1

1. INTRODUCTION

1.1 : Overview

High salinity, drought and extreme temperatures are among the most common

abiotic stresses for plants influencing growth and productivity (Boyer 1982). Soil

salinity affects plant growth and development by way of osmotic stress and injurious

effects of higher concentration of Na+ and Cl

- ions. Adaptive response to salinity is

multigenic in nature. During salinity stress a number of tolerance mechanism are

affected, such as various compatible solutes/osmolytes, polyamines, reactive oxygen

species, antioxidant defense mechanism and ion transport and compartmentalization.

Mechanisms of conferring salt tolerance vary with the plant species; however the basic

strategy works toward the maintenance of Na+ homeostasis in the cytosol (Blumwald

2000). Na+ homeostasis is maintained either by active exclusion through plasma

membrane Na+/H

+ antiporter (Shi et al 2000; 2003), or by sequestration of excess

sodium into the vacuoles via vacuolar Na+/H

+ antiporters. When grown in saline

environment, all plants will accumulate Na+ ions to some extent, except for some

halophytic species that are able to effectively maintain very low Na+ net influx (Taji et

al 2004; Zahran et al 2007; Munns and Tester 2008). The accumulation of Na+ inside

vacuoles is a strategy used by many plants to survive salt stress, an active vacuolar

antiporter utilizes the proton motive force generated by vacuolar ATPases and

pyrophosphatases to sequester excess Na+ into the vacuole, thereby reducing the toxic

effects of Na+ inside the cytosol and utilizing these ions for maintenance of turgor in

the vacuole for cell expansion and growth (Niu et al 1995; Blumwald et al 2000;

Munns and Tester 2008). 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

2

stress in some plant species (Maathuis and Amtmann 1999; Chauhan et al 2000;

Munns and Tester 2008). Other plant species tend to limit Na+

accumulation in shoots

by reduced transport from root to shoot, recirculation of Na+ out of the shoots and

storage in root or stem cell vacuoles (Maathuis and Amtmann 1999; Zörb et al 2004;

Munns and Tester 2008).

It has been reported that several isoforms 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. 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 IC clade can be further divided into two main groups

denoted as Class-I and Class-II (Pardo et al 2006). In Arabidopsis, members of the

Class-I category (AtNHX1-4) are 56–87% similar, whereas AtNHX5 and 6 (Class-II)

are 79% similar but only 21–23% similar to Class-I isoforms (Yokoi et al 2002). All

NHX proteins of Class-I, characterized to date, are localized in the vacuolar membrane

and form a separate clade within the IC group that is composed exclusively of plant

exchangers. By contrast, Class-II members are found in endosomal vesicles of plants

and homologous proteins with various endosomal localizations are also present in

animals and fungi (Pardo et al 2006). The plant vacuolar NHE clade is abundantly and

exclusively represented in plants. The absence of ATP-powered plasma membrane

sodium intracellular pumps in plants may be the reason for development of the

3

specialized clade of vacuolar NHE in plants, which act to store high concentrations of

salt and water in the vacuole. These NHE are critical determinants of salt tolerance and

osmoregulation in plants.

Over 800 million hectares of land throughout the world are affected by salt (FAO

2008; http:www.fao.org/ag/agl/agll/spush/). In addition to natural causes, such as salty

rainfalls near and around the coasts, contamination from parental rocks and oceanic

salts, cultivation practices have also exacerbated the growing concentration of salts in

rhizospheres (Mahajan and Tuteja 2005). Crop productivity is greatly affected by

various environmental stresses. High salt concentration in soil causes yield reduction

in wide variety of crops all over the world. 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.

Only few plants species are able to adapt and survive under these conditions. Among

these, Salicornia brachiata is one which thrives well on these soils. S. brachiata is

highly salt tolerant plant than most other genera of halophytes. It can grow optimally

in sea water and is also capable of growth in soil having salinity 3-4 times higher than

sea water. The unique feature of Salicornia to defy salt and its abundance in the area

4

makes it a naturally adapted higher plant model to study the molecular mechanism of

salt tolerance and also an important source of genes for abiotic stress tolerance (Jha et

al. 2009).

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. There is a need to develop plants that can

tolerate adverse conditions such as high salinity. The success in getting stress 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 and mutant generation. These strategies have evolved as one of the most

promising methods for improving stress tolerance in plants. Recent advances in

understanding crop abiotic stress resistance mechanisms and the introduction of

molecular biology techniques allow us to address these issues more efficiently than in

the past. Improved resistance to salinity, drought and extreme temperatures has been

observed in transgenic plants that express/overexpress genes regulating osmolytes,

specific proteins, antioxidants, ion homeostasis, transcription factors and membrane

composition.

With the burgeoning population and greater urbanization the arable land area is

decreasing gradually. Competition for fresh water between industry and farmers and

possible global environmental changes have necessitated the need for additional

strategies by which food, feed and fiber supply can be guaranteed. The vast unutilized

coastal areas could be tapped as one of the alternatives. In these areas fresh water is a

precious commodity. Moreover, the increased productivity achieved in irrigated

5

agriculture has also contributed towards salinization following prolonged irrigation.

These considerations have evolved strong interest in studying plant abiotic stress

response and understanding the meaning of stress tolerance as a biological

phenomenon.

The naturally adapted salt tolerant plants (halophytes) like Salicornia brachiata

may play an important role in isolating salt-responsive gene(s) and subsequently

engineering salt tolerance in glycophytes. 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 which could withstand higher salinity.

Generally, it is known that halophytes imply Na+ compartmentalization in the vacuole

that is channelized by the membrane targeting proteins. Therefore, cloning,

characterization and finally genetic transformation of Na+/H

+ antiporter (NHX1) gene

from Salicornia brachiata may be utilized to develop salt tolerant plants for

sustainable agriculture in salt affected areas.

1.2: Salt stress and its effect on plant growth and productivity

The environmental stresses caused by non-living components are cumulatively

known as abiotic stress (physicochemical stress) like light (high intensity and low

intensity), temperature [high and low (chilling or freezing)], water [deficit (drought)

and excess (flooding)], radiation (IR, visible, UV and ionizing (X-ray and γ-ray),

chemicals (salts, ions, gases, herbicides, heavy metals) and mechanical factors (wind,

pressure). Abiotic stress negatively affects crop growth and productivity around the

world. Abiotic stress is the primary cause of crop loss worldwide, reducing average

yields of major crop plants by more than 50% (Vinocur and Altman, 2005).

6

Sodium and Potassium, constituting the sixth and seventh most abundant

elements on earth play essential roles for all living organisms. In plants, physiological

studies and thermodynamic considerations have indicated the presence of K+/H+

antiporter systems at the plasma membrane, tonoplast, mitochondrial and chloroplast

membranes and intracellular membranes of the secretory pathway (Walker et al 1996;

Song et al 2004; Sze et al 2004). K+/H

+ antiporters are suggested to be responsible for

the active accumulation of K+ inside vacuoles, essential to maintain turgor and drive

cell expansion (Leigh and Wyn Jones 1984; Walker et al 1996). At the same time,

although high cytoplasmic Na+

concentrations are toxic, plants activate high affinity

Na+

uptake mechanisms in conditions of K+

deficiency, indicating that the more

ubiquitous Na+

can to some extend functionally replace K+

(Garciadeblás et al 2003;

Horie et al 2007) at least as osmoticum inside the vacuole. Clearly in conditions of

high salinity this becomes evident, as an important mechanism to survive salt stress

relies on the accumulation of excess cytoplasmic Na+ in vacuoles, reducing the amount

in the cytoplasm and providing osmotic pressure (Niu et al 1995, Blumwald et al

2000).

Salt stress causes multifarious adverse effects in plants. Salinity immensely

affects plant growth and development and is a major constraint for crop production.

Plants need essential mineral nutrients for proper growth and development. However,

excessive soluble salts in the soil are harmful to most plants. Salinity is generally

defined as the presence of excessive amount of soluble salt 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 with ECe

more than 4 dSm-1

equivalent to 40 mM NaCl, ESP less than 15 % and pH below 8.5

7

(Waisel 1972; Abrol 1986; Szabolcs 1994). 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 in terms of molarity Na+ is about 500 mM. Salinity at a particular area is

influenced by microclimate of that area like amount of evaporation (leading to increase

in salt concentration), or the amount of precipitation (leading to decrease in salt

concentration). In India, the total cultivable land area is about 183.95 m ha of which

8.6 m ha is salt affected (FAO, 2005). Gradually the problem of soil salinization is

getting more severe. Soil salinity is steadily increasing mostly due to repetitive

seawater invasion, erroneous irrigation (greater exploitation of ground water for

agricultural purpose), degradation of native saline parent rock and the ingression of

salinity in the costal and canal areas (Ashraf, 1994). Globally, approximately 22% of

the agricultural land is saline (FAO, 2005).

Adverse effects of salinity on plant growth are due to (1) Disruption of ionic

equilibrium: Influx of Na+ dissipates the membrane potential and facilitates the uptake

of Cl- down the chemical gradient, reduction in growth, inhibition of cell division and

expansion, (2) Sodium toxicity: Na+ is toxic to cell metabolism and has deleterious

effect on the functioning of some of the enzymes (Niu et al 1995). High Na+ levels also

lead to reduction in photosynthesis and production of reactive oxygen species.

Different plants employ different mechanisms to minimize the damage from Na+ e.g.

minimize initial entry, maximize efflux, minimize loading to the xylem, maximize

recirculation out of the shoot in phloem, intercellular compartmentalization and secret

salt from the leaf surface (Tester and Davenport 2003).

The salt stress is complex trait and causes a number of detrimental effects.

Among these ionic and water constraints constitute the most important. The water

constraint even called osmotic pressure is characterised by difficulties to absorb water.

8

Salt and drought stresses are quantitative in nature and are regulated by polygenes. The

osmotic stress induces water deficiency, while the ionic resultant induces on the one

hand ionic toxicity due to Cl–

and Na+ accumulation and on the other hand indirect

toxicity due to the difficulty of essential nutrient elements uptake. All these constraints

are perceived and send to the genome which activates appropriate mechanisms to re-

establish water transport, limit Na+ and Cl

– uptake or lowers their concentration in

cytoplasm and allowing the absorption of ions indispensable for growth. Tolerance

depends on a range of physiological, biochemical and molecular adaptations activated

by the genome to survive in salt effected soil. Plants response to salt stress involves

numerous processes that function in coordination to alleviate both cellular

hyperosmolarity and ion disequilibrium.

Salinity causes suppression of growth in all plants, but their tolerance levels and

rate of growth reduction at higher concentration differ widely among different plant

species. Generally, salt stress reduces water potential, causes ion imbalance or

disturbances in ion homeostasis and also causes ion toxicity, which inhibits enzymatic

functions of key biological processes (Zhang and Blumwald 2001; Blumwald et al

2004). Along with these primary effects, secondary stresses, such as oxidative damage,

occur because high concentrations of ions disrupt cellular homeostasis (Dat et al

2000).

The injurious effects of high salinity on plants can be observed at the whole plant

level, such as significant reduction in plant growth, reduction in productivity and even

the death of plants. The accumulation of Na+ in leaf tissues usually results in the

damage of old leaves, which decreases the life time of individual leaf, thus reducing

the net productivity and crop yield (Munns 1993; 2002). Increased NaCl levels result

in drastic decrease in root length, shoot length, leaf biomass and an increase in

9

root/shoot ratio. Salinity increases epidermal thickness, mesophyll thickness, palisade

cell length, palisade diameter and spongy cell diameter in leaves of bean, cotton and

Atriplex (Longstreth and Nobel 1979). Salinity causes detrimental effect on

ultrastructure of grana and thylakoids of chloroplasts (Keiper et al 1998; Khavarinejad

and Mostofi 1998; Parida et al 2003) and reduces plant leaf area as well as stomatal

density (Romero-Aranda et al 2001).

Nitrate uptake and nitrate reductase activity in leaves decreases in many plants

under salt stress (Flores et al 2000; Silveira et al 2001). The reduction of NO3− to NO2

−

catalyzed by nitrate reductase is considered to be the rate-limiting step in nitrogen

assimilation (Srivastava, 1990; Lea 1997). The primary cause of the reduction in

nitrate reductase activity in the leaves is the presence of a high concentration of Cl−

and Na+, which leads to decrease in NO3

− uptake and accordingly a lower NO3

−

concentration in the leaves. This may lead to severe consequences for whole plant

nitrate assimilation. Therefore, a decrease in nitrate reductase activity and reduced

nitrate level under high salinity condition may be accountable for reduction in plant

growth and biomass production. Increased accumulation of Na+ is generally coupled

with reduced Ca++

and Mg++

uptake (Delfine et al 1998) and sometimes with decline in

carbon assimilation (Parida et al 2004a).

Plant growth, such as biomass production, is a key measure of net

photosynthesis. Salt stress dramatically reduces the rate of photosynthesis (Kawasaki

et al., 2001), which in turn, retards plant growth. Net photosynthetic rate (PN)

declines with increasing salinity (Li et al 2008). Some studies have shown that salt

stress inhibits PSII activity (Bongi and Loreto 1989; Mishra et al 1991; Masojidek and

Hall 1992; Belkhodja et al 1994; Everard et al 1994). Salt stress also inhibits the repair

of PSII via suppression of D1 (quinone-binding protein) protein synthesis at the

10

transcriptional and translational levels (Allakhverdiev et al 2002). Salinity decreases

CO2 assimilation into carbohydrate through reductions in leaf area (Papp et al 1983;

Munns et al 2000), stomatal conductance (Brugnoli and Lauteri 1991; Agastian et al

2000; Ouerghi et al 2000; Parida et al 2003), mesophyll conductance (Delfine et al

1998) and the efficiency of photosynthetic enzymes (Seemann and Critchley 1985;

Yeo et al 1985; Seemann and Sharkey 1986; Brugnoli and Bjorkman 1992; Reddy et al

1992). The harmful effects of salinity on plant growth are usually associated with low

osmotic potential of soil solution and toxic levels of sodium ion, which cause

unfavourable multiple effects on plant metabolism, growth and development at

molecular, biochemical and physiological levels (Gorham et al 1985; Winicov 1998;

Munns 2002; Tester and Davenport 2003).

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,

either by uptake of soil solutes or by synthesis of metabolically compatible solutes.

The synthesis of compatible osmolytes in plants under high salt stress could be

considered as a sacrifice of resources in exchange for plant survival. A decrease in

plant fertility involving aborting ovules and pollen, shifts resources from reproductive

activities into metabolic reactions and increases stress tolerance (Davidonis et al 2000;

Asch and Wopereis 2001). One of the important biochemical mechanisms by which

mangroves counter the high osmolarity of salt is accumulation of compatible solutes

(Parida et al 2004c). Furthermore, the success in engineering of metabolic pathways

for compatible solutes such as glycine betaine, sorbitol, mannitol, trehalose and proline

11

have been reported in transgenic plants which display increased tolerance to high

salinity, drought stress and cold stress (Chen and Murata 2002).

1.3: Salt tolerance mechanisms and role of the Na+/H

+ antiporter

genes

In recent past wide range of research activities have been initiated to identify the

genes that are regulated under salt stress. Salt shock experiments with several

unicellular model organisms as well as with higher plants have been taken up to

understand the molecular responses and to develop plants with enhanced salt tolerance.

Cellular and molecular response of plants to salinity has been reviewed by Hasegawa

et al (2000). Further, Vinocor and Altman (2005) have recently reviewed the progress

in engineering the plant tolerance to abiotic stresses, particularly salinity and drought,

and have emphasized the need for the detailed molecular analysis underlying salt

tolerance in salt-tolerant model species. Plant responses to salinity stress have been

discussed with emphasis on molecular mechanisms of signal transduction and on the

physiological consequences of altered gene expression that affects biochemical

reactions downstream to stress sensing. Results obtained with model unicellular

organisms such as bacteria, yeast and algae have been used to draw comparisons and

to provide an understanding of higher plant salt tolerance (Gustin et al 1998; Serrano et

al 1999a; 1999b; Bohnert et al 2001). Dunaliella has been extensively studied to

understand the molecular mechanisms of adaptation to higher salt concentration. A

large number of expressed sequence tag (EST) have been recorded for the halotolerant

algae Dunaliella salina (Alkayal et al 2010; Kim et al 2010; Mishra and Jha 2011).

Though researches on model unicellular organisms have been highly applicable to, and

will continue to provide, greater insight into plant salt tolerance, it is obvious that cell

12

differentiation and integrative hierarchical functioning among cells, tissues, and organs

make salt tolerance in higher plants difficult and complex. Atriplex and

Mesembryanthemum crystallinum are two higher plants in halophyte category, which

have been used to study the response of halophytes to stress and adaptive responses.

Salt tolerance mechanisms in mangroves have recently been reviewed by Parida and

Jha (2010).

The advances in understanding the effectiveness of stress responses are

increasingly based on transgenic plant and mutant analysis, in particular the analysis of

Arabidopsis mutants defective in elements of stress signal transduction pathways

(Bohnert et al 2001; Rus et al 2001; Shi et al 2002; Rus et al 2004). Global gene

expression profiles monitored under salt stress conditions in Synechocystis,

Sachharomyces cerevisiae, Dunaliella Salina, Mesembryanthemum, Arabidopsis, etc.

have been analysed. More than 4,00,000 T-DNA tagged lines of A. thaliana have been

generated, and lines with altered salt stress responses have been obtained. Among the

cDNA libraries that have been established for many plant species, very few have been

generated with tissues from stressed plants.

Salinity tolerance depends on a range of adaptations, including ion

compartmentation, osmoregulation, selective transport and uptake of ions,

maintenance of a balance between the supply of ions to the shoot, and capacity to

accommodate the salt influx. The tolerance to a high saline environment is also tightly

linked to the regulation of gene expression. Salt accumulators accumulate high

concentration of salts in their cells and tissues and avoid salt damage by efficient

sequestering of ions to the vacuoles in the leaf, translocation outside the leaf, possible

cuticular transpiration and efficient leaf turnover to salt shedding (Tomlinson 1986;

Aziz and Khan 2001b). Species of Lumnitzera and Excoecaria accumulate salts in leaf

13

vacuoles and become succulent. Salt concentrations in the sap may also be reduced by

transferring the salts into senescent leaves or by storing them in the bark or roots

(Tomascik et al 1997; Perry et al 2008). The most direct way to maintain low

cytoplasmic Na+ is to sequester it in the vacuoles within each plant cell. The pumping

of Na+ into the vacuole is catalyzed by a vacuolar Na

+/ H

+ antiporter, the difference in

H+ being initially established by H

+ pumping ATPase and pyrophosphates proteins

(Blumbald et al 2000). Na+/ H

+ antiporter activity can increase upon addition of Na

+

and this induction was found to be much greater in the salt- tolerant species, Plantago

maritima, then in the salt sensitive species, P. media (Staal et al 1991). Consistently,

salinity did not induce tonoplast Na+/ H

+ antiport activity in salt-sensitive rice (Fukuda

et al 1998). This induction reflected increase transcript levels of some members of the

Arabidopsis AtNHX gene family which encode vacuolar Na+/ H

+ antiporters (Yokoi et

al 2002). Salinization also induces activity of both vacuolar primary H+ ion pumps,

although this appears to occur in both Na+ -tolerant and Na

+- sensitive species

(Hasegawa et al 2000). The central importance of vacuolar sequestration has recently

been underlined by experiments in which constitutive over expression of vacuolar

transporters has greatly increased salinity tolerance of a range of species. Over

expression of an Arabidopsis vacuolar Na+/ H

+ antiporters (NHX 1) increase salinity

tolerance of Arabidopsis (Apse et al 1999), tomato (Zhang and Blumwald 2001) and

Brassica napus (Zhang et al 2001), and over expression of a native vacuolar H+

translocating pyrophosphate gene (AVP1 ) increases salinity tolerance of Arabidopsis

(Gaxiola et al 2001). The apparent K+ ion transporting activity of the Arabidopsis Na

+/

H+ antiporters (Zhang and Blumwald 2001; Venema et al 2002) does not appear to

cause perturbations in cytosolic K+ ion homeostasis.

14

Plants growing in a particular habitat are often well adapted to the environmental

stress conditions at that place. Halophytes are important plant growing on or surviving

in saline conditions, such as marine estuaries and salt marshes. They respond to salt

stress at three different levels; cellular, tissue and the whole plant level. The advances

in physiology, genetics, and molecular biology have greatly improved our

understanding of plant responses to stresses. Understanding of the molecular processes

regulating these metabolic adaptations will facilitate engineering of salt stress

tolerance. When a plant is subjected to salt stress, a number of genes are turned on,

resulting in increased levels of several metabolites and proteins, some of which may be

responsible for conferring a certain degree of protection to these stresses. Realizing the

importance of halophyte for elucidating the salt tolerance mechanism, a number of

EST data bases have been developed for halophytes like Sueda salsa (Zhang et al

2001a), Tamarix androssowii (Wang et al 2006), Thullungella halophilla (Wang et al

2004a), Mesembryanthum crystallinum (Kore-eda et al., 2004) Avicina marina (Mehta

et al 2005) and Salicornia brachiata (Jha et al 2009). 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. CSMCRI, Bhavnagar is working on Salicornia brachiata, an extreme

halophyte growing commonly in the coastal areas in India. The differential EST

database from Salicornia generated 930 sequences, out of which 789 ESTs showed

matching with different genes in NCBI database. 4.8% ESTs belonged to stress-

tolerant gene category and approximately 29% ESTs showed no homology with

known functional gene sequences, thus classified as unknown or hypothetical (Jha et al

2009). In addition, several important functional and regulator genes from Salicornia

have been isolated and being utilised to develop transgenic crop plants for salt

tolerance (Gupta et al 2010; Jha et al 2011).

15

Genome sequencing projects have now shown that plants contain a very large

number of putative Cation/Proton antiporters, the function of which is only beginning

to be studied. The intracellular NHX transporters constitute the first Cation/Proton

exchanger family studied in plants. The founding member, AtNHX1, was identified as

an important salt tolerance determinant and suggested to catalyze Na+

accumulation in

vacuoles. According to the classification made by Saier et al (1999) (http://

www.tcdb.org/index.php), Cation/Proton antiporters can be grouped into the CPA1

and CPA2 families. The CPA1 family has evolved from ancestral NhaP genes in

prokaryotes (Brett et al 2005). The Arabidopsis plasma membrane Na+/H

+ antiporter

AtSOS1 gene is related to the NhaP genes, and representative SOS or NhaP like

sequences can be found in all phylae of the plant kingdom (SOS-Like). The most

extensively studied family of the CPA1 proteins are the plasma membrane NHE

antiporters present only in vertebrates (PM-NHE). The more recently discovered

intracellular NHE/NHX sequences that can be found in plants, animals and fungi, have

evolved separately from the plasma membrane NHE sequences, and constitute a very

diverse group (IC-NHE/NHX). This family was subdivided into Class-I and Class-II

sequences (Pardo et al 2006), that share only about 20–25% identity, as well as the

NHE8-like family found in animals only (Brett et al 2005). Class-I sequences are very

divergent from other IC-NHE/NHX sequences and have so far been identified in

monocotyledonous and dicotyledonous angiosperms as well as gymnosperms.

The genes induce by salt stress are classified into two groups, the functional and

regulator genes. Functional genes generally modify the single metabolite; these include

osmolytes, transporters/ channel protein, antioxidative enzymes, lipid biosynthesis

genes, polyamines etc. The second class of genes consists of regulatory protein like

16

Figure 1.1: Showing phylogenetic tree of different proteins of monovalent cation

proton antiporter (CPA 1) family of Plant (adapted from Rodríguez-Rosales et al

2009).

bZIP, DREB, MYC/MYB and NAC, which control the expression of many salt stress

tolerant genes. Na+ homeostasis is maintained either by active exclusion through

plasma membrane Na+/H

+ antiporter (Shi et al 2003), or by sequestration of excess

17

sodium into the vacuoles via vacuolar Na+/H

+ antiporters. 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 Arabidopsis thaliana AtNHX1 conferred 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 (Rajagopala et al 2007). Na+/H

+

antiporter have also been isolated from different halophytes such as

Mesembryanthemum crystallinum (Chauhan et al 2000), Atriplex gmelini (Hamada et

al 2001), Suaeda salsa (Ma et al 2004), Beta vulgaris (Xia et al 2002) and Salicornia

europia (Zhou et al 2008). The vacuole is a major salt accumulating cabin in the cell; it

basically compartmentalizes Na+ and Cl

- into its vicinity and thus maintains ion

homeostasis. Na+/H

+ antiporter plays an important role in plant salt tolerance, it

extrudes Na+ from cell energized by the proton gradient generated by the plasma

membrane H+-ATPase and/or compartmentalizes Na

+ in vacuole energized by the

proton gradient generated by the vacuolar membrane H+-ATPase and H

+-Ppiase.

18

Figure 1.2: Schematic drawing showing the activity of Na+/H

+ at plasmemembrane

and tonoplast. Na+

influx occurs by passive (nonselective cation channel(s) (NSCC),

HKT1 transport system. The Na+ efflux is active, H

+ driven Na

+ antiporter SOS1

extrudes Na+

outside of cell. Na+ - influx in tonoplast is established by H

+ driven Na

+

antiporter NHX. For the efficient activity proton gradient by the (V-type) H+-ATPase

and pyrophosphatase is maintained.

Regulation of K+ uptake and/or prevention of Na

+ entry, efflux of Na

+ from the

cell, and utilization of Na+ for osmotic adjustment are strategies commonly used by

plants to maintain desirable K+/Na

+ ratios in the cytosol. Osmotic homeostasis is

established either by Na+

compartmentation into the vacuole or by biosynthesis and

accumulation of compatible solutes. A high K+/Na

+ ratio in the cytosol is essential for

normal cellular function of plants. Na+competes with K

+ uptake through Na

+–K

+ co-

transporters, and may also block the K+-specific transporters of root cells under salinity

(Zhu 2003). This results in toxic levels of sodium as well as insufficient K+

K

+

Plasma Membra

ne

polyols proline betaine

Na

+

Cl-

Tonoplast

OH-*-scavenging

pero

x

cp

mt

Na+/H

+

K+

H+

H+

Na+

pH 5.5

pH 7.5 pH 5.5

-120 to -200

mV

+20 to +50 mV H

+

H+ PP

i

H+

AT

P

K+(Na+)

H+

Cl-

H+ Cl

-

AT

P

Na+

H+

Na+

Cl-

19

concentration for enzymatic reactions and osmotic adjustment. Under salinity, sodium

gains entry into root cell cytosol through cation channels or transporters (selective and

nonselective) or into the root xylem stream via an apoplastic pathway depending on

the plant species (Chinnusamy et al 2005). Silica deposition and polymerization of

silicates in the endodermis and rhizodermis block Na+ influx through the apoplastic

pathway in the root (Yeo et al 1999). Restriction of sodium influx either into the root

cells or into the xylem stream is one way of maintaining the optimum cytosolic K+/Na

+

ratio of plants under high salinity. In saline conditions, cellular potassium level can be

maintained by activity or expression of potassium-specific transporters. In

Mesembryanthemum crystallinum L., high affinity K+ transporter-K

+ uptake genes are

up-regulated under NaCl stress (Su et al 2001; 2002). Sodium efflux from root cells

prevents accumulation of toxic levels of Na+ in the cytosol and transport of Na

+ to the

shoot. Molecular genetic analysis in Arabidopsis have led to the identification of a

plasma membrane Na+/H

+ antiporter, SOS1 (Salt Overly Sensitive 1), which plays a

crucial role in sodium extrusion from root epidermal cells under salinity (Chinnusamy

et al 2005). Sodium efflux by SOS1 is also vital for salt tolerance of meristem cells

such as growing root-tips and shoot apex as these cells do not have large vacuoles for

sodium compartmentation (Shi et al 2002). The expression of SOS1 is ubiquitous, but

stronger in epidermal cells surrounding the root-tip, as well as parenchyma cells

bordering the xylem. Thus, SOS1 functions as a Na+/H

+ antiporter on the plasma

membrane and plays a crucial role in sodium efflux from root cells and the long

distance Na+ transport from root to shoot (Shi et al 2002). Sodium efflux through

SOS1 under salinity is regulated by SOS3–SOS2 kinase complex (Chinnusamy et al

2005). Vacuolar sequestration of Na+ is an important and cost effective strategy for

osmotic adjustment that also reduces the Na+

concentration in the cytosol. Na+

20

sequestration into the vacuole depends on expression and activity of Na+/H

+ antiporters

as well as V-type H+- ATPase and H

+- PPase. These phosphatases generate the

necessary proton gradient required for activity of Na+/H

+antiporters. Salt accumulation

in mangroves occurs with the sequestration of Na+ and Cl

- into the vacuoles of the

hypodermal storage tissue of the leaves (Werner and Stelzer 1990; Aziz and Khan

2001a; Kura-Hotta et al 2001; Mimura et al 2003). Cram et al (2002) reported two

subsequent phases of salt accumulation in leaves of Bruguiera cylindrica, Avicennia

rumphiana and A. marina. The first phase is the rapid increase in leaf salt

concentration, as it grows from bud to maturity followed by a slower but continuous

change in salt content via changes in ion concentration and/ or in increased leaf

thickness. Compartmentalizing NaCl into the vacuole is likely to depend on Na+/H

+

antiporter systems (Garbarino and DuPont 1988; Fu et al 2005), H+-coupled Cl

-

antiport (Schumaker and Sze 1987) or ion channels (Pantoja et al 1989; Maathuis and

Prins 1990). Ion compartmentation in the vacuole would limit excessive salt

accumulation in the symplast, thus protecting salt-sensitive enzymes in the cytoplasm

and chloroplasts. Hence, the ability to maintain lower Na+ and Cl

- in the symplast may

be an underlying determinant of the tolerance (Li et al 2008).

The conventional breeding strategies have been useful in some cases for the

genetic improvement of crop plants. However, the integration of genomic portions

often brings undesirable agronomic characteristics from the donor parents. Therefore,

the development of genetically engineered plants by the overexpression of selected

genes is better choice for avoiding undesirable traits as well as in fastening the

breeding of ‘‘improved’’ plants. Genetic engineering would be a faster way to insert a

particular gene than by conventional or molecular breeding. Various transgenic

technologies like Agrobacterium tumefaciens mediated, particle bombardment, PEG

21

mediated and electroporation have been used commonly to transfer the genes in plants.

By the easy availability of the super virulent strains and also very economic the

Agrobacterium now remains the best choice for the transfer of foreign gene (s) in host

plant. Nevertheless, the task of generating transgenic cultivars is not only limited to the

success in the transformation process, but also proper incorporation of the stress

tolerance. Evaluation of the transgenic plants under stress conditions, and

understanding the physiological effect of the inserted genes at the whole plant level

remain as major challenges to overcome.

Keeping in view the above facts, it is envisaged to isolate and clone Na+/H

+

antiporter (NHX1) gene from Salicornia brachiata and its detailed transcript profiling

under salt stress. Finally, functional analysis of the cloned gene will be carried out

using transgenic approach.