Tolerance Mechanism of Salt and Drought Stresses in Plants: Genes and their

roles
Jogendra Singh, Vijayata Singh, SK Sanwal, Awtar Singh and PC Sharma

INTRODUCTION
Agricultural productivity faces climate challenges i.e. drought and salinity due to their high magnitude of
impact and wide distribution. Crop plants often suffer from salt and drought stress resulting in a significant
reduction in both yield and quality. In some areas, the soil and weather conditions are so much unfavorable
that cultivation of certain crops becomes very difficult and non-profitable. Under such situations, it is
essential either to develop stress resistant cultivars or to modify the environment to the possible extent.
Breeding of crop cultivars resistant to stress environment is the most practical way of solving such problems.
There are two types of approaches are followed for salinity and drought tolerance breeding that is (1)
improving yield level of salt and drought tolerant cultivars, and (2) transfer of salt tolerant genes to high
yielding cultivars. In the first case, traditional cultivars of salt and drought affected areas are improved for
their productivity without affecting their salt and drought tolerance ability. In the second approach, salt and
drought tolerance genes from locally adapted (salt tolerant) cultivars are transferred to high yielding
cultivars.
Drought and salt signaling encompasses three important parameters (Mahajan and Tuteja, 2005).
First one is reinstating osmotic as well as ionic equilibrium of the cell to maintain cellular homeostasis under
the condition of stress. Secondly, control as well as repair of stress damage by detoxification signaling.
Thirdly, signaling to coordinate cell division to meet the requirements of the plant under stress.
Salt and water stress lead to cellular dehydration, which causes osmotic stress and removal of water
from the cytoplasm into the extracellular space resulting in a reduction of the cytosolic and vacuolar
volumes. Another consequence is the production of reactive oxygen species which then, in turn, affects
cellular structures and metabolism negatively. Early responses to water and salt stress are largely identical
except for the ionic component. These similarities include metabolic processes such as, for example, a
decrease of photosynthesis or hormonal processes like rising levels of the plant hormone ABA. Adaptation to
salinity and drought is undoubtedly one of the complex processes, involving numerous changes including
attenuated growth, the activation/increased expression or induction of genes, transient increases in ABA
levels, accumulation of compatible solutes and protective proteins, increased levels of antioxidants and
suppression of energy-consuming pathways. With the advancement of high throughput DNA technologies,
several hundred stress-induced or upregulated genes have been identified. However, the function of only a
limited number of gene products is known (Ingram and Bartels, 1996; Bray, 1997; Shinozaki and
Yamaguchi-Shinozaki, 1997; Hasegawa et al., 2000; Ramanjulu and Bartels, 2002). In this article, the
responses of plants to salinity and drought stress are explained, gene network and their regulatory
mechanism which assists the plants to adapt with salt and water stress.
SALT AND DROUGHT STRESS SIGNALING PATHWAYS
Salt and drought stresses affect virtually every aspect of plant physiology and metabolism. Knowledge of the
signaling pathway of stress is important because they represent suitable targets for genetic suppression to
improve salt and drought stress tolerance. In nature, for a plant to sacrifice a part of its structure constitutes
an adaptive strategy to survive in a stress condition. For adaptive or presumed adaptive responses, it may be
helpful to conceptually group them into three aspects: (a) homeostasis that includes ion homeostasis, which
is mainly relevant to salt stress, and osmotic homeostasis or osmotic adjustment; (b) stress damage control
and repair, or detoxification; and (c) growth control (Zhu et al. 2000). Accordingly, salt and drought stress
signaling can be divided into three functional categories: ionic and osmotic stress signaling for the
reestablishment of cellular homeostasis under stress conditions, detoxification signaling to control and repair
stress damages, and signaling to coordinate cell division and expansion to levels suitable for the particular
stress conditions. Homeostasis signaling negatively regulates detoxification responses because, once cellular
homeostasis is reestablished, stress injury would be reduced, and failure to reestablish homeostasis would
aggravate stress injury. Homeostasis and detoxification signaling leads to stress tolerance and is expected to
negatively regulate the growth inhibition response, i.e., to relieve growth inhibition. It is not enough to know
only the components or elements of signaling pathways (Xiong et al. 2001). Good comprehension requires
knowledge of precise inputs and outputs of the pathways. It is here that water stress signaling is most poorly
understood. Just because certain changes occur upon drought stress treatment, one cannot assume that
drought is the direct input signal, nor can one assume that the output is any of the adaptive responses. For the
ionic aspect of salt stress, a signaling pathway based on the SOS (Salt Overly Sensitive) genes has been
established. The input of the SOS pathway is likely excess intracellular or extracellular Na+, which
somehow triggers a cytoplasmic Ca2+ signal (Zhu et al. 2000). The outputs are expression and activity
changes of transporters for ions such as Na+, K+, and H+. The input for osmotic stress signaling is likely a
change in turgor. Several osmotic stress-activated, SOS independent protein kinases probably mediate
osmotic stress signaling, but no output is known (Zhu et al. 2001). Possible outputs of osmotic signaling
pathways include gene expression and/or activation of osmolyte biosynthesis enzymes as well as water and
osmolyte transport systems. Most of the other changes induced by salt or drought stress can be considered as
part of detoxification signaling. These include (a) phospholipid hydrolysis; (b) changes in the expression of
LEA/dehydrin-type genes, molecular chaperones, and proteinases that remove denatured proteins; and (c)
activation of enzymes involved in the generation and removal of reactive oxygen species and other
detoxification proteins. The input signal(s) for the detoxification pathways is most likely not an ionic or
osmotic change, but a product of stress injury, e.g., reactive oxygen species or protein denaturation. Water
stress generally inhibits plant growth, and indirect evidence suggests active signaling to cell division and
expansion machinery (Zhu et al. 2001). Slower cell division under water stress is probably a result of
reduced cyclin-dependent kinase (CDK) activity (Schuppler et al.1998). Reduced CDK activity may be a
result of combined effects of transcription suppression of cyclins and CDKs (Burssens et al. 2000) and
induction of CDK inhibitors. The direct input signal(s) for the CDK regulation is unclear but can be a
product of stress injury or any of the primary or intermediary signals involved in the homeostasis and
detoxification pathways.

TOLERANCE MECHANISM OF SALT AND DROUGHT STRESSES IN PLANTS VIA

ADOPTING TWO DIFFERENT MECHANISMS:

The SOS regulatory pathway for ion homeostasis and salt tolerance

Ion homeostasis is one of the most important strategies adopted by the plant for mitigating toxic impact\
caused by salinity and drought. Due to similar responses of both stresses, plants accumulate diverse groups
of osmoprotectants, which provide osmotic adjustment and ion homeostasis via the ion exchange activity
(Ranganayakulu et al. 2013).
Molecular mechanism of salt tolerance revealed in the model plants will facilitate identification of
candidate genes and the development of transgenic plants with salt tolerance in Brassica crops. Over-
expression of genes encoding enzymes related to abiotic stresses enhanced crop salt tolerance. The SOS
signaling pathway governing salt tolerance in Arabidopsis well clarifies the mechanisms of salt tolerance
(Fig.3). The SOS pathway comprises three family members, i.e., the plasma membrane Na+/H+ antiporter
SOS1, the protein kinase SOS2, and the Ca2+ binding protein SOS3. An increase of Na+ concentration leads
to an elevation of intracellular Ca2+, and SOS3 binds Ca2+ and activates SOS2 to form a compound which
phosphorylates the plasma membrane-localized SOS1. Finally, over-expression of SOS1 results in an efflux
of more Na+ (Martinez-Atienza et al. 2007). SOS1 and SOS3 are constitutively expressed in Brassica crops,
while the expression pattern for SOS2 amongst Brassica species was found to be very unique (Kumer et al.
2009). SOS2 may be upregulated by salinity stress in the roots of all the Brassica species except for B.
juncea, which maintains high SOS2 transcripts even under non-stress conditions, indicating a very unique
feature of B. juncea (Kumer et al. 2009). The strong correlation between transcript abundance for SOS
pathway related genes and salinity stress tolerance was observed in Brassica crops (Chakraborty et al. 2012,
Kumer et al. 2009).
Recently studies of salinity tolerance in plants have ranged from genetic mapping to molecular
characterization of salt induced genes. Increasing understanding of biochemical pathways and mechanisms
that participate in plant stress responses has made it evident that many of these responses are common
protective mechanisms that can be activated by salt, drought and cold, even if sometimes through different
signaling pathways. Salt stress is perceived by an unknown receptor present at the plasma membrane (PM)
of the cell. This induces a cytosolic calcium perturbation, which is sensed by SOS3 and accordingly changes
its conformation in a Ca2+-dependent manner and interacts with SOS2. This interaction relieves SOS2 of its
auto-inhibition and results in activation of the enzyme. Activated SOS2, in complex with SOS3
phosphorylates SOS1, a Na+/H+ antiporter resulting in efflux of excess Na+ ions. SOS3–SOS2 complex
interacts with other salt mediated pathways resulting in ionic homeostasis. This complex inhibits HKT1
activity (a low affinity Na+ transporter) thus restricting Na+ entry into the cytosol. SOS2 also interacts and
activates NHX (vacuolar Na+/H+ exchanger) resulting in sequestration of excess Na+ ions, further
contributing to Na+ ion homeostasis. CAX1 (H+/Ca+ antiporter) has been identified as an additional target for
SOS2 activity reinstating cytosolic Ca2+ homeostasis (Mahajan and Tuteja (2005).

Fig. 1. Regulation of ion homeostasis by SOS and related pathways in relation to salt stress adaptation
(Mahajan and Tuteja, 2005).
Antioxidant defense systems
In response to severe oxidative stress caused by salinity and drought, plants trigger a complex enzymatic
antioxidant enzymes including superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and
ascorbate peroxidase (APX) and some other nonenzymatic low molecular weight antioxidants like
glutathione (GSH), ascorbate (ASC) and carotenoids which provide protection via quenching of toxic ROS
(Gill and Tuteja 2010a; Hossain and Fujita 2010; Kadioglu et al. 2011; Vardharajula et al. 2011; Kaya et al.
2013; Kubis et al. 2014). In a series of detoxifying mechanisms, plants enhanced the production of
metalloenzyme SOD which is responsible for the conversion of O2 _- to H2O2 and further CAT and PODs
catalyze breakdown of H2O2 (Gill and Tuteja 2010a). Although CAT is apparently absent in the chloroplast,
however, H2O2 can be detoxified in a reaction catalyzed by an ascorbate specific peroxidase often present in
high levels in this organelle through the ascorbate-glutathione cycle. Ascorbate can also be oxidized via
direct reaction with O2- or by serving as a reductant of a chromoxyl radical of oxidized a tocopherol (Gill and
Tuteja 2010a). Recently, Vardharajula et al. (2011) and Wu et al. (2012) have also reported enhanced
activities of a diverse range of antioxidant enzymes including SOD, POD, CAT and APX in Solanum
melongena seedlings and maize plant under salinity and drought stresses. CATA, CATB, and POX1 are
playing role in the excessive generation of ROS due to decreased stomatal conductivity under low water
potential, over- reduction of electron transport in cellular organelles, accumulation of Na and Cl ions. It also
saves the plants from oxidative damage to lipid membranes and other essential macromolecules including
pigments, proteins, DNA, and RNA.
ABA AND OSMOTIC STRESS SIGNALING
Role of ABA in Water Stress Tolerance
Although ABA has broad functions in plant growth and development, its main function is to regulate plant
water balance and osmotic stress tolerance. This point is best illustrated by plant mutants that cannot produce
ABA. Several ABA-deficient mutants have been reported for Arabidopsis, namely aba1, aba2, and aba3
(Koornneef et. al 1998). There are also ABA-deficient mutants for tobacco, tomato, and maize (Liotenberg et
al. 1999). Without water or temperature stress, ABA-deficient mutants grow and develop relatively normally
(Koornneef et. al 1998). The mutants, such as the Arabidopsis aba1, aba2, and aba3, have slightly smaller
statures, which may be caused by unavoidable stress even under the best growth conditions. Additionally, the
smaller stature of the aba mutants may also be due to ABA regulation of the cell cycle and other cellular
activities. Under drought stress, ABA-deficient mutants readily wilt and die if the stress persists.
Under salt stress, ABA-deficient mutants also perform poorly (Xiong et al. 2001). The role of ABA
in drought and salt stress is at least twofold: water balance and cellular dehydration tolerance. Whereas the
role in water balance is mainly through guard cell regulation, the latter role has to do with the induction of
genes that encode dehydration tolerance proteins in nearly all cells. In addition, ABA is required for freezing
tolerance, which also involves the induction of dehydration tolerance genes (58, Xiong et al. 2001).
SALT AND DROUGHT STRESS REGULATION OF ABA BIOSYNTHESIS AND DEGRADATION
Osmotic stress induction of ABA accumulation is a well-known fact. Recently, some of the underlying
molecular mechanisms became clear. Osmotic stress-induced ABA accumulation is a result of both the
activation of synthesis and inhibition of degradation. Several ABA biosynthesis genes have now been cloned
(Figure 5). Zeathanxin epoxidase (known as ABA2 in tobacco and ABA1 in Arabidopsis) catalyzes the
epoxidation of zeaxanthin and antheraxanthin to violaxanthin (Marin et al. 1996). The 9-cis-epoxycarotenoid
dioxygenase (NCED) gene was first cloned from the maize vp14 mutant (99). ABA aldehyde oxidase
catalyzes the last step. ABA3, also known as LOS5, encodes a sulfurylase that generates the active form of
the molybdenum cofactor required by ABA aldehyde oxidase (Xiong et al. 2001). With the cloning of these
biosynthesis genes, it has been possible to determine which of them may be activated by osmotic stress.
Biochemical studies suggested that the rate-limiting step is the reaction catalyzed by NCED (Koornneef et. al
1998). Indeed, when VP14 and its homologous genes became available, their expression was seen to be
upregulated by drought stress (Iuchi et al. 2001, Qin et al. 1999, Thompson et al. 2000). It is surprising,
however, that other ABA biosynthesis genes are also upregulated by osmotic stress. This is true for the
Arabidopsis ABA1 (Liotenberg et al. 1999), for AAO, and also for ABA3 (Xiong et al. 2001). Admittedly, the
protein amount or activity has not been shown to increase in response to osmotic stress for all these genes.
Nevertheless, it is evident that ABA biosynthesis is subjected to osmotic stress regulation at multiple steps
(Figure 5). To date, genes responsible for ABA degradation have not been identified. Biochemical studies
suggest that a cytochrome P450 monooxygenase catalyzes the first step in the oxidative degradation of ABA
(Krochko et al. 1998). It should not be long before this gene, which is expected to be induced by ABA but
repressed by osmotic stress, is identified. Nothing is known about the signaling between osmotic stress
perception and the induction of ABA biosynthesis genes. It should not be long before this gene, which is
expected to be induced by ABA but repressed by osmotic stress, is identified. Nothing is known about the
signaling between osmotic stress perception and the induction of ABA biosynthesis genes. Presumably, it
involves calcium signaling and protein phosphorylation cascades.

CONCLUSION AND FUTURE PERSPECTIVES

Environmental challenges such as salinity and drought are major factors limiting plant growth and
productivity. The proportionately higher role of genes pertaining to osmotic and ionic modules of salt
drought stress tolerance is clearly evident in imparting tolerance. We can develop and utilize specific marker
for those genes which are pertaining to the osmotic, signaling modules and emphasize the better protection
of cellular membranous network and membrane-bound macromolecules under salt stress), and development
and/or improvement of new drought and salinity-tolerant accessions.
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