indicate osmotic adjustment, protection of cellular macro­
molecules, the storage form of nitrogen, maintaining cellular pH,
detoxification of the cells, and scavenging of free radicals. Similarly,
physiological traits such as RWC, root length, electrolyte leakage (EL),
and osmotic potential levels also changed during stress conditions. The
sensitive cultivars show maximum alteration compared to tolerant cul­
tivars. Therefore, these traits are also useful to screen cultivars for
drought stress. Overall, our results provide useful biochemical (proline,
MDA, glycine betaine, and antioxidant enzymes), and physiological
markers that may be suitable for the screening of different cotton ge­
notypes for drought tolerance. Therefore, a similar future study with a
larger number of genotypes will certainly provide better insights for the
use of the parameters estimated in the present study, for their role as
potential markers for drought tolerance. This will pave way for the
breeding of cotton genotypes for drought tolerance that might lead to
the development of drought-tolerant cotton cultivars.
Declaration of Competing Interests
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
Research Scientist, Main Cotton Research Scientist, Navsari Agri­
cultural University, Surat, Gujarat is gratefully acknowledged for
providing necessary experimental fields and laboratory facilities for the
conduction and execution of this study.
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