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, H 2 O 2 , hydroxyl radical and singlet oxygen. Photosynthetic efficiency of plants is severely damaged through a combination of superoxide and H 2 O 2 -mediated oxidation (Herna´ndez et al., 1995). Plants adapt to environmental 1
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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
1
Chapter 1
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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Chapter 1
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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Chapter 1
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
7
Chapter 1
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.
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).
13
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
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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