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1. INTRODUCTION
1.1 Overview
Environmental stresses affecting crop productivity are categorized mainly into biotic
stress and abiotic stress. Biotic stress includes the infection or competition by other
organisms. The major abiotic stress includes the unfavourable environmental
conditions such as high salinity, drought, temperature extremes, water logging, high
light intensity or mineral deficiencies. These abiotic stresses can delay growth and
development, reduce productivity and in extreme conditions, cause the plant to die.
Abiotic stresses are the primary causes of crop loss worldwide, reducing average
yields of major crop plants by more than 50% (Vinocur and Altman, 2005).
High salinity is one of the most serious abiotic stresses that adversely affect
crop productivity and quality (Chinnusamy et al., 2005). The productivity of over one-
third of the arable land in the world is affected by the salinity of the soil (Epstein and
Bloom, 2005). More than 800 million ha of land worldwide are salt-affected (FAO,
2008). High salinity adversely affects plant growth and development by disturbing the
intracellular ion homeostasis, which results in membrane dysfunction, attenuation of
metabolic activity and secondary effects that inhibit growth and induce cell death
(Hasegava et al., 2000). Activities of all the enzymes involved in various metabolic
pathways are severely reduced at NaCl concentrations above 0.3 M because of
disruption of the electrostatic forces that maintain protein structure (Wyn Jones and
Pollard, 1983). NaCl stress also induces generation of various reactive oxygen species
such as superoxide, H2O2, hydroxyl radical and singlet oxygen. Photosynthetic
efficiency of plants is severely damaged through a combination of superoxide and
H2O2-mediated oxidation (Herna´ndez et al., 1995). Plants adapt to environmental
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stresses via a plethora of responses, including the activation of molecular networks
that regulate stress perception, signal transduction and the expression of both stress-
related genes and metabolites. Plants have stress-specific adaptive responses as well as
responses which protect the plants from more than one environmental stress (Huang et
al., 2011). Numerous abiotic stress-related genes, as well as transcription factors and
regulatory sequences in plant promoters, have been characterized (Agarwal and Jha,
2010). Plants employ three different strategies to prevent and adapt to high Na+
concentrations: (i) active Na+ efflux, (ii) Na+
compartmentalization in vacuoles, and
(iii) Na+ influx prevention (Niu et al., 1995; Rajendran et al., 2009). Antiporters are an
important group of genes that plays a pivotal role in ion homeostasis in plants. Na+/H+
antiporters (NHX1 and SOS1) maintain the appropriate concentration of ions in the
cytosol, thereby minimising cytotoxicity. NHX1 are located in tonoplast and reduce
cytosolic Na+ concentration by pumping in the vacuole (Gaxiola et al., 1999), whereas
SOS1 is localized at the plasma membrane and extrudes Na+ in apoplasts (Shi et al.,
2002a). Both of these are driven by proton motive force generated by the H+-ATPase
(Blumwald et al., 2000).
The discovery of, and pioneer studies on, sos mutants in Arabidopsis thaliana
uncovered a new pathway for ion homeostasis that promotes tolerance to salt stress.
The sos mutants were specifically hypersensitive to high external concentrations of
Na+ or Li+ and were unable to grow at low external K+ concentrations (Wu et al.,
1996; Zhu et al., 1998). The SOS pathway consists of three proteins: SOS3 (Salt
Overly Sensitive 3), a calcium sensor protein (Liu and Zhu, 1998); SOS2 (Salt Overly
Sensitive 2), a serine/threonine protein kinase (Liu et al., 2000); and SOS1 (Salt
Overly Sensitive 1), a plasma membrane Na+/H+ antiporter that excludes Na+ by
taking H+ into the cytoplasm (Shi et al., 2000). During salt stress, cellular Ca2+ levels
2
Introduction
are altered and CBL (Calcineurin B-like proteins) and CBL-interacting protein kinases
(CIPK) are activated. The CBL participate in salt stress-mediated signal transduction
to control the influx and efflux of Na+ (Pardo et al., 1998). The calcineurin B-like
(regulatory) Ca2+ sensor SOS3 has been cloned from A. thaliana (Liu and Zhu, 1998).
SOS3 interacts with and activates the serine/threonine protein kinase SOS2 (Halfter et
al., 2000; Liu et al., 2000). This interaction has been reported to recruit SOS2 to the
plasma membrane where it interacts with SOS1 (Qiu et al., 2002). The A. thaliana
SOS1 gene was ectopically expressed for the first time in Arabidopsis and suppressed
the accumulation of Na+ in the presence of salt (Shi et al., 2003b). Similar results were
obtained when SOD2 and NhaA, which are plasma membrane Na+/H+ antiporters from
Schizosaccharomyces pombe and Escherichia coli, respectively, were overexpressed
in Arabidopsis (Gao et al., 2003) and rice (Wu et al., 2005). Heterologous expression
of different plant SOS1 genes suppressed the Na+ sensitivity of the yeast mutant
(AXT3K) (A. thaliana, Shi et al., 2002a; A. thaliana, Quintero et al., 2002;
Cymodocea nodosa, Garciadeblás et al., 2007; Oryza sativa, Martinez-Atienza et al.,
2007; and Solanum lycopersicum, Olias et al., 2009). Additionally, Wu et al. (2007)
and Garciadeblas et al. (2007) showed that the expression of Populus euphratica
PeSOS1 and C. nodosa CnSOS1 partially suppressed salt-sensitive phenotypes of
EP432 bacterial strain (nhaAnhaB), which lacks the activity of two Na+/H+ antiporters
EcNhaA and EcNhaB. These studies suggest that SOS1 gene could be employed to
develop salt-tolerant transgenic crops.
In the mission to meet food demand for the ever increasing world population,
the adverse environmental factors are becoming a major challenge for the scientific
community. If crops can be redesigned to cope up with abiotic stresses, agricultural
production could be increased dramatically. So there is a need of hour to develop
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plants that can tolerate adverse conditions such as high salinity. The success through
traditional breeding approaches in transferring the desirable traits from the wild
relatives to cultivated varieties has been limited due to reproductive barriers and
frequent failures of the inter-specific crosses. Genetic engineering can serve as better
tool to introduce the desired genes in the crops of interest across the taxa. Utilization
of naturally adapted salt tolerant plants (halophytes) like Salicornia species may play a
paramount role in genetic engineering of salt tolerance in glycophytes, because
halophytes have strong Na+ compartmentalization and active efflux mechanism to
manage low salinity (Na+ concentration) in the cytosol. Genetic engineering
approaches i.e. transfer of genes, which display a vital role in stress tolerance in other
plants could be used for development of transgenic crop plants that could withstand
higher salinity. The transgenic technology presages the great potential of genetically
engineered plants that are capable of growing in high saline soil and improving
agricultural productivity.
Since last two decades, the major studies on molecular mechanism of salt
tolerance is concentrated on glycophytes, however limited studies have been
performed on halophytes. Only two recent studies have performed in planta
overexpression of the SOS1 gene from halophytes: Thellungiella halophila (Oh et al.,
2009) and Puccinellia tenuiflora (Wang et al., 2011). The study of the salt tolerance
mechanisms of halophytic plants has emerged as an important area because these
species are well-adapted to and can overcome soil salinity more efficiently than
glycophytic plants (Gong et al., 2005). The halophytes have a unique genetic makeup
allowing them to grow and survive under salt stress conditions (Agarwal et al., 2010).
The experimerimental studies in our laboratory concentrated on an extreme halophyte,
Salicornia brachiata Roxb., in an effort to identify and characterize novel genes that
4
Introduction
enable salt tolerance. S. brachiata (Amaranthaceae), a leafless succulent annual
halophyte, commonly grows in the salt marshes of Gujarat coast in India. Salicornia
can grow in a wide range of salt concentrations (0.1–2.0 M) and can accumulate
quantities of salt as high as 40% of its dry weight (Agarwal et al., 2010). This unique
characteristic provides an advantage for the study of salt tolerance mechanisms.
Salicornia accumulates salt in the pith region, which reflects the fact that antiporter
genes are necessary to maintain homeostasis in extreme salinity. This plant may serve
as a model plant to study the salt responsive genes. Moreover, there is no report in the
literature about SOS1 gene from Salicornia. Therefore, the major objective of the
proposed work is “Cloning and characterization of the Salt Overly Sensitive 1 (SOS1)
gene from Salicornia brachiata Roxb. and its overexpression in tobacco plant for
functional validation.”
1.2 Review of literature
1.2.1 Salinity: The major environmental concern
High salinity is one of the most serious environmental factor limiting the plant
productivity (Allakhverdiev et al., 2000). Plants need essential mineral nutrients (ions)
to grow and develop. Salinity is generally defined as the presence of excessive amount
of soluble ions that hampers the normal functions essential for plant growth. It is
measured in terms of electric conductivity (ECe), or of the exchangeable Na+
percentage (ESP) or with the Na+ absorption ratio (SAR) and pH of saturated soil
paste extract. Therefore, saline soils are those having ECe more than 4 dS m-1
equivalent to 40 mM NaCl, ESP less than 15% and pH below 8.5 (Abrol, 1986;
Szabolcs, 1994; IRRI 2011). Most of the glycophytes are salt sensitive and cannot
grow even in < 4 dS m-1 ECe. Sea water contains approximately 3-3.5% of NaCl and
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in terms of molarity Na+ is about 500 mM. The productivity of over one-third of the
arable land in the world is affected by the salinity of the soil (Epstein and Bloom,
2005). According to FAO (2008) more than 800 million ha of land is salt-affected
worldwide. Globally, approximately 22% of the agricultural land is saline (FAO,
2005). In India salt-affected area is about 8.6 million ha (FAO, 2005).
The problem of soil salinization is getting more serious due to scanty rainfall,
repetitive seawater invasion, heavy utilization of ground water for agricultural and
industrial purposes, and degradation of saline parent rock (Mahajan and Tuteja, 2005).
Increasing soil salinity is a major problem in several states of our country. Gujarat is
having 1600 km long coastline and together with more than 15 km stretch of landward
zone makes an area of about 25000 sq. km. This vast coastal area largely consists of
sandy loam and mud flats and falls under semi-arid climatic zone. India produces ca.
18 MT of salt annually and more than 70% of it is produced in Gujarat. Salt
production in Gujarat is based entirely on solar energy, utilizing either sea brine or sub
soil brine. Due to extensive salt farming, scanty rainfall and heavy utilization of
ground water for industrial purposes, the entire coastal area of Gujarat is becoming
increasingly saline and salt ingress has become a common feature. Soil salinity of
coastal area is increasing day by day. The area under cultivation is fast getting
depleted and becoming unsuitable for agricultural crops (Jha, 2011).
1.2.2 Adverse effects of salinity on plants
Salt stress causes multifarious adverse effects in plants (Figure 1.1). High Salinity
immensely affects plant growth and development and is a major constraint for crop
production. It has been mentioned that the salinity stress first causes the rapid osmotic
stress that inhibits the growth of young leaves, followed by slow ionic stress that
6
Introduction
accelerates senescence of mature leaves (Munns and Tester, 2008; Horie et al., 2012).
Salinity causes suppression of growth in all plants, but their tolerance levels and rate
of growth reduction at higher concentration of salt differ widely among different plant
species (Dat et al., 2000). When cytoplasmic Na+ concentration increases, potassium
(K+) levels decreases, which in turn is directly correlated with lower growth rate (Ben-
Hayyim et al., 1987; Katsuhara and Tazawa, 1986). NaCl stress also significantly
damages photosynthetic mechanisms through a combination of superoxide- and H2O2-
mediated oxidation (Herna´ndez et al., 1995). Reduction in photosynthesis ultimately
arrests plant growth. There are several reports of inhibition of photosynthesis in
different plants under salt stress (Qiu et al., 2003a; Sudhir and Murthy, 2004; Koyro,
2006; Munns et al., 2006; Chaves et al., 2009). Salinity decreases CO2 assimilation
into carbohydrate through reductions in leaf area (Munns et al., 2000; Parida et al.,
2004), stomatal conductance (Ouerghi et al., 2000; Agastian et al., 2000; Parida et al.,
2004; Gorham et al., 2009), mesophyll conductance (Delfine et al., 1998; Parida et al.,
2004), and the efficiency of photosynthetic enzymes (Brugnoli and Bjorkman, 1992).
The detrimental effects of high salinity on plants can be observed at the whole plant
level, such as a significant reduction in plant growth, decrease in productivity, and
even the death of plants. The accumulation of Na+ in leaf tissues usually results in the
damage of old leaves due to ion toxicity, which shortens the lifetime of individual
leaves, thus reducing the net productivity and crop yield (Munns, 2002; Munns and
Tester, 2008; Gorham et al., 2009). Leaf senescence is one of the most limiting factors
to both biological and economic yields of a plant species under salinity (Ghanem et al.,
2008, Pe´rez-Alfocea et al., 2010). Increased NaCl levels result in a significant
decrease in root, shoot, and leaf biomass and an increase in root/shoot ratio in cotton
(Meloni et al., 2001). In addition, salt stress can also induce or accelerate senescence
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of the reproductive organs. Salinity reduces the yield of rice approximately by 45%,
which mainly results from spikelet sterility and reduced seed weight (Asch and
Wopereis, 2001). In field-grown cotton, salinity stress was a major reason for seed
abortion, leading to both yield loss and bad fiber quality (Davidonis et al., 2000).
Nearly 90% of the ovules of Arabidopsis aborted and smaller fruits resulted when
roots were incubated for 12 hrs in a hydroponic medium supplemented with 200 mM
NaCl (Sun et al., 2004). High soil salinity also substantially decreases seed
germination and seedling growth (Hasegawa et al., 2000). It has been reported that
salinity delays and reduces germination and emergence, decreases cotton shoot
growth, and finally leads to reduced seed cotton yield and fibre quality (Khorsandi and
Anagholi, 2009). Shoji et al., (2006) demonstrated that high salinity also affects
cortical microtubule organization and helical growth in Arabidopsis.
Salinity increases epidermal thickness, mesophyll thickness, palisade cell
length, palisade diameter, and spongy cell diameter in leaves (Parida et al., 2004). It
has been reported that salinity reduces plant leaf area and stomatal density in tomato
(Romero-Aranda et al., 2001) The NaCl treated plants revealed disorganized
thylakoidal structure, increased number and size of plastoglobuli and decreased starch
content by the electron microscopy (Hernandez, 1999; Parida et al., 2003).. In the
mesophyll of sweet potato leaves, thylakoid membranes of chloroplast are swollen and
most are lost under severe salt stress (Mitsuya et al., 2000). In potato, salt stress
reduces the number and depth of the grana stacks, and causes a swelling of the
thylakoid along with larger starch grains in the chloroplasts (Bruns and Hecht-
Buchholz, 1990).
8
Introduction
Figure 1.1: Schematic diagram of salinity stress effects on plant. (Reproduced from Horie et al., 2012)
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The transmission electron microscopy (TEM) data demonstrated that in leaves of salt
treated plants, the chloroplasts are aggregated, the cell membranes are distorted and
wrinkled, and there are no signs of grana or thylakoid structures in chloroplasts
(Tomato, Khavarinejad and Mostofi, 1998; Eucalyptus microcorys, Keiper et al., 1998;
Bruguiera parviflora, Parida et al., 2003).
Nitrogen metabolism is also affected by high salinity. It has been reported that
both nitrate uptake and nitrate reductase (NR) activity in leaves decrease in many
plants under salt stress (Abdelbaki et al., 2000; Flores et al., 2000, Meloni et al., 2004,
Parida and Das, 2004). The primary cause of the reduction in NR activity in the leaves
is the presence of a high concentration of Cl− and Na+, which leads to a decrease in
NO3− uptake and accordingly a lower NO3
− concentration in the leaves (Silveira et al.,
2001; Flores et al., 2000). This may lead to severe consequences for whole plant
nitrate assimilation. Therefore, a decrease in NR activity and reduced nitrate level
under high salinity condition may be responsible for a reduction in plant growth and
biomass production under salt stress (He, 2005).
1.2.3 Mechanism of salinity tolerance
High salinity interferes with plant growth and development and can also lead to
physiological drought conditions and ion toxicity (Zhu, 2002). Plant adaptation to
salinity stress involves a plethora of genes involved in ion transport and
compartmentalization (ion homeostasis), compatible solutes/osmolytes synthesis,
reactive oxygen species, antioxidant defence mechanism. Based on the capacity to
grow on a high salt medium, plants are usually categorized into glycophytes and
halophytes. The maximum NaCl limit that glycophytes can tolerate is up to 50 mM.
Halophytes are remarkable plants that tolerate salt concentrations that kill 99% of
10
Introduction
other species and can grow in the environment where the salt (NaCl) concentration is
200 mM or more (Flowers et al., 1986). Some halophytes can even tolerate the salinity
of more than twice the concentration of seawater (Flowers et al., 1977). Salinity
tolerance is multigenic trait and involves a network of genes for successful tolerance.
Several salt tolerant genes are isolated from wide variety of plants and their functional
analysis by the transcript expression and overexpression in homologous or
heterologous system has been studied. The genetic transformation of genes from signal
perception to ion homeostasis have resulted salt stress tolerance in various plants.
Since last two decades, the major studies on molecular mechanism of salt tolerance is
concentrated on glycophytes, however limited studies have been performed on
halophytes. The study of the salt tolerance mechanisms of halophytic plants has
emerged as an important area because these species are well-adapted to and can
overcome soil salinity more efficiently than glycophytic plants (Gong et al., 2005).
Halophytes luxuriantly grow in coastal marshes area and are well-adapted to salinity.
Halophyte has unique genetic makeup that provides an advantage for the study of salt
tolerance mechanisms. Halophytes maintain low salt concentration inside the cytosol
by extrusion of Na+ outside the cell membrane or sequestration in vacuoles and
secretion of salt outside the plant (bladders, salt glands). Halophyte accumulate Na+
and Cl- in vacuoles and synthesize organic osmolytes in the cytoplasm, which
facilitates water uptake into the plant and enhances turgor-driven growth at low to
moderate salinity levels (Bell and O'Leary, 2003).
Realizing the importance of halophyte for elucidating the salt tolerance
mechanism, recently a number of EST data bases have been developed for halophytes
like Sueda salsa (Zhang et al., 2001), Mesembryanthemum crystallinum (Kore-eda et
al., 2004), T. halophila (Wang et al., 2004), Avicennia marina (Mehta et al., 2005),
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Chapter 1
Limonium sinense (Chen et al., 2007), Aleuropus littoralis (Zouri et al., 2007),
Spartina alterniflora (Baisakh et al., 2008), Macrotyloma uniflorum (Reddy et al.,
2008), S. brachiata (Jha et al., 2009), Tamarix hispida (Li et al., 2009), Alfalfa (Jin et
al., 2010) and Chenopodium album (Gu et al., 2011). In total, the gene pool obtained
by the EST data base or by total sequencing, provides a list of the genes involved in
stress tolerance.
The advances in physiology, genetics, and molecular biology have greatly
improved our understanding of plant responses to salt stress. Understanding of the
molecular processes regulating these metabolic adaptations will facilitate engineering
of salt stress tolerance. Plants employ basically three different strategies to prevent and
adapt to high Na+ concentrations are: (i) Na+
compartmentalisation in vacuoles, (ii)
Active Na+ efflux outside the plasma membrane and (iii) Synthesis of compatible
solutes (osmolytes) (Figure 1.1, 1.2).
1.2.3.1 Sodium compartmentalization into vacuoles
The central vacuole is the largest compartment of a mature plant cell and may occupy
80% of total cell volume. The strategy of accumulation of Na+ inside vacuoles is used
by many plants to survive under salinity stress, an active vacuolar antiporter (NHX1)
utilizes the proton motive force generated by vacuolar H+-ATPases and H+-
pyrophosphatases to sequester excess Na+ into the vacuole, thereby reduce the toxic
effects of Na+ inside the cytosol (Munns and Tester, 2008; Niu et al., 1995; Blumwald
et al., 2000). In this way, the translocation and storage of Na+ inside vacuoles in the
shoot are suggested to be key factors for sustained growth during salt stress in some
plant species. Other plant species tend to limit Na+ accumulation in shoots by reducing
12
Introduction
Figure 1.2: Regulation of ion homeostasis by SOS signaling pathway for salt stress adaptation. Salt stress induce Ca2+ signal that activates the SOS3/SOS2 protein kinase complex, which then phosphorylates a plasmamembrane Na+/H+ antiporter SOS1, and regulates the expression of some genes as well. SOS2 also activates tonoplast Na+/H+ antiporter sequestering Na+ into the vacuole (NHX1). ABI1 regulates the gene expression of NHX1 whereas ABI2 interacts with SOS2 and negatively regulates ion homeostasis either by inhibiting SOS2 kinase activity or the activities of SOS2 targets. CAX1 (H+/Ca+ antiporter) is an additional target for SOS2 activity restoring cytosolic Ca2+ homeostasis. SOS3 and SOS2 complex negatively regulate the activity of AtHKT1. SOS4 gene encodes a pyridoxal (PL) kinase that is involved in the biosynthesis of PL-5-phosphate (PLP), which contributes Na+ and K+ homeostasis by regulating ion channels and transporters. SOS5 is involved in the maintenance of cell expansion. Dashed arrow shows SOS3-independent and SOS2-dependent pathway. PM: Plasma membrane (adapted from Turkan and Demiral, 2009).
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Chapter 1
transport from root to shoot, recirculation of Na+ out of the shoots and storage in root
or stem cell vacuoles (Munns and Tester, 2008). It has been reported that several
isoform of Na+/H+ antiporters exist in Arabidopsis, rice and mammalian systems.
These isoforms show differences in tissue specificity, expression patterns and
regulation. The role of NHX antiporters in ion accumulation and salt tolerance have
been obtained by overexpression or silencing of the genes, or by studying NHX gene
expression and ion accumulation in different species, differing in salt tolerance (Jha et
al., 2011).
The eukaryotic NHE (Na+/H+ hydrogen exchangers) gene family is divided into two
major clades, the intracellular (IC, endosomal/TGN, NHE8-like, and plant vacuolar)
and plasma membrane (PM, recycling and resident) on the basis of cellular location,
ion selectivity, inhibitor specificity, and protein sequence similarity (Brett et al.,
2005). The vacuolar NHE clade is abundantly and exclusively presented in plants. The
absence of ATP powered plasma membrane sodium intracellular pumps in plants may
be the reason for development of the specialized clade of vacuolar NHE in plants,
which act to store high concentrations of salt and water in the vacuole (Jha et al.,
2011). These NHE are critical determinants of salt tolerance and osmoregulation in
plants. Although physiological and biochemical data since long suggested that Na+/H+
and K+/H+ antiporters are involved in intracellular ion and pH regulation in plants, it
has taken a long time to identify genes encoding antiporters that could fulfill these
roles. A gene, encoding a protein with homology to animal plasma membrane Na+/H+
antiporters of the NHE family and the yeast ScNHX1 gene was first identified from
Arabidopsis genome and named AtNHX1 (Gaxiola et al., 1999). Na+/H+ antiporters,
NHX1 have been cloned from several plant species and its overexpression showed
greater tolerance in sensitive plants. Overexpression of A. thaliana AtNHX1 conferred
14
Introduction
enhanced salt tolerance in Arabidopsis (Apse et al., 1999) and several other plant
species such as tomato (Zhang and Blumwald, 2001), Brassica napus (Zhang et al.,
2001), Triticum aestivum (Xue et al., 2004) and Brassica juncea (Rajagopal et al.,
2007). Vacuolar Na+/H+ antiporter have also been isolated from different halophytes
such as M. crystallinum (Chauhan et al., 2000), Atriplex gmelini (Hamada et al., 2001),
S. salsa (Ma et al., 2004), Beta vulgaris (Xia et al., 2002), and S. brachiata (Jha et al.,
2011).
1.2.3.2 Active sodium efflux outside the plasma membrane
Salt Overly Sensitive (SOS) pathway is involved in Na+ exclusion from the plasma
membrane (Figure 1.2). The SOS pathway consists of three proteins with one proton
pump (PM H+-ATPase): SOS3, a calcium sensor protein (Liu and Zhu, 1998); SOS2, a
serine/threonine protein kinase (Liu et al., 2000); and SOS1, a plasma membrane
Na+/H+ antiporter that excludes Na+ by taking H+ into the cytoplasm (Shi et al., 2000).
The SOS pathway is regulated by Ca2+-dependent protein kinase signaling
(Rodrı´guez-Rosales et al., 2009). Ca2+ signaling is perceived by SOS3, a calcium
binding protein. SOS3 activates SOS2, a protein kinase that activates SOS1 by its
phosphorylation. SOS pathway also regulates vacuolar Na+/H+ antiporter exchange
activity and Na+ compartmentalization (Qiu et al., 2004). Further studies have shown
the functional conservation of SOS pathway in rice (Martínez-Atienza et al., 2007),
tomato (Olías et al., 2009) and poplar (Tang et al., 2010).
1.2.3.2.1 Salt Overly Sensitive 3 (SOS3) gene
The SOS3 (CBL4) locus was identified by root-bending assays on fast neutron-
mutagenized M2 Arabidopsis seedlings (Liu and Zhu, 1997). Further, Liu and Zhu
15
Chapter 1
(1998) determined that SOS3 gene encodes a 222 amino acid residue protein encoded
by an 8 exon and 7 intron coding region. SOS3 encodes a Ca2+-binding protein with an
N-myristoylation motif and four Ca2+-binding EF hands. The amino acid sequence of
SOS3 shows significant similarity to the regulatory subunit of yeast calcineurin and
animal neuronal Ca2+ sensors (Ishitani et al., 2000). A loss-of-function mutation that
reduces the Ca2+-binding capacity of SOS3 (sos3-1) renders the mutant plant to salt
sensitive. This mutant (sos3-1) defect can be partially rescued by high levels of Ca2+ in
the growth medium (Liu and Zhu, 1998). Compared to other Ca2+ sensors like
calmodulin and caltractin, SOS3 binds Ca2+ with a relatively low affinity. This
difference in the affinity may be an important factor in distinguishing and decoding
various Ca2+ sensors (Ishitani et al., 2000).
During salt stress, cellular Ca2+ levels are altered and CIPK and CBL
interacting proteins are activated. SOS3 (CBL protein) participate in salt stress-
mediated signal transduction to control the influx and efflux of Na+ (Pardo et al.,
1998). SOS3 has been cloned from Arabidopsis (Liu and Zhu, 1998). SOS3 interacts
with and activates the serine/threonine protein kinase SOS2 (Halfter et al., 2000; Liu et
al., 2000).
1.2.3.2.2 Salt Overly Sensitive 2 (SOS2) gene
SOS2 gene was isolated through the genetic screening of Arabidopsis mutants
oversensitive to salt stress. SOS2 is a Ser/Thr kinase of the SnRK3/CIPK family
(Kolukisaoglu et al., 2004) with a C-terminal regulatory domain and an N-terminal
catalytic domain (kinase domain) (Liu et al., 2000). The regulatory region of SOS2 has
an auto-inhibitory role and contains FISL (21-amino acid sequence motif) and PPI
(phosphatase interaction) motifs where a positive regulator SOS3 and the negative
16
Introduction
regulator type 2C protein phosphatase ABI2 bind, respectively (Ohta et al., 2003). The
function of ABI2 in the sodium regulation pathway is to dephosphorylate and
deactivate SOS2 or SOS1 (Ohta et al., 2003). SOS2 is normally inactive, presumably
because of an intramolecular interaction between the catalytic domain and the
autoinhibitory regulatory domain (Guo et al., 2001). SOS2 is active in substrate
phosphorylation only when plants are exposed to salt stress. Ca2+-activated SOS3
physically interacts with and activates SOS2 through a FISL conserved motif (Liu et
al., 2000). The SOS3/SOS2 kinase complex phosphorylates and activates the plasma
membrane Na+/H+ exchanger SOS1, thus leading to Na+ extrusion out of the cell
(Quintero et al., 2002, 2011; Shi et al., 2002a). Recently, it was shown that the SOS3
(CBL4)-SOS2 interaction occurs in the root, while SOS2 interacts with the SOS3
homolog SOS3-like CAlcium Binding Protein 8 (SCABP8)/Calcineurin B-Like 10
(CBL10) in the shoot (Kim et al., 2007; Lin et al., 2009). SOS2 transcription is up-
regulated by salt treatment (Liu et al., 2000; Gong et al., 2002).
It has been demonstrated that SOS2, independently of SOS3 or together with
SOS3 in the SOS2-SOS3 complex, can interact with proteins other than SOS1, and
regulate the several enzyme activities. In this respect, SOS2 play some role in the
regulation of the Na+/H+ and Ca2+/H+ exchange at the tonoplast because the activation
of their transport activities under salt stress requires SOS2 (Cheng et al., 2004; Qiu et
al., 2004; Kim et al., 2007). It has been also shown that there is a direct interaction
between SOS2 and vacuolar H+-ATPase and SOS2 promotes the transport activity of
H+-ATPase and also enhances salt tolerance (Batelli et al., 2007). Additionally, a
connection between SOS2 and reactive oxygen species (ROS) signalling was
established on the basis of the interaction found between SOS2 and the nucleoside
diphosphate kinase 2 (NDPK2) and between SOS2 and catalases 2 and 3 (Verslues et
17
Chapter 1
al., 2007). The 2C-type protein phosphatase ABI2 also interacts with SOS2, inhibiting
its activity as a result of the binding (Ohta et al., 2003), thus connecting the SOS
pathway to abscisic acid (ABA) responses.
1.2.3.2.3 Salt Overly Sensitive 1 (SOS1) gene
The plasma membrane Na+⁄H+ antiporter, SOS1 has been identified as a major
contributor to Na+ efflux in higher plants (Blumwald et al., 2000; Shi et al., 2000,
2003b; Qiu et al., 2002, 2003b; Xiong et al., 2002). Ethylmethane sulfonate (EMS)-
treated A. thaliana salt sensitive plants indicated that mutations in the SOS1
(GenBank: NM_126259) gene rendered the Arabidopsis plants extremely sensitive to
high Na+ or Li+ and low K+ environments. This experiment showed that SOS1 locus is
essential for Na+ and K+ homeostasis (Wu et al., 1996; Shi et al., 2000). The
Arabidopsis AtSOS1 gene contains 23 exons and encodes a plasma membrane protein
of 1146 amino acids with a calculated molecular mass of 127-kDa. Hydrophobic plot
analysis of AtSOS1 predicted 12 transmembrane domains in the N-terminal part and a
long hydrophilic cytoplasmic tail in the C-terminal part. The transmembrane region of
SOS1 has significant sequence similarities to plasma membrane Na+/H+ antiporters
from bacteria, fungi and animals (Shi et al., 2000). However, the C-terminal
hydrophilic domain was unique for SOS1 and no similarities were found with other
known antiporters in the NCBI GenBank database. In fact, the long C-terminal
hydrophilic tail makes SOS1 the largest known Na+/H+ antiporter sequence (Mahajan
et al., 2008). Sequence analysis of various SOS1 mutant alleles revealed several
residues and regions, which are essential for SOS1 function. The sos1-3 and sos1-12
alleles contain point mutations in the membrane spanning region. These mutations are
R to C and G to E, respectively. Both these mutations affect residues that are
18
Introduction
conserved in all antiporters and presumably abolish the antiport activity of SOS1 (Shi
et al., 2000). Two other point mutations (sos1-8 and sos1-9) are found in the
hydrophilic tail region. A 7-base-pair deletion resulting in a frame shift that truncated
the last 40 amino acids from the C terminus was found in sos1-11 allele obtained from
T-DNA mutagenesis. sos1-2 and sos1-6 mutations truncate the cytoplasmic tail of
SOS1 by a stop codon mutation. It is in fact interesting to note that these and other
mutations do not affect the transmembrane region and thus reveal that both N and C
domains may be essential for the function of SOS1 (Shi et al., 2000). The C-terminal
tail of SOS1 may play a vital role in interaction with various regulators of the antiport
activity of SOS1 and these mutations may disrupt the direct interaction of the
regulators with SOS1. In a recent study, some of the important genes, that control Na+
entry (HKT1) and exit (SOS1) from the cells, or help in the compartmentalization of
excess Na+ ions in the vacuole (NHX1, NHX5, AVP1 and AVP2) were targeted for
comparative analysis in the model plant Arabidopsis (a dicot) and evolutionary distant
monocot species such as rice and wheat. It was interesting to explore that the majority
of exons in Arabidopsis, rice and wheat orthologues of NHX1, NHX5 and SOS1 were
conserved except for those at the amino and carboxy terminal ends (Mullan et al.,
2007). However additional exons were also identified in predicted NHX1 and SOS1
genes of rice and wheat when compared with Arabidopsis, which indicates gene
rearrangement during evolution from a common ancestor (Mullan et al., 2007).
Confocal imaging of a SOS1–GFP fusion protein in transgenic Arabidopsis
plants indicated that SOS1 is localized in the plasma membrane. Analysis of SOS1
promoter–β-glucuronidase transgenic Arabidopsis plants revealed preferential
expression of SOS1 in epidermal cells at the root tip and in parenchyma cells at the
xylem/symplast boundary of roots, stems, and leaves (Shi et al., 2002a).
19
Chapter 1
SOS1 is expressed at low basal levels but is upregulated in the presence of
NaCl stress and has been shown to regulate other genes in response to salt stress (Shi
et al., 2000; Gong et al., 2001). This up-regulation is abated in sos3 or sos2 mutant
plants, suggesting that it is controlled by the SOS3/SOS2 regulatory pathway (Shi et
al., 2000). Consistent with its specific role in Na+ tolerance, AtSOS1 gene expression
was not up-regulated by cold stress or ABA (Shi et al., 2000). AtSOS1 mRNA was
more abundant in roots than in shoots. In both roots and shoots, AtSOS1 expression
was up-regulated by NaCl stress (Shi et al., 2000). In response to salt stress (200 mM)
the level of PeSOS1 protein in the leaves of P. euphratica was significantly up-
regulated, while the mRNA level in the leaves remained relatively constant (Wu et al.,
2007). Expression of Chrysanthemum crassum CcSOS1 in the roots was sensitive to
salinity stress, while in the leaves CcSOS1 was down-regulated in the presence of
abscisic acid. CcSOS1 transcript abundance was reduced in both roots and leaves of
plants exposed to low temperature, while it was increased in leaves (but not in roots)
after drought stress. CcSOS1 expression was not regulated in the presence of CaCl2
(Song et al., 2012).
A number of reports highlights that salt tolerance genes are constitutively
expressed in halophytic plants, however, they are stress inducible in glycophytes. This
implies that stress-inducible signalling pathways are active in stress tolerance plants
under unstressed conditions. The SOS1 transcript in halophytes shows more mRNA
abundance under salt stress as compared to glycopytes (Oh et al., 2009). SOS1 gene
expression was found up-regulated by NaCl stress in shoot tissue of many halophytes
(Thellungiella halophila, Kant et al., 2006; Chenopodium quinoa, Maughan et al.,
2009; Puccinellia tenuiflora, Wang et al., 2011). The SOS1 mRNA preferentially
accumulates higher in the root compared to shoot. T. halophila ThSOS1 transcript
20
Introduction
expression was found 7-fold in roots relative to shoots under salt stress (Kant et al.,
2006), whereas in C. quinoa CqSOS1 expression was high at low salt concentration in
the root tissue, which indicates that the SOS1 gene is hyper-inducible in the roots of
halophytic plants at even low salt concentrations (Maughan et al., 2009). The nitric
oxide (NO) treatment showed higher expression of SOS1 in Avicennia marina plants
(Chen et al., 2010).
In earlier studies, Na+/H+ exchange activity in wild-type (WT) and sos1 plants
was compared using highly purified plasma membrane vesicles. The result
demonstrated that plasma membrane Na+/H+ exchange activity was present in WT
plants treated with 250 mM NaCl, however this transport activity showed a reduction
by 80% in the similarly treated sos1 plants (Qiu et al., 2002). The addition of activated
SOS2, in vitro, increased Na+/H+ exchange activity in salt-treated WT plants by 2-fold
relative to transport activity without the addition of SOS2. Further, addition of
activated SOS2 in the vesicles of sos2 and sos3 plants increased their Na+/H+
exchange activity. These studies laid the foundation for addressing the role of plasma
membrane transporters in relation to salt stress and overall plant growth and
physiology (Qiu et al., 2002).
In another report, salt cress (T. halophila) was used as a system to identify
biochemical mechanisms that enable plants to grow under saline conditions. Salt
treatment increased the H+ transport and hydrolytic activity of the H+-ATPase in both
the plasma membrane as well as tonoplast of T. halophila. An increased expression of
SOS1 was observed in the plasma membrane isolated from control- and salt-treated
roots and leaves of this plant (Vera-Estrella et al., 2005).
21
Chapter 1
Shabala et al. (2005) reported on the electrophysiological data using the non-
invasive ion flux (MIFE) technique and compared the net K+, H+, Na+ fluxes from
elongation and mature root zones of Arabidopsis wild-type Columbia background and
sos1 mutants. Their study revealed that sos1 mutation affects the functioning of the
entire root and not just the root apex, sos1 mutation affects the H+ transport even in the
absence of salt stress. sos1 mutation also affects the intracellular K+ homeostasis with
a plasma membrane depolarization-activated outward rectifying K+ channel being a
likely target. Moreover, it was also suggested that H+ pump might also be a target of
SOS pathway signalling. Studies have indicated that SOS1 also functions to protect
plasma membrane K+ transport during salinity stress. It was observed that the K+-
uptake ability of the sos mutant root cells measured electro-physiologically was
normal in control conditions. However, in the presence of mild salt stress (50 mM
NaCl), root-cell K+ permeability was strongly inhibited in sos1 mutant but not in WT
plants. Alternatively, increasing K+ availability partially rescued the sos1 growth
phenotype. Therefore, it appears that in the presence of Na+, the Na+/H+ antiport
activity of SOS1 is necessary for protecting the K+ permeability on which growth
depends (Qi and Spalding, 2004). Guo et al. (2009) showed that during the first 15 min
after NaCl application, sos1 mutants showed net H+ efflux and intracellular
alkalinization in the root meristem zone, whereas wild-type (WT) showed net H+
influx and slight intracellular acidification in the root meristem zone.
Martı´nez-Atienza et al. (2007) have identified a rice plasma membrane
Na+/H+ exchanger that, on the basis of genetic and biochemical criteria, is the
functional homolog of the Arabidopsis SOS1 protein. The rice transporter, OsSOS1,
showed a capacity for Na+/H+ exchange in plasma membrane vesicles of yeast
(Saccharomyces cerevisiae) cells and reduced their net cellular Na+ content. The
22
Introduction
Arabidopsis protein kinase complex SOS2/SOS3, which positively controls the
activity of AtSOS1, phosphorylated OsSOS1 and stimulated its activity in vivo and in
vitro. Moreover, OsSOS1 suppressed the salt sensitivity of a sos1-1 mutant of
Arabidopsis. Putative rice homologs of the Arabidopsis protein kinase SOS2 and its
Ca2+-dependent activator SOS3 were also identified. OsCIPK24 and OsCBL4 acted
coordinately to activate OsSOS1 in yeast cells and they could be exchanged with their
Arabidopsis counterpart to form heterologous protein kinase modules that activated
both OsSOS1 and AtSOS1 and suppressed the salt sensitivity of sos2 and sos3 mutants
of Arabidopsis. These results suggest that the SOS salt tolerance pathway also operates
in cereals and provides evidences regarding high degree of structural conservation
among the SOS proteins from dicots and monocots.
Shi et al. (2002a) studied that SOS1 functions in retrieving Na+ from the xylem
stream under severe salt stress, whereas under mild salt stress it may function in
loading Na+ into the xylem. These results suggested that SOS1 is critical for
controlling long-distance Na+ transport from root to shoot. Olias et al. (2009)
demonstrate that S. lycopersicum SlSOS1 antiporter is also critical for the partitioning
of Na+ between plant organs. The ability of tomato plants to retain Na+ in the stems,
thus preventing Na+ from reaching the photosynthetic tissues, is largely dependent on
the function of SlSOS1. Katiyar-Agarwal et al. (2006) has reported that SOS1 interacts
with RCD 1 (regulator of oxidative stress responses) via its predicted cytoplasmic tail
to regulate the expression of ROS-scavenging genes. AtSOS1 mRNA is unstable at
normal growth conditions, but its stability is substantially increased under salt stress
and other ionic and dehydration stresses. Stress-induced SOS1 mRNA stability is
mediated by reactive oxygen species (ROS). The cis-element required for SOS1
23
Chapter 1
mRNA instability resides in the 500-bp region within the 2.2 kb at the 3′-End of the
SOS1 mRNA (Chung et al., 2008).
The Arabidopsis SOS1 gene was ectopically expressed for the first time in
Arabidopsis and suppressed the accumulation of Na+ in the presence of salt (Shi et al.,
2003b). Similar results were obtained when SOD2 and NhaA, which are plasma
membrane Na+/H+ antiporters from S. pombe and E. coli, respectively, were
overexpressed in Arabidopsis (Gao et al., 2003) and rice (Wu et al., 2005). Further,
few studies have performed in planta overexpression of the SOS1 gene from
Arabidopsis (Yue at al., 2012), Thellungiella (Oh et al., 2009) and P. tenuiflora (Wang
et al., 2011). Oh et al. (2009) also reported that SOS1 suppressed ThSOS1-RNAi
transgenic lines of Thellungiella salsuginea showed high salt sensitivity compared to
WT plants.
Instead of transforming single stress-responsive gene, some researchers have
tried manipulation of a combination of two or more genes in plants. Yang et al. (2009)
tested overexpression of multiple genes to improve salt tolerance in Arabidopsis. They
produced six different transgenic Arabidopsis plants overexpressing AtNHX1, SOS1,
and SOS3 alone or in different combinations (AtNHX1 + SOS3, SOS2 + SOS3, SOS1 +
SOS2 + SOS3). Surprisingly, the AtNHX1 alone did not show significant salt tolerance.
In 220 mM NaCl treatment for 3 days, less than 20% of the control and transgenic
plants overexpressing only AtNHX1 survived, but over 80% of the transgenic plants
overexpressing SOS1, SOS3, SOS2 + SOS3, AtNHX1 + SOS3, or SOS1 + SOS2 + SOS3
survived.
Heterologous expression of different plant SOS1 genes suppressed the Na+
sensitivity of the yeast mutant (AXT3K) (A. thaliana, Shi et al., 2002a; A. thaliana,
24
Introduction
Quintero et al., 2002; C. nodosa, Garciadeblás et al., 2007; O. sativa, Martinez-
Atienza et al., 2007; T. salsuginea, Oh et al., 2009; Reed plants, Takahashi et al.,
2009; S. lycopersicum, Olias et al., 2009; and C. crassum, Song et al., 2012).
Additionally, Wu et al. (2007) and Garciadeblas et al. (2007) showed that expression
of P. euphratica PeSOS1 and C. nodosa CnSOS1 partially suppressed the salt-sensitive
phenotypes of the EP432 bacterial strain (nhaAnhaB), which lacks activity of the two
Na+/H+ antiporters EcNhaA and EcNhaB. PeSOS1 expressing bacterial cells
maintained lower Na+ and higher K+ levels compared to vector alone, resulting in an
increase in the K+/Na+ ratio. Garciadeblas et al. (2007) also reported that CnSOS1 is
an excellent low-affinity K+ and Rb+ transporter and mediated a transient, extremely
rapid K+ and Rb+ influx in E. coli. These studies suggest that SOS1 gene could be used
to develop salt-tolerant crops.
1.2.3.2.4 Salt Overly Sensitive 4 (SOS4) gene
The SOS4 encodes pyridoxal (PL) kinase which is involved in the biosynthesis of
pyridoxal-5-phosphate (PLP), an active form of vitamin B. Besides being essential
cofactor for many cellular enzymes, PLP and its derivatives are also function as
ligands that regulate the activity of certain ion transporters in animal cells (Shi and
Zhu, 2002). The expression of SOS4 cDNAs complements an E. coli mutant defective
in pyridoxal kinase. Supplementation of pyridoxine but not pyridoxal in the growth
medium can partially rescue the sos4 defect in salt tolerance. SOS4 is expressed
ubiquitously in all plant tissues. As a result of alternative splicing, two transcripts are
derived from the SOS4 gene, the relative abundance of which is modulated by
development and environmental stresses. The sos4 mutant plants were hypersensitive
to Na+, Li+ and K+ but not to Cs+ and were not hypersensitive to general osmotic stress
25
Chapter 1
caused by mannitol. SOS4 seems to be involved in Na+ and K+ homeostasis in plants
as under NaCl stress sos4 mutant plants accumulate more Na+ and retain less K+
compared with the WT plants (Shi et al., 2002b). Therefore, SOS4 constitutes a novel
regulatory determinant of Na+ and K+ homeostasis in plants.
Shi and Zhu (2002) have discussed the possible role of SOS4 in ethylene and
auxin biosynthesis. The root growth of sos4 mutant plant is slower than that of the
WT. Microscopic observations revealed that sos4 mutant do not have root hairs in the
maturation zone. The sos4 mutation block the initiation of most root hairs, and impair
the tip growth of those that are initiated. The root hairless phenotype of sos4 mutants
was complemented by the WT SOS4 gene. SOS4 promoter-β-glucuronidase analysis
showed that SOS4 is expressed in the root hair and other hair-like structures.
Consistent with SOS4 function as a PL kinase, in vitro application of pyridoxine and
pyridoxamine, but not PL, partially rescued the root hair defect in sos4 mutant. 1-
Aminocyclopropane-1-carboxylic acid and 2,4-dichlorophenoxyacetic acid treatments
promoted root hair formation in both WT and sos4 mutant plants, indicating that
genetically SOS4 functions upstream of ethylene and auxin in root hair development.
1.2.3.2.5 Salt Overly Sensitive 5 (SOS5) gene
The sos5 mutant was isolated in a screen for Arabidopsis salt hypersensitive mutants
using the root bending assay. In response to salt stress, the root tips of sos5 mutant
plants swell and the root growth and elongation was arrested. The root tip of sos5 plant
shows certain phenotypic abnormalities such as thinner walls, reduced middle lamella
and abnormal cell expansion (Shi et al., 2003a). SOS5 also contains two alternatively
organized fasciclin-like domains and two putative AGP-like (arabinogalactan proteins-
like) domains. The presence of fasciclin-like domains, which are typically found in
26
Introduction
animal cell adhesion proteins, suggests a role in cell to cell adhesion in SOS5 protein.
The AGP-like domains are rich in Hyp residues for the addition of O-linked
arabinogalactan chains. SOS5 is predicted to contain a N-terminal signal peptide for its
plasma membrane localization and a C-terminal signal sequence for the addition of
GPI (glycosylphosphatidylinositol) lipid anchor. The N as well as C-terminal signal
sequences are expected to be cleaved after post-translational processing and thus the
mature SOS5 protein would contain two fasciclin-like domains, two AGP like
domains with attached carbohydrate chains and a GPI anchor to the C terminus (Shi et
al., 2003a). SOS5 has been suggested to function in cell-to-cell adhesion and
maintenance of cell wall integrity (Shi et al., 2003a) and architecture to sustain cell
expansion even under salt-stressed conditions (Mahajan et al., 2008).
The SOS5 transcript was detected in roots, leaves, stem, flowers and siliques,
with relatively higher abundance in leaves and flowers. The level of SOS5 transcript in
young seedlings was up-regulated slightly by ABA, cold and drought treatments.
SOS5 promoter-GUS (β-glucuronidase) analysis revealed strong GUS staining at the
root tip. In mature roots, strong GUS staining was detected in cortical cells as well as
vascular tissues (Shi et al., 2003a). Immunological characterization suggests that SOS5
probably is highly glycosylated and located mainly on the outer surface of the plasma
membrane.
1.2.3.2.6 The plasma membrane PM H+-ATPase
The PM H+-ATPase is an important plasma membrane bound protein. Basically, it is a
proton pump, its activity is to generate a proton gradient that gives rise
electrochemical gradient and pH difference across the membrane. This
electrochemical energy is the motive force for a large set of secondary transporters
27
Chapter 1
(like Na+/H+ antiporter) that move their ions against a concentration gradient
(Palmgren, 2001). The PM H+-ATPase involves in various physiological processes,
including those related to salinity stress tolerance, intracellular pH regulation, stomatal
opening and cell elongation (Arango et al., 2003). High salinity is one of the most
serious abiotic stress, which affects adversely on growth and productivity of the crops.
The PM H+-ATPase play a crucial role in ion-homeostasis under salinity stress by
regulating ion transporters across the PM to maintain a low Na+ concentration in
cytoplasm (Serrano, 1989; Niu et al., 1995). Several studies demonstrated that salt
stress enhances PM H+-ATPase activity in plants (Braun et al., 1986; Niu et al., 1993a,
b; Kerkeb et al., 2001; Sibole et al., 2005).
1.2.3.3 Synthesis of compatible solutes (osmolytes)
Under conditions of increased Na+ concentration, whether Na+ is compartmentalized
into the vacuole or excluded out of the cell to keep cytosolic Na+ at an optimal level,
the osmotic potential in the cytoplasm must be stabilized with that in the vacuole and
extracellular environments to ensure the maintenance of cell turgor and water uptake
for cell growth. This requires an increase in osmolytes in the cytosol. Osmolytes are
organic metabolites of low molecular weight known as compatible solutes and do not
interfere with normal biochemical reactions. The osmolytes such as glycine betaine,
fructans, trehalose, mannitol, sorbitol, ononitol, and pinnitol play prominent role as
osmoprotectants (Bohnert and Jensen, 1996; Ramanjulu and Bartels, 2002; Hasegawa
et al., 2000; Zhifang and Loescher, 2003). The primary function of compatible solutes
is to maintain lower water potential inside cells and thus generate the driving force for
water uptake (Carpenter et al., 1990). It has been also reported that compatible solutes
can also act as free-radical scavengers or chemical chaperones by directly stabilizing
28
Introduction
membranes and/or proteins (Akashi et al., 2001; Hare et al., 1998; Bohnert and Shen,
1999; McNeil et al., 1999; Diamant et al., 2001).
The synthesis of compatible osmolytes in plants under high salt stress could be
considered as a sacrifice of resources in exchange for plant survival. Furthermore,
genes for many osmolytes have been cloned and introduced into many plants (Agarwal
et al., 2012). Generally, this resulted in higher accumulation of osmoprotectants and
enhanced salt and drought tolerance. It is thus likely that compatible solute
biosynthesis is another important mechanism which enables plants to survive under
high salt conditions (Agarwal et al., 2012).
1.3 Rationale for studying SOS1 gene from the extreme halophyte Salicornia
brachiata and its overexpression in tobacco plant for salt tolerance
The world population is increasing rapidly and may reach 6 to 9.3 billion by the year
2050, whereas the crop production is decreasing rapidly because of the negative
impact of various environmental stresses; therefore, it is now very important to
develop stress tolerant varieties to cope with this upcoming problem of food security
(Mahajan et al., 2008). High salinity is one of the major abiotic stresses that adversely
affect crop productivity and quality (Chinnusamy et al., 2005). So it is need of the
hour to develop plants that can tolerate adverse conditions such as high salinity. The
success in getting salinity tolerant plant through conventional breeding has not been
very encouraging. Recent advances in the tools and techniques of molecular biology
have made it possible to study genetic structure, gene function, its regulation and
expression and finally culminating in transgenic generation. These strategies have
evolved as one of the most promising methods for improving salinity tolerance in
plants. Improved resistance to salinity, drought and extreme temperatures has been
29
Chapter 1
observed in transgenic plants that express/overexpress genes regulating osmolytes,
specific proteins, antioxidants, ion homeostasis, transcription factors and membrane
composition. Keeping the view of problem of salinity and drought, transgenic
technology holds a great potential of genetically engineered plants that are capable of
growing in soil of high salinity, drought and improving agricultural productivity.
Excess salt (NaCl) disturbs intracellular ion homeostasis in plants, leading to
membrane dysfunction, attenuation of metabolic activity, and secondary effects that
cause inhibition of growth and photosynthesis and, ultimately, cell death (Hasegawa et
al., 2000). In plant, the SOS signal transduction pathway is responsible for Na+ ion
homeostasis and salinity tolerance by maintaining favourable K+/Na+ ratios in the
cytoplasm through the action of the plasma membrane Na+/H+ antiporter SOS1, which
mediates Na+ extrusion out of the root cell and long-distance Na+ transport from roots
to shoots (Shi et al., 2000, 2002a; Zhu, 2002). Olias et al. (2009) demonstrate that
SlSOS1 antiporter is also critical for the partitioning of Na+ between plant organs.
AtSOS1 over-expression has been shown to markedly suppress the accumulation of
Na+ and enhanced salinity tolerance in Arabidopsis (Shi et al., 2003b). Similar results
were obtained when T. salsuginea and P. tenuiflora SOS1 gene were overexpressed in
Arabidopsis (Oh et al., 2009; Wang et al., 2011). Based on the studies described
above, SOS1 seems to be a key regulatory component of salt tolerance and therefore
can be considered a candidate to enhance crop salinity tolerance.
In the light of above facts and importance of genetic engineering for enhancing
salinity tolerance, we have cloned and characterised Salt Overly Sensitive 1 (SbSOS1)
gene from S. brachiata and overexpressed in tobacco plant for functional validation.
Accordingly, the following experiments were envisaged: (1) Cloning of full length
SbSOS1 gene from S. brachiata. (2) Expression analysis of SbSOS1 under salinity
30
Introduction
31
stress (transcript profiling). (3) Development of transgenic tobacco overexpressing
SbSOS1 gene. (4) Analysis of transgenic tobacco plants for salt tolerance and
functional validation of SbSOS1 gene.