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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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

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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.

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).

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Introduction 

Figure 1.1: Schematic diagram of salinity stress effects on plant. (Reproduced from Horie et al., 2012)

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Chapter 1

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

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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

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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.