Drought coping strategies in cotton: increased crop per drop

Abstract

The growth and yield of many crops, including cotton, are affected by water deficit. Cotton has
evolved drought specific as well as general morpho-physiological, biochemical and molecular
responses to drought stress, which are discussed in this review. The key physiological responses
against drought stress in cotton, including stomata closing, root development, cellular
adaptations, photosynthesis, abscisic acid (ABA) and jasmonic acid (JA) production and reactive
oxygen species (ROS) scavenging, have been identified by researchers. Drought stress induces the
expression of stress-related transcription factors and genes, such as ROS scavenging, ABA or
mitogen-activated protein kinases (MAPK) signalling genes, which activate various drought related
pathways to induce tolerance in the plant. It is crucial to elucidate and induce drought tolerant
traits via quantitative trait loci (QTL) analysis, transgenic approaches and exogenous
application of substances. The current review article highlights the natural as well as engineered
drought tolerance strategies in cotton.

Full text

Review
Drought coping strategies in cotton: increased crop per
drop
Abid Ullah, Heng Sun, Xiyan Yang* and Xianlong Zhang
National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, Hubei, China
Received 2 November 2016;
revised 6 December 2016;
accepted 27 December 2016.
*Correspondence (Tel +8613627260281;
fax +86 27 87283955; email
yxy@mail.hzau.edu.cn)
Keywords: ABA, cotton, drought
stress, MAPK, ROS.
Summary
The growth and yield of many crops, including cotton, are affected by water deficit. Cotton has
evolved drought specific as well as general morpho-physiological, biochemical and molecular
responses to drought stress, which are discussed in this review. The key physiological responses
against drought stress in cotton, including stomata closing, root development, cellular
adaptations, photosynthesis, abscisic acid (ABA) and jasmonic acid (JA) production and reactive
oxygen species (ROS) scavenging, have been identified by researchers. Drought stress induces the
expression of stress-related transcription factors and genes, such as ROS scavenging, ABA or
mitogen-activated protein kinases (MAPK) signalling genes, which activate various drought-
related pathways to induce tolerance in the plant. It is crucial to elucidate and induce drought-
tolerant traits via quantitative trait loci (QTL) analysis, transgenic approaches and exogenous
application of substances. The current review article highlights the natural as well as engineered
drought tolerance strategies in cotton.
Introduction
Cotton is grown as a leading commercial crop in more than 30
countries of world with major shares from China, India, the
United States and Pakistan, and is predominantly cultivated in
warmer regions (Riaz et al., 2013). According to statistics, China,
India, the United State, Pakistan and Brazil were the top 5
cotton-producing countries in 2014–2015, generating 6.5 M,
5.4 M, 3.5 M, 2.3 M and 1.5 M tones, respectively (Statista,
2015). As a glycophyte, cotton shows higher tolerance to abiotic
stresses than other major crops. However, extreme environmen-
tal conditions, such as drought affect growth, productivity, and
fibre quality of cotton (Parida et al., 2007). According to a press
release from the United States Department of Agriculture
(USDA), cotton production is expected to decline due to drought
stress (USDA, 2015). Similarly in Pakistan, cotton production
declined by 34% to just 9.68 M bales against the production of
14.4 M bales from previous year because of drought and high
temperature (Dawn news, 2016). In addition to cotton, other
crops were also affected by drought, as approximately 67% of
crop losses were due to drought stress over the last 50 years in
the United States (Comas et al., 2013). The impacts of drought
on cotton are widespread and varied, which makes it difficult to
determine accurate financial estimates (Table 1). As shown in
Figure 1, world cotton production was very low in 2008 and
2009, which led to a significant decrease in stocks in 2009.
Therefore, cotton prices were increased in 2010 and 2011,
resulting in the cotton consumption decline of 10% in 2011.
Cotton production was higher than demand from 2010 to 2013;
however, the production decreased from 2011, with a signifi-
cant decrease of 6.5% in 2015 from 2014, while consumption is
increasing by approximately 6.5 million bales annually (Figure 1).
Thus, we need to establish policies for the production and
consumption of the cotton. Moreover, it is also necessary to
produce stress-tolerant varieties of cotton due to the uncertain
conditions in the future. On the other hand, the emphasis should
not be only on stress-tolerant variety of cotton, although plant
survival is very critical in the early stages of growth, stress-
tolerant variety should therefore be based on stability of yield. It
is known that improving of yield and maintaining yield stability
of cotton crop, under normal as well drought stress conditions, is
essential for the growing global population.
Despite the complexity of drought tolerance mechanism in
cotton, tremendous progress has been made in understanding
the drought tolerance mechanism. Morpho-physiological, bio-
chemical and molecular adaptations by nature or by genetic
engineering can lead to the drought-tolerant variety of cotton.
The current review discusses effective techniques to alleviate the
negative effects of drought stress in cotton and maintain the
productivity as well as fibre quality. Moreover, the mechanisms of
drought tolerance in cotton and strategies to induce tolerance to
drought are also discussed.
Morpho-physiological mechanism of cotton in
responses to drought stress
Drought stress causes a wide range of morpho-physiological and
biochemical changes that adversely affect the development as
well as the productivity of the cotton (Figure 2). Generally,
drought stress severely restricts cotton growth and development,
such as affecting plant height, leaf dry weight, stem dry weight,
leaf area index, node number, fibre quality, canopy and root
development (Loka et al., 2011). Specifically, net photosynthetic
rate, transpiration rate, stomata conductance, carboxylation
efficiency and water potential of cotton leaves decrease signif-
icantly during drought conditions (Kumar et al., 2001). Recently,
Hejn
ak et al. (2015) studied the detrimental effects of drought
stress on cotton. According to their results, 50% dry matter
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This is an open access article under the terms of the Creative Commons Attribution License, which permits use,
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271
Plant Biotechnology Journal (2017) 15, pp. 271–284 doi: 10.1111/pbi.12688

accumulation of Gossypium barbadense (G. barbadense) was
limited under drought stress. Moreover, the stomata conduc-
tance, photosynthetic rate and transpiration rate were also
decreased under water deficit. Like other plants, cotton has
acquired a wide range of morpho-physiological, biochemical and
molecular mechanisms in response to multiple stresses that
enable them to avoid and/or tolerate these stress factors and
survive in harsh environments.
The plant drought tolerance mechanisms can be divided into
four strategies: drought avoidance, drought escape, drought
tolerance and drought recovery (Fang and Xiong, 2015).
Drought avoidance and drought tolerance are the two major
strategies of plants against drought stress. Drought avoidance is
the maintenance of key physiological processes, such as
stomata regulation, root system development and others,
during moderate drought conditions. Drought tolerance is the
capability of plants to withstand severe dehydration through
specific physiological activities, such as osmotic adjustment via
osmoprotectants (Luo, 2010). Drought escape is the ability of
plants to adjust their growth period or lifecycle, such as the
cotton variety with a short life cycle, to avoid the seasonal
drought stress (Manavalan et al., 2009). Drought recovery of
plants is the capability to resume growth and yield after
exposure to severe drought stress. Cotton has evolved several
common morpho-physiological strategies against drought stress,
which have been discussed in this section, such as stomata
Table 1 Direct and indirect impacts of drought on cotton and its management
Direct impacts Indirect impacts Management
Damage plants systems Food scarcity Drought-tolerant varieties should develop
Reduce crop productivity Reduce income of farmers and agribusiness Effective impact assessment procedures should develop
Reduce water level Increase prices of foods and goods Pro-active risk management measures
Increase insect infestation Increase unemployment (companies
dealing with agriculture will stop working)
Make plans aimed at increasing the coping capacity
Increase plant diseases Increase crime and insecurity Efficient emergency response programs should be planned which can be used for
reducing the impacts of drought
Cause pollution in the concern area Meetings should conduct on national and international level about drought stress
Migration Early warning system should develop to make decision earlier
Figure 1 Unstable world cotton production and their consumption since
2007.
Figure 2 Numerous effects of drought stress on cotton and their responses.
ª2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 15, 271–284
Abid Ullah et al.272

regulation, root development, photosynthetic response and
osmotic adjustment.
Stomata regulation
Reduction of water loss through leaves is a crucial phenomenon
in cotton plants under drought stress. Wilting and rolling of leaves
result in less radiation and thus reduced water loss (Fang and
Xiong, 2015). Plants often show various xeromorphic characters
and have structures that promote drought tolerance, such as
thicker and smaller leaves, a thicker cuticle epidermis, more
epidermal trichomes, thicker palisade tissues, smaller and denser
stomata, a high ratio of palisades to spongy parenchyma
thickness, and a developed vascular bundle sheath (Hetherington
and Woodward, 2003; Iqbal et al., 2013). For example, the
cotton variety, Gossypium hirsutum (G. hirsutum) YZ1, has
smaller leaves as compared to G. hirsutum Y668. Stomata
regulation plays a pivotal role in gas exchange between tissues
and the atmosphere. It is one of the key mechanisms that allow
plants to produce energy and maintain cellular function. Ninety
per cent of water losses (transpiration) from plants occur though
stomata openings (Wang et al., 2009). In cotton, closure of the
stomata is the first step to reduce water loss during drought
conditions, when the rate of transpiration is very high. Stomata
conductance could be a potential indicator of drought tolerance
in cotton as there is a negative correlation between drought
tolerance and stomata conductance.
Root development
All root traits are potentially important in the drought stress;
however, hydraulic conductance and plant allometry have been
of particular interest to researchers. Various scientists have
reviewed the potential function of roots under drought stress
(Comas et al., 2013). More profuse (higher root length density)
and deeper root systems in the soil are often proposed as
desirable characteristics for drought adaptation. In a case, Luo
et al. (2016) reported that mild and initial-stage drought stress
enhanced root length in cotton, but long-time water deficit
reduced the root activity as compared to control plants. In
another study, transgenic cotton plants were more tolerant to
drought stress, with a better root system than in wild type (Liu
et al., 2014). Similarly, the transgenic cotton plants harboured
Arabidopsis that enhanced drought tolerance 1/homodomain
glabrous 11 (AtEDT1/HDG11) gene had well-developed roots in
addition to other drought-tolerant features (Yu et al., 2015).
Photosynthesis
Drought stress causes stomata closure, which leads to the
decreased CO
2
intake, affecting the rate of photosynthesis and
consequently reduces growth and yield (Chaves et al., 2009).
However, in some cases, stomata conductance is not always
associated with the rate of photosynthesis, but this still needs to
be elucidated (Von Caemmerer et al., 2004; Xu et al., 2010).
Photosynthesis is severely affected along with growth as the
water deficit increases gradually in the field of cotton. For
example, it was found that photosynthesis as well as transpiration
was affected under drought conditions in cotton (Deeba et al.,
2012; Li et al., 2012). Interestingly, it has been reported that
young leaves of cotton are photosynthetically more tolerant to
drought and heat as compared to mature leaves. When young
leaves were subjected to high temperature (37 °C), no decline
was observed in net photosynthesis. In contrast, mature leaf net
photosynthesis declined 66% under the same conditions
(Chastain et al., 2016). In another field study of cotton for two
consecutive growing seasons, a decreased lint yield was observed
as net photosynthesis declined under water-deficit conditions in
the first growing season. However, no change was observed in
the yield of drought-treated field due to high rainfall in the next
growing season (Chastain et al., 2014). These studies revealed
that drought stress reduces photosynthesis in cotton which in
turn affects growth and yield.
Osmotic adjustment
At the cellular level, water deficit affects turgidity and osmotic
balance in the cell. Osmotic adjustment is a critical adaptation to
reduce the effects of drought-induced damage in crop plants.
Plant defence mechanisms also include osmoprotectants or
osmolytes that regulate homoeostasis following drought and
salinity stress on a cellular level. Drought stress has negative
effects on osmotic balance, and therefore, plants accumulate
different organic and inorganic substances to reduce the osmotic
potential in response to drought stress (Fang and Xiong, 2015).
Numerous organic compounds, including amino acids (proline,
glycine), sugars (trehalose, fructan), sugar alcohols (mannitol,
sorbitol, D-ononitol), amines and polyamines (polyamine,
betaines), polyols, ectoine, alkaloids and inorganic ions, known
as osmoprotectants/osmolytes, are involved in osmotic adjust-
ment (Fang et al., 2015; Singh et al., 2015). These solutes assist
in protecting proteins and membranes from the damage due to
high concentrations of inorganic ions and oxidative damage
under drought stress (Chen and Murata, 2011) and multiple
stresses, such as drought and salinity (Khan et al., 2015). The
exogenous application of osmoprotectants (proline and glycine-
betaine) has been shown to be effective in reducing the harmful
effects of drought stress in cotton (Noreen et al., 2013). The
transgenic cotton plants with enhanced glycinebetaine accumu-
lation were more tolerant to drought stress than control plants
and had increased photosynthesis, higher relative water content,
increased osmotic adjustment, lower lipid membrane peroxida-
tion and a lower percentage of ion leakage (Lv et al., 2007).
Ectopic expression of a mustard annexin gene, AnnBj1, enhanced
proline content and sucrose, which increased drought tolerance
in cotton (Divya et al., 2010). Moreover, overexpression of
GhAnn1, a cotton annexin gene, enhanced the tolerance to
drought and salt by increasing the activity of superoxide
dismutase (SOD) and elevated levels of proline and soluble sugars
(Zhang et al., 2015).
Biochemical and molecular mechanism of
drought tolerance in cotton
Similar to animals, plants also have a defence mechanism through
which they respond to various biotic and abiotic stresses. The
drought tolerance mechanism is very complex because it is a
multigenic system that is related to various morpho-physiological,
biochemical and molecular processes (Figure 3). Other than
morpho-physiological mechanism, cotton has evolved several
signal transduction pathways in response to drought stress.
Abscisic acid (ABA)
ABA is one of the most important stress hormones and
participates in various crucial physiological processes during the
plant life cycle, including stress responses, development and
reproduction. Studies indicate that osmotic stress occurs due to
high drought conditions or salt stress or when water availability is
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Drought coping strategies in cotton 273

reduced through water loss and turgor pressure (Boudsocq and
Lauriere, 2005). Osmotic stress promotes the synthesis of ABA,
which activates gene expression and adaptive physiological
changes (Yamaguchi-Shinozaki and Shinozaki, 2006). After stress
signal perception by the plasma membrane, ABA synthesis is
initiated, which occurs mostly in the plastids, with the exception
of xanthoxin conversion to ABA, which takes place in the
cytoplasm (Seo and Koshiba, 2002). Generally, ABA synthesis
occurs in the roots. It is then transported via vascular tissues, and
it shows stomatal closure responses in a variety of cells, such as
guard cells (Kuromori et al., 2010). As in other plants, perception
and signal transduction of ABA in cotton are mediated by two
pathways, which are ABA-dependent and ABA-independent.
ABA-dependent signalling pathways play a critical role in stress-
responsive gene expression during various stresses, especially
osmotic stress. ABA receptors are important elements for ABA
signal transduction. Various receptors have been identified in
different subcellular compartments, including the plasma mem-
brane, nucleus, cytosol and chloroplast envelope. Under normal
conditions, ABA content is low, and sucrose nonfermenting
1-related protein kinase 2 (SnRK2) protein activity is inhibited by
protein phosphatase 2C (PP2C), which leads to dephosphoryla-
tion. When plants suffer drought stress, the cellular ABA level
increases, and ABA then binds to PYR/PYL/RCARs, which in turn
bind and inactivate PP2Cs. The SnRK2s are autoactivated when
they dissociate from PP2Cs. Activated SnRK2s phosphorylate
downstream targets and trigger ABA-induced physiological and
molecular responses (Danquah et al., 2014; Dong et al., 2015;
Mehrotra et al., 2014; Yoshida et al., 2014)). ABA regulates
many stress-related genes to enhance drought tolerance in cotton
plants (Figure 4). Overexpressing an ABA-induced cotton gene
GhCBF3 in Arabidopsis enhanced drought and salinity tolerance
in transgenic lines, with higher proline content, relative water
content and chlorophyll content in transgenic lines than those in
wild type. In the presence of ABA, stomatal aperture was smaller
in transgenic lines, and expression level of AREB1 and AREB2 was
remarkably higher than wild type. They suggested that GhCBF3
enhance drought and salt tolerance via ABA signalling pathway
(Ma et al., 2016).
Jasmonic acid (JA)
Jasmonic acid (JA) is another phytohormone derived from
a-linolenic acid. JA and its active derivatives, which are known
as jasmonates, have a significant role in regulating stress
responses of plants to various biotic as well as abiotic stresses.
In addition to plant growth and development, JA is also involved
in root growth, fruit ripening, tendril coiling and viable pollen
production (Wasternack, 2007). JA has been shown to participate
in the response to drought conditions. Genomewide functional
analyses of cotton were performed to analyse the molecular
mechanism of drought resistance, and they identified various
genes related to JA signalling pathways (Chen et al., 2013). Tan
Figure 3 Various signalling pathways connectively enhance drought tolerance in cotton. These pathways work together to maintain their normal activities
under drought stress.
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Abid Ullah et al.274

et al. (2012) reported that JA application inhibited fibre elonga-
tion in cotton. Similarly, several studies have also shown that
exogenous application of jasmonates enhances plant resistance to
water-deficit conditions (Bandurska et al., 2003). Similar to ABA,
various studies have shown that jasmonates also participate in the
regulation of stomatal closure (Riemann et al., 2015).
Although JA signalling pathway has not been fully elucidated,
its biosynthesis and signalling pathway have been reviewed
extensively in the last few years (Ahmad et al., 2016; Kombrink,
2012; Wasternack and Hause, 2013). The jasmonate-zim domain
(JAZ) repressor proteins have a key role in the JA signalling
pathway—they function as a switch for JA signalling. In normal
conditions, when JA is absent, jasmonate-insensitive/jasmonate-
zim (JAI3/JAZ) proteins bind to various transcription factors,
including MYC2 (Myelocytomatosis), and limit their activity.
However, during stress conditions, when JA and its derivatives
are present, degradation of JAZ proteins occurs as described
above, resulting in active transcription factors (MYC2) that
up-regulate genes involved in stress responses (Chini et al.,
2007). The signalling pathway and JAZ protein interactions in this
pathway have been comprehensively reviewed (Wager and
Browse, 2012). Generally, plant hormones do not function in
discrete pathways but rather depend on each other at different
stages to control environmental as well as developmental
pathways. This results in signal transduction that can assimilate
various processes and respond to the stress in a complex way
(Riemann et al., 2015). The jasmonates, similar to ABA signalling,
act as a hub where different processes are initiated to appropri-
ately respond to drought stress.
Reactive oxygen species (ROS)
Partial reduction of atmospheric O
2
leads to the production of
ROS, also known as active oxygen species (AOS) or reactive
oxygen intermediates (ROI). Cellular ROS basically consist of four
forms, hydrogen peroxide (H
2
O
2
), the hydroxyl radical (HO•),
superoxide anion radical (O
2) and singlet oxygen (
1
O
2
). Two of
these forms are especially very reactive, that is HO•and
1
O
2
. They
can harm and oxidize various components of the cell, such as
lipids, proteins, DNA and RNA. Eventually, they can result in cell
death if the oxidation of cellular components is not controlled
(Fang et al., 2015). Subcellular locations, such as the mitochon-
dria, plasma membrane, cell wall, chloroplast and nucleus, are
responsible for the production of ROS (Gill and Tuteja, 2010).
Under drought stress, the production of these ROS increases in
various ways. For example, a reduction in CO
2
fixation leads to
decreased NADP
+
regeneration during the Calvin cycle, which will
reduce the activity of the photosynthetic electron transport chain.
Moreover, during drought conditions, there is excessive leakage
of electrons to O
2
by the Mehler reaction during photosynthesis
(Carvalho, 2008). The Mehler reaction reduces O
2
to O
2by
donation of an electron in photosystem I. O
2can be converted to
H
2
O
2
by SOD which can be further converted to water by
ascorbate peroxidase (Heber, 2002). However, it is difficult to
evaluate the levels of ROS produced during the Mehler reaction
compared to those generated through photorespiration. Drought
conditions also enhance the photorespiratory pathway, particu-
larly when RuBP oxygenation is high due to limited CO
2
fixation.
Noctor et al. (2002) found that approximately 70% of total H
2
O
2
production occurs through photorespiration under drought
stress.
Plants have developed complicated scavenging mechanisms
and regulatory pathways to monitor the ROS redox homoeostasis
to prevent excess ROS in cells. Alterations in antioxidant enzyme
metabolism could influence drought tolerance in cotton. The
defence mechanism against ROS has been reviewed in detail by
Das and Roychoudhury (2014). The antioxidant machinery has
been developed by the plants to ensure survival (Figure 5). It has
two arms, (i) enzymatic components, such as catalase (CAT),
Figure 4 ABA mediated signalling pathway
during normal and stress conditions. Under
normal conditions, ABA content is low, and
SnRK2 protein kinase activity is inhibited by PP2C
phosphatases. Under drought stress, the cellular
ABA level increases, and ABA then binds to PYR/
PYL/RCARs, which in turn bind and inactivate
PP2Cs. The SnRK2s autoactivate when they
dissociate from PP2Cs. Activated SnRK2s
phosphorylate downstream targets and trigger
ABA-induced physiological and molecular
responses.
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Drought coping strategies in cotton 275

SOD, ascorbate peroxidase (APX), glutathione reductase (GR),
guaiacol peroxidase (GPX), dehydroascorbate reductase (NADH)
and monodehydroascorbate reductase (MDAR), and (ii) nonen-
zymatic antioxidants, such as reduced glutathione (GSH), ascorbic
acid (AA), a-tocopherol, flavonoids, carotenoids and the
osmolyteproline (Figure 5). To scavenge ROS, these two arms/
components work together (Das and Roychoudhury, 2014;
Heiber et al., 2007; Wu et al., 2015). APX, along with MDAR,
NADH and GR, removes H
2
O
2
via the Halliwell–Asada pathway
(Uzilday et al., 2012). APOX reduces H
2
O
2
to water by oxidizing
ascorbate to MDHA and thus plays a key role in the ascorbate–
glutathione cycle (de Azvedo Neto et al., 2006). MDHA is then
reduced to ascorbate by MDHAR. However, two molecules of
MDHA can be nonenzymatically converted to MDHA and
dehydroascorbate, which is further reduced to ascorbate via the
NADH and GR cycle (Szalai et al., 2009). In this cycle, glutathione
(GSH) is reduced by GR oxidation to oxidized glutathione at the
expense of NADPH (nicotinamide adenine dinucleotide phos-
phate). Glutathione reductase activity increased during drought
stress to keep oxidized and reduced glutathione ratios at
adequate level (Chan et al., 2013). The balance between ROS
production and antioxidative enzyme activities determines
whether oxidative signalling and/or damage will occur (Zhang
et al., 2014c). The antioxidative capability of different cotton
cultivars determines the resistance capability to drought stress.
The drought-tolerant cultivar M-503 has constitutively active
antioxidative enzymes, including SOD, APX, CAT and POX, which
decrease the oxidative stress induced by lipid peroxidation
(Sekmen et al., 2014). In cotton, drought induced the production
of ROS, but on the other hand, the APX and GR activities also
increased and maintained the ROS scavenging process until the
plant recovered from stress conditions (Ratnayaka et al., 2003).
Supplemental Zn in cotton contributed to alleviating oxidative
injuries under polyethylene glycol-simulated (PEG) drought stress
because it enhanced SOD, CAT, APX activities and the content of
nonenzymatic antioxidants (Wu et al., 2015). In another example,
Zhang et al. (2014c) conducted an experiment on the cotton
cultivars: drought-resistant (CCRI-60) and drought-sensitive
(CCRI-27). They found that the CCRI-60 cultivar was drought
tolerant due to increased root length and vigour, antioxidant
enzyme activities and significantly increased GR activity and
proline content. CCRI-60 has the ability to scavenge free radicals
and provides better protection compared to CCRI-27; thus, it is
more resistant to drought and has increased growth. Down-
regulation of GbMYB5 in G. barbadense resulted in decreased
antioxidant enzyme activities such as, SOD, peroxidase (POD),
CAT and glutathione S-transferase (GST), and increased oxidative
stress under drought conditions (Chen et al., 2015a). These
results show that cotton has numerous genes involved in the
antioxidant enzyme-related pathways that need to be explored in
drought-tolerant cultivars. Moreover, other factors are also
involved in improving the antioxidant machinery of cotton plants,
such as Zn, (Wu et al., 2015).
MAPK signalling pathway
Plants have developed various adaptations to environmental
stresses that function through a series of molecular networks
consisting of stress perception, signal transduction and expression
of specific stress-related genes. The mitogen-activated protein
kinase (MAPK) cascade is one of the key strategies developed by
plants against multiple biotic and abiotic stresses that participates
in signal transduction of extracellular stimuli and regulates
responses. MAPK pathway is a highly conserved central regulator
of various processes, including developmental programs, hor-
monal responses, cell division and apoptosis, proliferation and
stress responses. A MAPK cascade is minimally composed of at
least three distinct protein kinases, that is MAPKKK, MAPKK and
MAPK, which activate each other in a sequential manner via
phosphorylation (Ichimura et al., 2002). An activated MAPKKK
first phosphorylates two serine and/or threonine residues in a
conserved S/T-X3-5-S/T motif located within the activation loop of
the MAPKK. The activated MAPKK in turn phosphorylates MAPK
on threonine and tyrosine residues in the invariant T-X-Y motif in
the activation loop, and then MAPK phosphorylate specific
targets and modulate the activity of other kinases, transcription
factors, phospholipases, cytoskeletal proteins and microtubule-
associated proteins, whose altered activities mediate an extensive
range of response (Danquah et al., 2014; Nakagami et al., 2005;
Popescu et al., 2009).
To date, many reports have confirmed that MAPKs are involved
in plant signal transduction in response to abiotic stresses, such as
drought, salinity, cold and oxidative stress, in Arabidopsis and rice
(Ning et al., 2010; Shen et al., 2012; Teige et al., 2004; Xing
et al., 2008, 2015). In recent years, several genes involved in the
MAPK pathway response to abiotic stresses have been identified
in cotton (Table 2). Transcriptome analysis revealed that MAPK
components are activated by diverse abiotic stresses, such as
ABA, cold, drought and pH treatments (Zhu et al., 2013).
Twenty-eight putative MAPK genes distributed on 11 chromo-
somes were identified in the G. raimondii genome by performing
a bioinformatics homology search. These MAPK genes are
classified into the four known A, B, C and D groups and have
Figure 5 ROS scavenging machinery having two
arms: enzymatic arm and nonenzymatic arm.
Enzymatic arm contents on various enzymes
which converting ROS into other substances.
Likewise, Nonenzymatic arm content on other
substances which scavenge ROS.
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Abid Ullah et al.276

diverse functions (Zhang et al., 2014b). From the above analyses,
we conclude that MAPK signalling pathways are involved in the
response to multiple environmental stresses in cotton. However,
there are no reports on improving plant stress tolerance by
engineering MAPK cascades in cotton thus far. We recommend
improving cotton stress tolerance by engineering MAPK cascades,
and we believed that MKK1, MKK3, MPK6 etc. (Table 2) are
effective candidates to improve the tolerance against abiotic
stresses because it have been evaluated in Arabidopsis and
tobacco.
Calcium signalling pathway
Calcium is a key regulator of various cellular and physiological
processes in higher plants. In signal transduction pathways,
Calcium (Ca
2+
) is a universal second messenger that regulates a
variety of physiological processes in cotton plants. In addition to
other stresses and hormones, drought and ABA are involved in
changes of cytoplasmic Ca
2+
concentration (Li et al., 2015). Plant
cellular calcium signals are detected and transmitted by three
major classes of Ca
2+
sensor molecules: calmodulin (CaM) and
CaM-related proteins, calcium-dependent protein kinase (CDPK)
and calcineurin B-like proteins (CBLs). Calmodulin is acidic
calcium-binding protein that contains four EF hand motifs
(helix-loop-helix structural domains that coordinate with Ca
2+
ions). When Ca
2+
bind to EF motif, conformational transforma-
tion undergoes in CaM that either promotes its own catalytic
activity or its interactions with downstream target proteins. As
long as calcium sensor genes related to CaM and CaM-related
proteins have been studied for cotton fibre elongation (Cheng
et al., 2016; Tang et al., 2014), there are no such reports found
on drought stress in cotton. It has been noted that only a few
CDPKs in cotton have been characterized extensively. GhCPK1
was identified for the first time that has role in calcium signalling
events associated with fibre elongation (Huang et al., 2008).
Wang et al. (2012) opened a new door after sequencing the draft
genome of G. raimondii. Last year, Li et al. (2015) identified 41
CDPKs gene from the G. raimondii genome. Their study revealed
that GhCDPK3,GhCDPK2,GhCDPK11,GhCDPK16,GhCDPK28,
GhCDPK35 and GhCDPK14 genes are involved in drought and
salt stress. Further, they noted that these genes also respond to
ABA. CBLs proteins are another group of calcium sensor which
are specific to higher plants and play a significant role in decoding
calcium transients by specifically interacting with and regulating a
unique family of CBL-interacting protein kinases (CIPKs). GhCIPK6
was induced by drought, salt and ABA treatments. In addition,
overexpression of GhCIPK6 in Arabidopsis significantly enhanced
tolerance to drought, salt and ABA stresses (He et al., 2013).
These reports indicate that change in the Ca
2+
concentration
transduce Ca
2+
signals via CaMs, CDPKs and CBLs, which
phosphorylate downstream targets and subsequently respond
to drought and other abiotic stresses.
Stress-related transcription factors
Transcription factors are master regulators of normal cellular
processes as well as respond to biotic and abiotic stresses. Plants
including cotton respond and/or adapt to various stresses, and
transcriptional modulation is one of the most important ways that
induce or repressed a number of genes in plants under biotic and
abiotic stresses. Transcription factors play a significant role in the
stress signalling, from the perception of drought to the stress-
responsive gene expression by interacting with cis-acting ele-
ments present in the promoter region of numerous drought
Table 2 List of cotton MAPK genes engineered in other plants
Name Induced by stress Transgenic plant Phenotype/Result Interaction References
GhMKK3 Drought N. benthamiana Enhanced drought tolerance GhMPK7 and GhPIP1 Wang et al. (2016)
GhMAP3K40 Low temperature, NaCl, PEG, H
2
O
2
N. benthamiana Enhanced drought and salt tolerance at the
germination stage but reduced drought and oxidative
stresses tolerance at the seedling stage
GhMKK4 Chen et al. (2015b)
GhMPK4 High salinity, osmotic stress A. thaliana Enhanced the sensitivity to salt, osmotic stresses and exogenous ABA –Wang et al. (2015)
GhMKK4 NaCl, mannitol, ABA N. benthamiana Had no significant effects on salt or drought tolerance –Li et al. (2014)
GhMPK17 NaCl, mannitol, ABA A. thaliana Enhanced plant tolerance to salt and osmotic stresses –Zhang et al. (2014a)
GbMPK3 NaCl, cold, heat, dehydration,
oxidative stress
N. benthamiana Enhanced drought and oxidative stress tolerance –Long et al. (2013)
GhMPK6a Cold, NaCl, PEG N. benthamiana Reduced drought and salt tolerance GhMKK4 Li et al. (2013)
GhMKK1 NaCl, drought, H
2
O
2
N. benthamiana Enhanced salt and drought tolerance –Lu et al. (2013)
GhMKK5 Low temperature, NaCl, Wounding N. benthamiana Reduced the tolerance to salt and drought stresses –Zhang et al. (2012)
GhMPK2 ABA, NaCl, PEG, dehydration N. tobacum Reduced sensitivity to ABA, enhanced drought and salt tolerance –Zhang et al. (2011)
GhMPK6 ABA, NaCl, drought stresses Arabidopsis mutant atmkk1 Recovers the wild-type phenotype of atmkk1 mutant –Luo et al. (2011)
GhMPK16 Low and high temperatures,
mannitol, NaCl
Arabidopsis Reduced drought tolerance –Shi et al. (2011)
ª2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 15, 271–284
Drought coping strategies in cotton 277

stress-responsive genes in the signal transduction processes. In
this way, transcription factors activate signalling cascade of entire
network of drought stress-responsive genes that operate
together in inducing plant tolerance to drought and other abiotic
stresses (Guo et al., 2016). As a model plant, more than 1500
transcription factors of the Arabidopsis genome are thought to be
involved in stress-responsive gene expression (Lata and Prasad,
2011).
To increase the tolerance in cotton against drought stress,
transcription factors are excellent candidates for the plant
scientists. Various transcription factors (such as MYB, WRKY,
ERF, NAC, bZIP) are involved in normal development as well as in
stress (drought) response (Table 3). These transcription factors
have been cloned and proven useful tool for stress tolerance in
cotton and/or in other plants. The genetic engineering of
transcription factor genes could activate drought tolerance
pathways and enhance drought tolerance in cotton. Recently, a
bZIP transcription factor gene, GhABF2, has been reported to be
involved in the drought and salt tolerance in Arabidopsis and
cotton. The transcriptomic analysis revealed that GhABF2-
regulating genes related to ABA. Overexpressing GhABF2 cotton
increased SOD and CAT activities as compared to wild-type
plants. Moreover, overexpressed plants showed better results in
the field and meanwhile its yield were recorded higher than wild-
type plants (Liang et al., 2016). In another case, an R2R3-type
MYB transcription factor gene, GbMYB5, positively involved in
response to drought stress in cotton. Overexpressing GbMYB5
tobacco reduced water loss by decreasing the stomatal size and
showed hypersensitivity to ABA, and survival rate was higher after
drought treatment. In addition, proline content and antioxidant
enzymes were enhanced, while malondialdehyde (MDA) produc-
tion was lower in transgenic lines than in wild-type plants.
Furthermore, the transcript level of drought-responsive genes
(NCED3,BG,RD26), antioxidant genes (SOD,CAT,GST) and
polyamine biosynthesis genes (ADC1,SAMDC) were generally
higher in GbMYB-overexpressing tobacco (Chen et al., 2015a).
Similarly, tobacco plants with ectopic-expressing GhWRKY41
gene showed higher antioxidant enzyme activity, enhanced
stomatal closure and reduced MDA content. In addition, the
expression of antioxidant genes was also up-regulated in trans-
genic plants exposed to osmotic stress. These characteristics of
transgenic plants enhanced plant tolerance to drought stress
(Chu et al., 2015).
Strategies to induce drought tolerance in cotton
To enhance plant tolerance as well as vigour against drought,
alternative solutions must be developed. In this way, we can
maintain crop yields under extreme environmental conditions to
overcome economic losses. Improvements in cotton productivity
are urgently needed, especially in the areas where water
availability is scarce. In this regard, cotton crops that require less
water but produce higher yields and better fibre quality will be
highly desirable. Cotton characteristics should be site-specific
according to the environmental conditions of that area for
instance, an area having less rainfall need drought-tolerant variety
of cotton but on other side, saline area need a salt-tolerant variety
of cotton. Along with traditional breeding, development in the
field of biotechnology can produce transgenic cotton that
performs better in current and future environmental conditions.
However, exogenous application of particular substances, includ-
ing growth regulators, specific osmoprotectants and required
Table 3 Transcription factors in cotton playing important role in drought and other abiotic stresses
Genes encoding
transcription factors Expressing plant Mode of expression
Environmental
condition
Beneficial features
under drought and
other abiotic stress Abiotic stress type References
GhABF2 (bZIP) A. thaliana and
G. hirsutum
Overexpressed
and silenced
Greenhouse
and field
Regulated genes related to ABA, Increased the activities of SOD and CAT Drought and salt Liang et al. (2016)
GhNAC2 A. thaliana and
G. hirsutum
Overexpressed Greenhouse Higher root length, Drought Gunapati et al. (2016)
GbMYB5 G. barbedensis
and N. tobacum
Overexpressed
and silenced
Greenhouse Reduced stomatal size, rate of its opening and water loss, while
proline content and antioxidant enzymes increased
Drought and salt Chen et al. (2015a)
GhWRKY41 N. benthamiana Overexpressed Greenhouse Induced stomatal closure, higher antioxidant activity and lower
malondialdehyde content
Drought and salt Chu et al. (2015)
GhWRKY17 N. benthamiana Overexpressed Greenhouse Impaired ABA-induced stomatal closure, Reduced ABA level,
decreased the expression of ROS scavenging genes,
reduced proline content, elevated electrolyte leakage,
and malondialdehyde
Drought and salt Yan et al. (2014)
GhNAC8-GhNAC17 G. hirsutum Up-regulation Greenhouse NA Drought, salt, heat and Cold Shah et al. (2013)
GhERF1 G. hirsutum Up-regulation Greenhouse Signal regulation during stress and ABA production Drought, salt and Cold Qiao et al. (2008)
GhERF4 G. hirsutum Up-regulation Greenhouse Signal regulation during stress and ABA production Drought, salt and Cold Jin and Liu (2008)
ª2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 15, 271–284
Abid Ullah et al.278

minerals, can enhance drought tolerance in otherwise susceptible
plants. The aim was to produce more cotton yield per drop, and
in this regard, crucial approaches are needed to identify and
enhance drought-tolerant traits, such as quantitative trait loci
(QTL) analysis, transgenic approaches and exogenous application
of substances.
Marker-assisted selection (MAS) based on drought-
related QTLs/genes
Various minor genes, that is polygenes, have stronger additive
effects against drought tolerance than other biotic and abiotic
stresses. Thus, the sections of DNA (locus) located on chromo-
somes carrying these genes are known as quantitative trait loci
(QTL). Natural genetic variability of a crop can be utilized either
via direct selection under stress environments whether simulated
or natural or through QTL mapping (polygenes) and subsequent
marker-assisted selection (MAS) (Ashraf et al., 2008; Ashraf,
2010). QTL mapping allows us to determine the location,
numbers, degree of phenotypic effects and gene action pattern
(Vinh and Paterson, 2005). The role of polygenes has been
extensively evaluated using traditional methods, but DNA markers
as well as QTL mapping have made it possible and convenient to
analyse complex traits (Ashraf, 2010). Biological and proteomics
analyses have identified drought tolerance-related QTLs and
proteins in crop plants. Furthermore, these drought-related QTLs
and proteins can be used as markers in breeding programmes to
develop drought-tolerant genotypes.
In cotton, using F3 families derived from the cross of
G. barbadense cv. F-177 and G. hirsutum cv. Siv’on, a subset
of 33 QTLs identified under water-deficit conditions, that is five
QTLs for different physiological traits, 11 for plant productivity
and 17 for fiber quality. Most of these QTLs were located on
chromosome c2, 6 and 14 (Saranga et al., 2001). Based on
marker-assisted selection, near-isogenic lines were produced by
exchanging QTL for drought- and some yield-related traits
between G. barbadense cv. F-177 and G. hirsutum cv. Siv’on
(Levi et al., 2009a,b). Moreover, metabolite and mineral anal-
yses were conducted for these two species with QTLs for
drought- and productivity-related traits. The G. hirsutum cv.
Siv’on showed higher levels of metabolites under drought and
well-water conditions compared to G. barbadense cv. F-177.
Under drought stress, Siv’on (Gh) had higher mineral and
metabolite content and greater water use efficiency. Moreover,
Siv’on also showed stable photosynthesis and a greater
assimilation rate than F-177 under drought conditions. For
most of the studied traits, Siv’on showed a marked adaptation
to drought (Levi et al., 2011). In another QTL study, five QTLs
for osmotic potential (two QTLs were on chromosome c1, while
rest of three were on c2, 6 and 25 each contained one), three
for chlorophyll (two were on c2 and one on c14), 25 for leaf
morphology and various others for yield and biotic stress were
identified in cotton. QTLs for leaf morphology were distributed
across the genome which is associated with leaf size and
shape. Most notably, chromosome c15 contained six QTLs, c17
contained four, c6 contained three and c1 and c9 contained
two QTLs each, while c2, c3, c4, c10, c12, c18, c22 and c25 all
contained one QTL each (Said et al., 2013). In addition, 106
microsatellite markers were used to investigate 323 G. hirsutum
germplasms, treated by drought and salt, and 15 markers were
found related to drought tolerance. For the drought tolerance,
12 markers showed negative allele affects and the remaining
markers showed positive allele effects (Jia et al., 2014).
Likewise, a field study conducted for two consecutive years
under water-deficit and well-water conditions, 11 physiological
and morphological traits were recorded. As a result of QTL
mapping, 67 and 35 QTLs were identified under water-deficit
and well-water conditions, respectively. Most notably, chromo-
some c16, c9 and c2 contained 13, 12 and 7 QTLs, respectively
(Zheng et al., 2016).
Transgenic approach
Plants respond to multiple abiotic stress conditions at the
molecular level by altering gene expression (up- or down-
regulation), which further regulates a number of proteins, and
as a result, various biological functions are altered (Deeba et al.,
2012). The regulation of genes involved in the stress response is
one of the key factors in plants that cope with abiotic stresses
and enhance tolerance against these conditions (Hozain et al.,
2012). There are thousands of genes in plants, and a number
of them are involved in drought stress. Different techniques
were used to identify specific genes such as, the amplified
fragment length polymorphism (AFLP) was used by Park et al.
(2012), who identified several genes expressed under drought
stress in cotton (G. hirsutum L.). In their study, heat-shock
protein-related and ROS-related transcripts were induced by
water deficit. In another case, various stress-related genes were
identified by constructing normalized cDNA libraries of
G. barabadense regarding drought-, salt-, heat-, cold- and
phosphorus-deficit stresses (Zhou et al., 2016). It is possible to
transfer specific traits or gene of interest, that is drought-
tolerant genes, from an organism of interest into another
organism to obtain the desired characteristic by genetic
engineering or biotechnology (Herdt, 2006). Recently, scientists
transformed various drought-tolerant genes into cotton, result-
ing in drought-resistant plants (Table 4). Overexpressing of
TsVP,anH
+
-PPase gene from Thellungiella halophile in cotton
improved shoot and root growth as compared to wild type. In
addition, transgenic lines had higher chlorophyll content,
improved photosynthesis and higher relative water content of
leaves, and cell membrane damage was observed less than wild
type. These properties improved root development and the
lower solute potential resulting from higher solute content such
as soluble sugars and free amino acids in the transgenic plants.
These beneficial features enhanced drought tolerance in trans-
genic cotton, and seed cotton yield was 51% higher than wild-
type cotton plants (Lv et al., 2009). In another study, a gene,
ScALDH21, from Syntrichia carninervis was transformed into
cotton (Yang et al., 2016). Transgenic plants were checked in
the green house as well as in the field for drought tolerance.
Under field conditions, overexpressed transgenic lines showed
greater plant height, larger bolls and greater fibre yield than
wild type during different treatments of drought stress. It is due
to improved proline and soluble sugars, greater photosynthetic
rate and reduced lipid peroxidation in transgenic cotton as
compared to wild type. These discoveries and other studies
have led scientists to engineer drought-tolerant plants (cotton)
using genetic engineering methods, which are an effective
technology at the present time. Thus, various transgenic plants,
including cotton, have already been produced by inserting
various stress-related genes and then examined the plants for
the specific traits, that is drought tolerance. However, field
applications still need to be assessed because most of these
experiments were performed in the laboratory and greenhouse
conditions and did not produce appreciable results in the field.
ª2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 15, 271–284
Drought coping strategies in cotton 279

Table 4 Successful stories of genetically modified cotton with enhanced yield under drought stress
Gene(s) Promoter
Plant from which
gene taken Environmental condition Abiotic stress type
Beneficial traits of transgenic cotton
against drought stress Effect on yield References
ScALDH21 CaMV 35S Syntrichia caninervis Greenhouse and field Drought Soluble sugar and proline content increased, higher
peroxidase activity, reduced loss of net photosynthetic rate,
reduced lipid peroxidation, greater plant height,
larger bolls
Yield increased Yang et al. (2016)
AtEDT1/HDG11 CaMV 35S A. thaliana Laboratory
Greenhouse and Field
Drought and salt Soluble sugar and proline content increased,
well-developed roots, low stomatal density,
increased ROS scavenging enzymes
43% higher seeds Yu et al. (2015)
SNAC1 CaMV 35S Rice Greenhouse Drought and salt Enhanced proline content and root development,
while transpiration rate decreased
131% more bolls Liu et al. (2014)
AVP1 CaMV 35S A. thaliana Greenhouse and field Drought and salt Enhanced sequestration of ions and sugars into
vacuole, reduced water potential, and
enhanced root biomass
Increased 20% Pasapula et al. (2011)
Osmotin CaMV 35S Tobacco Greenhouse Drought Higher relative water content and proline
level, while H
2
O
2
, lipid peroxidation, and
electrolyte leakage were reduced
57.6% more bolls Parkhi et al. (2009)
TsVP CaMV 35S Thellungiella halophila Greenhouse Drought Improved root and shoot growth, higher rate of
photosynthesis and relative water content,
while less cell membrane damaged
42%–61%
higher (Lumianyan
19) 27%–53%
higher (Lumianyan 21)
Lv et al. (2009)
betA CaMV 35S Eschercia coli Greenhouse Drought Increased photosynthesis, higher relative water
content, better osmotic adjustment, less ion
leakage and lipid membrane peroxidation
3%–12% higher Lv et al. (2007)
ª2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 15, 271–284
Abid Ullah et al.280

Exogenous application of substances
Exogenous application of osmoprotectants and various plant
growth regulators have been found effectively to enhance
drought tolerance in cotton. Foliar application of osmoprotec-
tants and plant hormones, including ABA, gibberellic acid (GA
3
),
salicylic acid (SA), proline, glycinebetaine and polyamines, has
been reported to relieve the effects of stress. These treatments
elevated osmotic adjustment to improve turgor pressure and
promoted accumulation of antioxidants to detoxify ROS, thus
maintaining the integrity of membrane structures, enzymes and
other macromolecules during drought conditions (Anjum et al.,
2011). For example, the exogenous application of proline and
glycinebetaine as a foliar spray has also been found to be
effective in reducing the adverse effects of drought stress on
cotton (Noreen et al., 2013). In this way, GA exogenous
application enhanced the net rate of photosynthesis, transpiration
rate and stomata conductance in cotton (Lichtfouse et al., 2009).
Similarly, Zhao et al. (2013) exogenously sprayed ABA, JA and
MeJA on cotton plants. GbRLK was differentially induced by JA
and MeJA, but it was gradually up-regulated when exposed to
ABA treatment.
Concluding remarks and future perspectives
Currently, drought stress is responsible for extensive crop loss and
will likely become worse in the future; thus, there is international
interest in increasing drought-tolerant crops. The goal of our
study was to explore the mechanism of cotton under water-
deficit conditions. Scientists are attempting to induce drought
tolerance in cotton as well as other important crops. The aim was
to identify and enhance drought-tolerant traits via QTL analysis,
transgenic approaches and exogenous application of substances.
Several genes have been identified and characterized by pro-
teomic, transcriptomic and other omics that are induced by
drought stress and the associated signalling and regulatory
pathways in cotton plants. In comparison with Arabidopsis, the
amount of data on drought-regulated genes and their functions
in cotton are inadequate. However, a few of these genes have
been studied in cotton for their response to water-deficit
conditions, which is still in early stages. Transgenic cotton plants
were mostly studied under greenhouse conditions or tested in the
field under natural water-deficit environments with a small
amount (Table 4). It should be study in more realistic environment
that is in the field that what really happens there. Usually,
transgenic lines are developed by single gene transformation,
which may not be as productive as if it had been developed by
transferring a number of drought-related genes. It seems
interesting to transfer a number of prominent genes response
to drought and yield in the same variety of cotton. The amount of
data on drought-associated cotton protein kinases is also limited.
Only a few cotton protein kinases have been engineered and
studied in Arabidopsis and tobacco (Table 2); however, there are
no reports by engineering in cotton for enhancing drought
tolerance. More work on cotton plants is needed, however, to
link physiology, system biology and field performance. Under-
standing traits in cotton plants are associated with root architec-
ture, stomatal conductance, photosynthesis and osmotic
adjustment in drought stress. It is important to enhance the
drought tolerance capability of cotton, and there is still much
work to be performed to secure future generations from the
upcoming crisis. Drought is a complex trait; however, rapid
advances in the omics technologies will make it possible to use a
system biology approach to understand cotton plants responses
to drought stress and introduce drought-tolerant cotton.
Acknowledgements
Funding was provided by the National Natural Science Foundation
of China (31371675) and the Fundamental Research Funds for
the Central Universities (2662016PY001). The funders had no role
in the study design, data collection and analysis, decision to
publish or preparation of the manuscript. There is no conflict of
interest.
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