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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.