4. discussion 4.1: na /h antiporter (sbnhx1) gene from...
TRANSCRIPT
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4. DISCUSSION
4.1: Na+/H
+ Antiporter (SbNHX1) gene from Salicornia brachiata
In plants, the uptake and translocation of cations from the soil play an essential
role in plant nutrition, signal transduction, growth, and development. Potassium (K+)
and sodium (Na+) are important as K
+ is an essential macronutrient and the most
abundant inorganic cation in plant cells, whereas Na+ toxicity is a principal component
of the deleterious effects associated with salinity stress. The genome of Arabidopsis
thaliana appears to encode 984 membrane transport proteins, 67% of which are
secondary active transporters (Nagata et al 2008). The plant membrane transporters are
multigene families, whose members often exhibit overlapping expression patterns and
a high degree of sequence homology. The highest sequence homology among NHXs
occurs in the N-terminal part that forms the membrane pore, whereas C-terminal
domains are more dissimilar. In mammals, ameloride competitively inhibits Na+/H
+
exchange with K+
of 1-100 M depending on the cell type restricted manner
(Counillon and Pouyssegur 2000). SbNHX1 shows the presence of this motif which is
highly conserved in yeast and plant NHX genes (Gaxiola et al 1999; Yokoi et al 2002).
NHX proteins may therefore readily be identified by the presence of the following
consensus sequence for the ameloride (sodium) binding site: FFXXLLPPI, where X
may be any amino acid. The tonoplast NHX members constitute a very diverse group.
This family is divided into Class-I and Class-II groups (Pardo et al 2006) that share
only 20-25% identity. The SbNHX1 gene showed higher similarity with Class-I type
NHX genes of halophytes. The sequences of Class-I members are very divergent from
other IC-NHE/NHX sequences and have been identified in monocotyledonous and
dicotyledonous angiosperms, gymnosperms as well as the moss Physcomitrella patens
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(Rodríguez-Rosales et al 2009). The Class-I type of NHX isoforms have shown
vacuolar membrane localization except in one case where the AtNHX1 antibody
showed the cross reactivity with plasma membrane and not tonoplast in Thellungiella
(Vera-Estrella et al 2005), which appears to be a unique feature of this Class.
The cDNA of Na+/H
+antiporter gene from Salicornia brachiata named SbNHX1
(accession number: EU448383) is 2,110 bp, consisting of a 3' – noncoding stretch of
427 bp. The open reading frame of SbNHX1 gene is 1,683 bp, encoding a polypeptide
of 560 amino acid residues with an estimated molecular mass 62.44 kDa and
isoelectric point 6.83. Amino acids blast showed the presence of a conserved NHE
(Na+/H
+ exchanger) domain. Prediction of topology of membrane spanning domains
revealed the presence of 11 strong transmembrane domains in SbNHX1, similar to
other halophyte plants like Salicornia europaea, Suaeda japonica, and
Mesembryanthemum crystallinum checked by TMpred analysis. Whereas, Salicornia
bigelovii, Kalidium foliate, Atriplex gmelini and Chenopodium glaucum have 12
transmembrane domains. The variation in the number of transmembrane domain is
intriguing and it seems plausible that these regions may be pertinent to antiporter
function. It has been suggested that glycosylation plays an important role in the proper
biosynthetic processing of transporter proteins in yeast and ScNHX1 is reported to be a
glycoprotein (Wells and Rao 2001). The putative glycosylation sites seem to be well
conserved in SbNHX1. This is the first report so far to demonstrate glycosylation of
plant NHX. In addition to glycosylation sites, several putative phosphorylation,
myristolyation sites and leucine zipper pattern are also predicted in SbNHX1.
None of the biochemical approaches has lead to the identification of the proteins
of the NHX antiporter family responsible for antiport activity. It is now clear that the
situation is much more complex than originally anticipated, with a total of at least 44
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sequences with homology to Na+/H
+ or K
+/H
+ antiporters identified in the Arabidopsis
genome, some of which are expressed in tonoplast, plasma membrane or internal
membranes of the endosomal pathway (Sze et al 2004). Sequence homology of
AtNHX1 with amiloride sensitive animal NHE antiporters suggests that this is the
protein responsible for the amiloride sensitive and salt stress induced specific Na+/ H
+
antiport found in tonoplast vesicles (Apse et al 1999). The activity of AtNHX1 was
assayed in vacuolar membranes obtained from transgenic Arabidopsis overexpressing
the protein (Apse et al 1999). Na+/H
+ antiport activity could be measured in the
transgenic plants, showing a Km of 7 mM for Na+ whilst activity in wild type plants
was very low. Disruption of AtNHX1 resulted in an even lower Na+/H
+ exchange
activity (Apse et al 2003). Although at first suggested to be specific for Na+, later
studies have shown that AtNHX1 expressed in plants also catalyzes K+/H
+ antiport,
albeit with lower affinity (Apse et al 1999; Zhang and Blumwald 200; Apse et al
2003). The fact that null mutants in AtNHX1 or lines overexpressing the gene have
substantially altered total antiporter activity, indicate that AtNHX1 is the major
contributor to vacuolar Na+/H
+ or K
+/H
+ antiporter activity, in spite of the presence of
5 more NHX isoforms and other proteins of the CPA1 and CPA2 family in
Arabidopsis. To be able to evaluate the structure-function relationships of the plant
NHX antiporters in more detail, avoiding interference with other plant antiporters, a
more convenient system is provided by heterologous expression in yeast. Darley et al
(2000) showed that AtNHX1 activity could be measured in vacuolar membrane
vesicles obtained from a Saccharomyces cerevisiae yeast strain in which the only
endogenous NHX isoform ScNHX1 gene was disrupted. The activity of the plant
enzyme appeared to be slightly higher than that of the endogenous yeast protein, with
Km values of 11 and 16 mM for Na+ respectively, whilst no antiport activity could be
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detected in vesicles obtained from the strain not expressing yeast or plant antiporter.
The antiport appeared also electroneutral as judged from experiments with the
membrane potential probe Oxonol V, and was shown to be sensitive to amiloride.
Using a similar yeast strain, Yamaguchi et al (2003) found that K+/H
+ antiport activity
was about two times higher than Na+/H
+ antiport activity in vacuolar vesicles obtained
from a yeast strain overexpressing the AtNHX1 protein, whilst Km values for K+ and
Na+ where 12 and 24 mM respectively. The discrepancies between specificity
measurements in plant or yeast could be due to plant specific regulatory mechanisms
not present in the heterologous system. In support of this observation it was found that,
removal of the C-terminal domain increases the Na+/H
+ antiport activity, whilst
binding of the AtCaM15 protein has the opposite effect (Yamaguchi et al 2003; 2005).
It was proposed that in plants, in normal conditions AtNHX1 functions in K+
accumulation, but that salt stress would activate the Na+/H
+ exchange mode, releasing
interacting partners like CaM15 from the C-terminal domain. Overexpression of
AtNHX1 would also induce mainly Na+/H
+ antiporter mode due to lack of interacting
partners. In yeast, the enzyme would be present in the unactivated K+/H
+ antiporter
mode. However, differences in expression levels for the mutant enzymes might also
have affected the results (Yamaguch et al 2003). More importantly, it was shown that
the main contributor to Na+/H
+ and K
+/H
+ antiport activity in the yeast vacuolar
vesicles is the Vnx1 protein, a protein with homology to Ca2+/H
+ and Ca2
+/Na
+
antiporters (Cagnac et al 2007). The earlier reports describing AtNHX1 activity in
yeast should thus be reconsidered as they have been affected by this major background
activity. Finally, ScNHX1 was shown to be involved in protein targeting and
prevacuolar/vacuolar biogenesis (Bowers et al 2000; Brett et al 2005) which
complicates the obtention of comparable vacuolar membrane preparations from wild
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type and ScNHX1 null mutants. For these reasons, the use of membrane vesicles or
intact vacuoles in which many unidentified ion transporters are still functional is not
ideal for structure function studies on the plant NHX antiporters. In this respect,
heterologous expression in yeast also facilitates protein purification using suitable
affinity tags. To avoid interference with other ion transporters such purified protein
can be reconstituted in artificial liposomes. Finally, the encapsulation of impermeant
pH indicator dyes inside the proteoliposomes during reconstitution, permits real
quantitative measurement of pH within the range of responsiveness of the dye. Using
this approach it was shown that the AtNHX1 protein catalyzes both Na+/H
+ and K
+/H
+
antiport with similar affinity of about 40 mM (Venema et al 2002). This antiport could
be inhibited by the amiloride analogs EIPA and benzamil.
The protein sequence of 85-LFFIYLLPPI-94 in SbNHX1 is highly conserved. In
mammals, this region was identified as the binding site of amiloride, which inhibits the
eukaryotic Na+/H
+ exchanger. These features confirm that the SbNHX1 is a
vacuolartype Na+/H
+ antiporter. The analysis of SbNHX1 showed four potential N-
glycosylation sites, nine N-myristoylation sites predicted by PPsearch. Further, there
are fifteen protein kinase phosphorylation sites of casein kinase II (8 sites) and Protein
kinase C (7 sites) and a Leucine zipper pattern were also found in the SbNHX1. The
predicted secondary structure using the PSIPRED protein structure prediction server
and reveals a structure of 24 coils, 22 alpha-helices and 5 beta-strands. To investigate
the molecular evolution and phylogenetic relationships among Na+/H
+ Antiporters in
plants, Na+/H
+ antiporter protein sequences from both glycophytes and halophytes
were aligned and phylogenetic tree was constructed. SbNHX1 gets clustered with
halophytes within the Class-I type of NHX proteins.
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Topological analysis and structure-function studies have so far only been
performed with the AtNHX1 protein. Hydropathy analysis of NHX indicates a domain
organization similar to NHE isoforms, suggesting that structural features are conserved
across the families. Typically, 12 hydrophobic regions that potentially constitute
transmembrane helices are predicted in the conserved hydrophobic N-terminal domain,
with a divergent hydrophilic C-terminal domain that would be involved in regulatory
interactions. To date, two different topological models are proposed (Yamaguch et al;
2003; Sato et al; 2005). Detailed in vitro translation experiments indicate that AtNHX1
topology closely resembles the model proposed for human NHE1 (Wakabayashi et al;
2000; Sato et al; 2005), with 11 transmembrane helices and an intramembraneous loop
corresponding to hydrophobic region (Sze 1983). The plant NHX isoforms, contrary
to most intracellular NHX isoforms of other organisms lack the first H1 hydrophobic
stretch that in NHE1 or NHE6 were shown to represent an N-terminal signal peptide
required for endoplasmic reticulum insertion (Sato et al; 2005). Even so, the first
transmembrane helix of AtNHX1, corresponding to transmembrane helix 2 in NHE1,
is inserted in the same orientation into the membrane, whilst the C-terminus is exposed
to the cytoplasm (Sato et al; 2005). Hydrophobic region 9 has a very similar topology
as compared to the characteristic H10 loop in the human NHE1 isoform, and wouldn’t
cross the membrane completely (Wakabayashi et al 2000; Sato et al 2005).
Mutagenesis of the corresponding region in the yeast ScNHX1 protein has revealed
that several amino acids that are uniquely conserved amongst the intracellular NHX
are essential for function (Mukerjee et al 2006). Still care has to be taken with these
data, as a 3D homology model of NHE1 based on the crystal structure of NhaA gives
slightly different results, notably concerning NHE1 TM helix 9 and the
intramembraneous loop H10 (Landau et al 2007). A different membrane topology for
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the Arabidopsis AtNHX1 protein was found based on insertion mutagenesis with a
3xHA epitope (Yamaguchi et al 2003). In this model the C-terminal domain would be
exposed to the vacuolar lumen, whilst the N-terminus would be cytoplasmic. In
accordance with this topology it was found that in the yeast ScNHX1 protein some
amino acids in the C-terminal domain are N-glycosylated, which indicates that at least
part of the C-terminal domain of ScNHX1 is exposed to the endosomal lumen at some
stage (Wells et al 2001). In this new topology model of AtNHX1, which predicts only
9 transmembrane helices, hydrophobic domain 3, containing the putative amiloride
binding domain, and the hydrophobic domains 5 and 6, containing residues that are
likely involved in Na+ or H
+ binding and transport, would not cross the membrane.
This would result in several transmembrane helices being inserted in the opposite
direction in the membrane, which was related to the fact that plant NHX enzymes have
an opposite transport direction as compared to the plamsa membrane located NHE
proteins (Yamaguchi et al 2003). Whilst animal plasma membrane NHE proteins are
activated by cytoplasmic acidification, and normally catalyze entry of Na+ coupled to
the extrusion of protons (Landau et al 2007) the plant enzymes are suggested to be
involved in extrusion (to the vacuole) of Na+ or K
+, causing cytoplasmic acidification.
The related bacterial (2H+/Na
+) NhaA protein is activated by internal alkalinization
and catalyzes the entry of protons coupled to the extrusion of Na+ (Taglicht et al 1991).
Electroneutral 1:1 Cation/Proton exchangers could also be fully reversible, as was
shown for instance for amiloride sensitive Na+/H
+ exchange in mamalian cells (Paris et
al 1983) and the Schizosaccharomyces pombe plasma membrane SOD2 Na+/H
+
antiporter (Hahnenberger 1996). Detailed mutagenesis studies for the human NHE1
protein and structural resolution of individual transmembrane helices, have pinpointed
residues in transmembrane segments 4, 7 and 9 (corresponding to 3, 6 and 8 in
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Arabidopsis AtNHX1) that could be directly involved in ion transport (Reddy et al
2008). These transmembrane regions are strongly conserved also in the intracellular
NHX family. A mechanism for ion translocation in NHE1 was proposed, based on
these mutagenesis data and an NHE1 homology model build according to the structure
of the bacterial (2H+/Na
+) antiporter NhaA (Landau et al 2007). This model shows
essential roles for P167, P168, E262, D267 and S351, which correspond to amino
acids P88, P89, E179, D185 and S271 in the Arabidopsis AtNHX1 sequence, and that
are conserved throughout the intracellular NHX family. Also most other residues in
NHE that are important for drug binding or activity, are conserved in the NHX
sequences. These data strengthen the idea that structure and functioning of the NHX
and NHE families is very similar, and that the transport direction is imposed by
regulatory domains. The activity of animal NHE proteins can be regulated by a variety
of regulatory mechanisms involving the long C-terminal tail. Preliminary experiments
have indicated that removal of the last 82 amino acids in the Arabidopsis AtNHX1
protein modifies the transport specificity of the protein, increasing especially Na+/H
+
antiport activity but not K+/H
+ antiport activity, indicating a regulatory role of this
domain (Yamaguchi et al 2003). Later it was shown, using a two-hybrid screen and
immuno precipitation assays, that the C-terminal domain interacts with a CaM-Like
protein AtCaM15 (Yamaguchi et al 2005). AtCaM15 was also found inside the
vacuole of transiently transformed Arabidopsis protoplasts and in yeast cells
expressing the protein. This localization would permit interaction with the C-terminal
domain of AtNHX1 within the vacuoles. Activity measurements using yeast vacuoles
obtained from cells expressing AtNHX1 and AtCaM15 indicated that the CaM15
binding inhibits Na+/H
+ antiport by AtNHX1, without a significant effect on the Km of
the transport reaction. AtNHX1 activity is possibly also regulated through interaction
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with the protein kinase SOS2.82 SOS2 is the pivotal kinase of the SOS (Salt Overly
Sensitive) pathway involved in regulation of ion transport under salt stress and in
regulation of several other stress responses (Batelli et al 2007). It was reported that
amiloride sensitive specific vacuolar Na+/H
+ antiporter activity in Arabidopsis
membrane vesicles was lower in vesicles obtained from sos2 knockout mutants, and
that this activity could be stimulated in vitro by the addition of activated SOS2 protein
(Qui et al 2004). The activity was further inhibited by AtNHX1 antibodies. It was later
shown by tandem affinity purification and yeast two-hybrid assays that SOS2 also
interacts with several vacuolar V-ATPase subunits and that vesicles isolated from sos2
knockout mutants show considerably lower V-ATPase dependent acidification (Batelli
et al 2007). Comparison of antiport activity in vesicles obtained from wild-type and
sos2 mutant plants is thus difficult, as the V-ATPase mediated vesicle acidification and
thus driving force for the antiport is not the same in the two cases. Structural or
regulatory mechanisms have not been studied for other plant NHX isoforms. The C-
terminus of the yeast NHX1 protein was shown to interact with the small GTPase
activating protein Gyp6 (Ali et al 2004). A model was proposed in which Gyp6
functions as a negative regulator of NHX. Inhibition of NHX1 would result in a more
acidic endsosome/prevacuolar compartment limiting retrograde traffic from the
prevacuolar compartment to the trans Golgi network or Golgi compartment. Such
inhibition would be relieved upon delivery by anterograde traffic of the small GTPase
protein GTP-Ypt6, as it will compete with NHX1 for Gyp6 binding. This would result
in endosome/prevacuolar alkalinization and termination of the Ypt6 signal, stimulation
of retrograde traffic permitting reactivation of Gyp6 by Ric1/Rgp1 in the trans Golgi
network or Golgi (Ali et al 2004). The C-terminus of the human trans Golgi network
localized NHE7 protein was shown to interact with several SCAMP (secretory carrier
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membrane protein) proteins, which would affect shutteling of NHE7 between
recycling vesicles and the trans Golgi network (Lin et al 2005). The C-terminus of the
NHE7 isoform was also shown to interact with caveolins, facilitating association of
NHE7 to caveole/lipd rafts (Lin et al 2007). The C-terminal domains of the human
isoforms NHE6, 7 and 9, but not NHE8 were found to interact with RACK1 (Receptor
for activated C Kinase 1).
4.2: Expression profiling of SbNHX1 gene
SbNHX1 gene showed very high expression with NaCl treatment. In the present
study we found a basal level of transcripts in plants without salt stress which increased
significantly with salt treatment. SbNHX1 transcripts increased gradually from 4-fold
to 12-fold by increasing the NaCl concentration up to 500 mM NaCl and thereafter
reduced. Plants were also treated at 0.5 M NaCl for different time period to see the
sequential pattern of transcript. The transcript was increased from 2 to 10 folds
concomitantly by increasing the duration treatment from 6 to 48 h, however at 72 h it
reduced further.
Involvement of NHX antiporters in maintaining ion homeostasis is indicated by
the induction of Na+/H
+ antiport activity or NHX gene expression in aerial parts or
roots of many plant species when grown in saline conditions. On comparing Melilotus
indicus, a halophyte growing up to 400 mM NaCl, with a glycophytic relative
Medicago intertexta, it was found that the halophytic species accumulated much less
Na+ and maintained higher levels of K
+. Na
+ accumulation and induction of very
similar NHX transcripts in response to salt stress were found only in the glycophytic
species, but not in the halophyte, indicating that NHX gene induction is related to the
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includer phenotype (Zahran et al 2007). Also comparing different maize varieties
differing in salt tolerance, it was observed that NHX transcripts were only induced in
roots of a variety known to exclude Na+ from the shoot (Zörb et al 2004). Similarly, it
was observed that HvNHX1 was mostly induced in roots of the relatively salt tolerant
monocot barley, whilst in rice, OsNHX1 induction is observed in shoots, suggesting
that the high salt tolerance in barley is related to accumulation of Na+
in root cell
vacuoles in order to limit transport to the shoot (Fukuda et al 2004a; 2004b). The
expression of most of the vacuolar NHX genes is induced under NaCl treatment,
including Arabidopsis AtNHX1 and rice OsNHX1. In cotton, the GhNHX1 gene was
strongly induced by salt stress in all organs, with the highest levels occurring in leaves
(Wu et al 2004). In wheat TaNHX1 transcripts also showed increased level of
expression in the seedlings after salt treatment (Wang et al 2002). Similarly, SbNHX1
gene showed very high expression with NaCl treatment.
Regulatory genes are quite important, since they can express wide array of genes
to control the multifarious environment conditions. The transcription factors interact
with cis-elements in the promoter regions of various abiotic stress-related genes and
up-regulate the expression of many secondary responsive genes resulting in abiotic
stresses tolerance. In Arabidopsis thaliana, cis-elements and corresponding binding
proteins, with distinct type of DNA binding domains, such as AP2/ERF (apetala 2/
ethylene responsive factor), basic leucine zipper, HD-ZIP (homeodomain leucine
zipper), MYC, MYB (myelocytomatosis, myeloblastosis ) and different classes of zinc
finger domains, have been identified (Shinozaki and Yamaguchi-Shinozaki 2000).
Genetic engineering of certain transcription factors can greatly influence plant stress
tolerance. Putative leucine zipper sites were also present in SbNHX1. Some
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transcription factors follow an ABA-dependent signal transduction pathway, while
others appear ABA-independent. The role of ABA in stress signalling and its
involvement in different regulatory systems during abiotic stress in ABA-dependent
and independent manner has been summarized in detail (Agarwal and Jha 2010).
The tonoplast localization of the plant Class-I NHX antiporters is well
documented (Rodriguez- Rosales et al, 2009). Subcellular localization studies have
been performed by immunoblotting using polyclonal antibodies against native proteins
(Apse et al 1999; Hamada et al 2001; Xia et al 2002; Fukuda et al 2004; Yoshida et al
2005) by transitory expression of fluorescent fusion proteins in onion epidermal cells
(Yokoi et al 2002; Ohnishi et al 2005; Yoshida et al 2005), by stable expression of
fluorescent fusion protein in BY2 cells30 and by immunogold labelling (Yoshida et al
2005). The only exception was found by Vera-Estrella et al (2005) who detected a 50
kDa protein that cross-reacted with a polyclonal antibody raised against the AtNHX1
protein in Plasma membranes but not in tonoplast from Thellungiella roots. For the
Class-II antiporters, subcellular localization studies have only been reported for the
tomato LeNHX2 and the Arabidopsis AtNHX5 proteins (Pardo et al 2006; Rodríguez-
Rosales et al 2008). For both proteins, transitory expression in onion epidermal cells of
fluorescent fusion proteins shows localization in small vesicles, indicative of a
prevacuolar or endosomal localization clearly distinct from the central vacuole or
ER/Golgi membranes, although the exact localization was not yet determined. The
localization is reminiscent of the prevacuolar localization of the yeast ScNHX1 protein
which suggests similar functions for the yeast and plant proteins as opposed to the
human isoforms found in recycling endosomes (NHE6, NHE9,15,33), and trans or
mid-trans Golgi (NHE7, NHE8,33,34). In all plants, several isoforms of NHX proteins
are found. Most of the isoforms are expressed in the absence of stress throughout the
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plant (Quintero et al 2000; Hamada et al 2001; Yokoi et al 2002; Fukuda et al 2004;
Wu et al 2004; Zhang et al 2008) and induced by salt stress in leafs (Quintero et al
2000; Venema et al 2003; Kagami and Suzuki 2005), both roots and leafs (Fukuda et
al 1999; Hamada et al 2001; Birni et al 2005; Zahran et al 2007), stems (Chauhan et al
2000) or roots (Fukuda et al 2004; Zörb et al 2004). Some isoforms are also reported to
be induced by ABA( Yokoi et al 2002; Venema et al 2003), KCl (Fukuda et al 1999;
Gaxiola et al 1999; Fukuda et al 2004; Wu et al 2004), dehydration stress (Li et al
2006) or hyper-osmotic stress (Fukuda et al 1999; Yokoi et al 2002; Fukuda et al
2004a; 2004b). The AtNHX1 isoform was reported not to be induced by cold or
drought (Shi and Zhu 2002). Finally, in Citrus, an isoform was discovered based on its
induction by heat (Porat et al 2002). In Arabidopsis, the expression of all 6 isoforms
was studied in more detail (Yokoi et al 2002). The predominant isoforms are AtNHX1
and AtNHX2, found in roots, shoots and seedlings (Yokoi et al 200; Shi and Zhu 2002;
Apse et al 2003). Expression levels of AtNHX3, 4 and 6 was much lower in these
tissues. AtNHX1 expression was shown to be upregulated in leaves but not roots by
NaCl or ABA (Quintero et al 2000). In seedlings, AtNHX1 and AtNHX2 were shown
to be induced by salt stress, hyper-osmotic shock and ABA treatment, whilst AtNHX5
was induced by salt stress only (Blumwald et al 2000). AtNHX1 and AtNHX2 were
not induced by NaCl in ABA deficient aba2-1 mutants, showing that NaCl induction of
these isoforms is dependent on ABA signalling (Shi and Zhu 2002; Yokoi et al 2002).
The tissue distribution of AtNHX1 was further studied by promotor-GUS
analysis in transgenic Arabidopsis (Shi and Zhu 2002) and by in situ hybridization
(Apse et al 2003), showing that the gene is expressed in all tissues except the root tip.
Especially high expression levels were observed in guard cells suggesting a role for
AtNHX1 in K+ accumulation in these cells (Shi and Zhu 2002). High GUS activity
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was also induced in response to salt stress not only in leaves, but also in root hair cells,
suggesting a role in Na+ accumulation in the enlarged vacuoles of these cells in
response to salt stress (Shi and Zhu 2002). High levels of expression were also
observed in floral tissues, and in cells closely associated to the vascular tissue in leaves
and inflorescence stems (Shi and Zhu 2002; Apse et al 2003). Some NHX isoforms
have been shown to have a more specific expression pattern in flower or fruit, related
to specific function. This is the case for the Ipomea Nil InNHX1 protein, found mainly
in flower limbs where it determines flower colour through vacuolar pH changes
(Ohnishi et al 2005) and the grape berry VvNHX1 protein that is highly expressed in
mature fruit where it is supposed to be involved in K+ accumulation and vacuolar
expansion during ripening (Hanana et al 2007). In the present study, uniformly high
level of GUS expression in leave tissue of transgenic tobacco was observed which is in
sync with the recent observation with different NHX1 genes.
Information about gene expression can also be obtained by exploring microarray
data available from high-throughput projects. User friendly interphases to these data
are provided at for instance http://wardlab.cbs.umn.edu/arabidopsis/or
http://bar.utoronto.ca/ efp/cgi-bin/efpWeb.cgi (Winter et al 2007). These microarray
data confirm the induction of Class-I AtNHX antiporters in leaves (AtNHX1, 2 and 4)
or roots (AtNHX3) by salt or osmotic stress. Like other tissue specific isoforms from
Ipomea Nil, grape or tomato (see above), AtNHX4 expression is detected mainly in
mature pollen and seeds. High levels of AtNHX1 are found in guard cells compared to
surrounding mesophyl cells (http://www-biology.ucsd.edu/labs/ schroeder/index.html),
in accordance with the promotor-GUS fusion experiments (Shi and Zhu 2002). Also
AtNHX2, 5 and 6 show a high level of expression in guard cells as compared to
surrounding mesophyl cells.
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4.3: Overexpression of SbNHX1 gene in transgenic tobacco
Plants in field have to re-establish Na+ homeostasis and ionic balance under
saline environments. Toxicity of Na+
is not due to toxic effect of Na+ but because of
disrupted K+ homeostasis. The Na
+ competes for binding sites for K
+. The main
mechanisms to prevent Na+ accumulation in plant cell is- restrict entry of Na
+ in the
cell, exclusion of Na+
out of the cell, or its compartmentalization within cellular
organelles like vacuole or in different tissues to facilitate metabolic functions of cells.
Transporter proteins are important candidates for genetic engineering to develop salt
tolerant plants.
Different NHX isoforms have been overexpressed in variety of plant species and
showed substantial salt tolerance (Rodríguez-Rosales et al 2009). There is no clear
difference in efficiency of the different isoforms, or whether they were obtained from
glycophyte or halophytes (Li et al 2007; Zhang et al 2008). In the present study,
SbNHX1 gene transformed into tobacco of showed overexpression consistently in T0
and T1 transgenic lines. Salt tolerance of the transgenic lines was greatly improved as
compared to wild type. Transgenic lines were healthy and normal flowering and seed
setting were observed. Earlier, Ohta et al (2002) reported that NHX from a halophyte,
Atriplex gmelini conferred salt tolerance in transgenic rice. Transgenic Arabidopsis
and tomato plants overexpressing AtNHX1 accumulated abundant quantities of the
transporter in the tonoplast and exhibited enhanced salt tolerance (Chauhan et al 2000;
Hamada et al 2001). Similar results with AtNHX1 were observed in Brassica (Ma et al
2004). Cotton orthologue, GhNHX1, when overexpressed in tobacco showed increased
salt tolerance (Wu et al 2004). PgNHX1 from Pennisetum glaucum has been reported
to confer high level of salinity tolerance when overexpressed in Brassica juncea, these
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plants could withstand 300mM salt stress till the seed setting stage (Rajagopal et al
2007). Overexpression of GhNHX1, AtNHX1 and SsNHX1 in Arabidopsis increased
tolerance to salt stress by the accumulation of carotenoids and anthocyanins (Liu et al
2010). In the present study, the overexpression of SbNHX1 gene in tobacco plants (T1)
showed NaCl tolerance (up to 200 mM) in pot experiments. This shows that SbNHX1
is a potential gene for salt tolerance, and can be used in future for developing salt
tolerant crops.
A number of salt tolerant transgenic plants have been produced by
overexpressing vacuolar antiporter proteins that control the transport functions. For
example, expressing the Yeast HAL1 gene showed a certain level of salt tolerance in
transgenic melon (Bordas et al 1997) and tomato (Gisbert et al 2000) plants and
retained more K+ than the control plants under salt stress. A vacuolar AtNHX1 (Na
+/H
+
antiporter) gene homologous to NhxI gene of yeast was cloned and overexpressed in
Arabidopsis that conferred salt tolerance by compartmentalizing Na+ ions in the
vacuoles. Transgenic Arabidopsis and tomato plants overexpressing AtNHX1
accumulated abundant quantities of the transporter in the tonoplast and exhibited
enhanced salt tolerance (Apse et al 1999; Zhang and Blumwald 2001). Similar results
with AtNHX1 were observed in Brassica (Zhang et al 2001b).
The proton pumps present at the cellular membrane work as driving force for
nutrient uptake (Serrano et al 1999). Three distinct proton pumps are responsible for
the generation of the proton electrochemical gradients (Sze et al 1999): (1) the plasma
membrane H-ATPase pump (PM H-ATPase), (2) Vacuolar type H+ATPase (V-
ATPase) and the vacuolar H-pumping pyrophosphatase (H-PPase). The PM H-ATPase
extrudes H+
from the cell and thus generates a proton motive force while V-ATPase
and H-PPase acidify the vacuolar lumen and other endomembrane compartments.
107
Overexpression of Avp1 encoding vacuolar H+- PPase showed enhanced salt tolerance
in transgenic Arabidopsis plants (Gaxiola et al 2001).
SOS (Salt Overly Sensitive) pathway was also found to be involved in Na+
exclusion. SOS1 is a plasmamembrane Na+/
H+
antiporter that excludes Na+
by taking
H+
in cytoplasm. Basically, it is activated by a sodium-induced rise in Ca2+
. The SOS
pathway is regulated by Ca2+
dependent protein kinase signalling (Zhu 2003). SOS
pathway involves SOS1, SOS2 and SOS3. Ca2+
signaling is perceived by SOS3, a
calcium binding protein. SOS3 activates SOS2, a protein kinase that activates SOS1 by
its phosphorylation. Recently, SOS4 and SOS5 have also been characterized (Shi et al
2002). SOS pathway also regulates vacuolar Na+/H
+ antiporter exchange activity and
Na+ compartmentalization (Qiu et al 2004). Salt Overly Sensitive 1 (SOS1) gene from
A. thaliana, which is similar to plasma membrane Na+/H
+ antiporter from bacteria and
fungi, was cloned and overexpressed using CaMV 35S promoter. The up-regulation of
SOSI gene was found to be consistent with its role in Na+ tolerance, providing a greater
proton motive force that is necessary for elevated Na+/H
+ antiporter activities (Shi et al
2000). Similar results were obtained when the plasma membrane Na+/H
+ antiporters,
SOD2 (Sodium 2) from Schizosaccharomyces pombe and nhaA from Escherichia coli,
were overexpressed in Arabidopsis (Gao et al 2003) and rice (Wu et al 2005),
respectively. Yang et al (2009) tested, whether, overexpression of multiple genes can
improve plant salt tolerance, they produced six different transgenic Arabidopsis plants
overexpressing AtNHX1, SOS1 and SOS3 alone or in combination, AtNHX1+SOS3,
SOS2+SOS3, and SOS1+SOS2+SOS3. Surprisingly the AtNHX1 alone did not show
significant salt tolerance. In 220 mM NaCl treatment for 3 d, less than 20% of the
control plants and transgenic plants overexpressing only AtNHX1 survived, but over
80% of the transgenic plants overexpressing SOS1, SOS3, SOS2 + SOS3,
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AtNHX1+SOS3, or SOS1+ SOS2+SOS3 survived. The SOS1 from Thellungiella
salsuginea suppressed the salt sensitive phenotype when expressed in the yeast cells.
This gene showed high salt stress tolerance in the Arabidopsis transgenic plants. When
the SOS1 gene was transformed with ThSOS1-RNAi vector in Thellungiella
salsuginea, the plants showed high salt sensitivity compared to wild type plants (Oh et
al. 2009).
Involvement of NHX antiporters in combating salt stress is indicated by the
induction of Na+/H
+ antiport activity or NHX gene expression in many plant species
when grown in saline environments as discussed above. When grown in saline
environment, all plants will accumulate Na+ ions to some extent, due to the strong
driving force for its entry. 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
et al 2008), the accumulation of Na+ inside vacuoles is a strategy used by many plants
to survive salt stress (Niu et al 1995; Blumwald et al 2000; Munns et al 2008). At the
cellular level, Na+ accumulation in vacuoles will lower the amount of toxic Na
+ ions in
the cytoplasm, and lower osmotic potential in the vacuole to maintain turgor pressure
and cell expansion in saline conditions. 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 (Maathuis and Amtmann 1999; Chauhan et al
2000; Munns et al 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 et al 2008). Information on the role of NHX antiporters in ion accumulation
and salt tolerance can be obtained by overexpression or silencing of the genes, or by
comparison of NHX gene expression and ion accumulation in closely related species
109
differing in salt tolerance. In this context, comparing Melilotus indicus, a halophyte
growing up to 400 mM NaCl, with a glycophytic relative Medicago intertexta, it was
found that the halophytic species accumulated much less Na+ and maintained higher
levels of K+. Na
+ accumulation and induction of very similar NHX transcripts in
response to salt stress could be found only in the glycophytic species, but not in the
halophyte, indicating that NHX gene induction is related to the includer phenotype
(Zahran et al 2007). Also comparing different maize varieties differing in salt
tolerance, it was observed that NHX transcripts were only induced in roots of a variety
known to exclude Na+ from the shoot (Zörb et al 2004). Similarly, it was observed that
HvNHX1 was mostly induced in roots of the relatively salt tolerant monocot barley,
whilst in rice, OsNHX1 induction is above all observed in shoots, suggesting that the
high salt tolerance in barley is related to accumulation of Na+ in root cell vacuoles in
order to limit transport to the shoot (Fukuda et al 2004a; 2004b). Expression pattern of
other isoforms was however not studied and the result could thus also have been due to
the fact that an isoform with a root specific induction pattern was studied in barley,
whilst a shoot induced isoform was studied in rice. Preliminary studies have shown
that AtNHX1 null mutants, or tomato plants in which the antiporter LeNHX2 is
silenced are more sensitive to salt stress, although no data on ion accumulation are
available (Apse et al 2003; Rodríguez-Rosales et al 2008). Much more research is to
be expected in the future studying individual Arabidopsis NHX knock-out mutants.
Ectopic overexpression of NHX genes has however received by far the most attention,
as it can be used as a biotechnological tool to improve crop salt tolerance. Care has to
be taken however in interpreting these data, as expression is no longer tissue specific
or stress inducible, and regulatory properties and even cellular localization of the
enzymes might be altered by the strong overexpression. Indeed, altered ion specificity
110
was suggested for overexpressed AtNHX1 protein (Yamaguch et al 2003; 2005). Most
of the published reports on overexpression of AtNHX1 or other plant NHX isoforms
in a variety of plant species show substantially increased salt tolerance (Zhang and
Blumwald 2001; Zhang et al 2001; Li et al 2006; Verma et al 2007; Zhao et al 2007;
Chen et al 2008; Liu et al 2008; Rodríguez-Rosales et al 2008; Shi et al 2008; Zhang et
al 2008). There is no clear difference in efficiency of the different isoforms, or
whether they were obtained from glycophytes or halophytes (Li et al 2007; Zhang et al
2008), and both Class-I and Class-II antiporters seem to have a similar effect on salt
tolerance (Rodríguez-Rosales et al 2008; Shi et al 2008). Differential salt tolerance
appears thus to be related to regulation of NHX gene expression, and not to differential
properties of the proteins.
In the present study, we observed that in the presence of NaCl both the WT and
transgenics showed growth retardation in a dose-dependent manner, but the retardation
was more in case of WT plants. At 100 mM NaCl both WT and transgenics survived
but growth of WT was less as compared to the transgenic lines. However, at 200 mM
and 300mM NaCl WT seedlings were greatly stunted, but the transgenics showed slow
growth. Nnumber and length of roots, leaf area and fresh weight of seedlings were
more in case of transgenics as compared to WT after 30 days. The chlorophyll content
in WT plant reduced significantly with increasing salt concentration. Transgenic lines
showed higher chlorophyll amount, and the chlorophyll content in 200 and 300 mM
salt did not get affected much. The transgenic lines L77 showed higher chlorophyll
content. There was a significant increase in the leaf area of transgenic plants even
when grown in 200 mM and 300 mM NaCl (Figure 3.26). Arabidopsis nhx1 null
mutants are reported to have smaller leaf area and epidermal cell size (Apse et al 2003)
111
which is possibly related to a vacuolar K+ deficit necessary for turgor generation and
cell expansion.
Increased salt tolerance is not always accompanied by increased vacuolar Na+
accumulation. In plants, all possible scenarios with respect to Na+ or K
+ can be found
(higher Na+, lower K
+ (Apse et al 1999; Chen et al 2008); higher Na
+ and K
+ (Brini et
al 2007; Zhao et al 2007; Shi et al 2008); only marginal differences (Fukuda et al
2004; Li et al 2007); higher K+ and lower Na
+ (Rodríguez-Rosales et al 2008; Zhang et
al 2008). As NHX transporters also transport K+, an effect on internal K
+
concentrations is to be expected, especially for the more K+ specific Class-II
antiporters. A decrease in Na+ content is more difficult to explain, but could result
from secondary mechanisms triggered by the improved K+ homeostasis in transgenic
plants (Rodríguez-Rosales et al 2008). Salicornia brachiata shows high accumulation
of NaCl in its stem tissues. About 30-35% of its dry weight is NaCl (Jha et al 2009).
Transgenic plants were also reported to have lowered leaf water potential, allowing
higher water uptake rates in saline or drought conditions (Lohaus et al 2000). In yeast,
disruption of ScNHX1 or overexpression of plant antiporters also affects intracellular
Na+ and K
+ concentrations. Especially yeast in which the main Na
+ efflux system
ENA1 is disrupted will accumulate large amounts of Na+ in response to salt stress, at
the cost of internal K+ (Gaxiola et al 1999). Disruption of ScNHX1 has some effect on
internal Na+ content, but above all causes a further diminution of internal K
+ (Quintero
et al 2000; Venema et al 2003). Overexpression of AtNHX1 or AtNHX2 strongly
increases intracellular K+ and Na
+ in ENA and ScNHX1 disrupted yeast cells grown in
the presence of NaCl (Yokoi et al 2002). Overexpression of the Class-II antiporters
AtNHX5 and LeNHX2 increases intracellular K+, but reduces intracellular Na
+ (Yokoi
et al 2002; Venema et al 2003). Also Gaxiola et al.(1999) showed that ScNHX1
112
mainly affects intracellular K+ accumulation, as overexpression of the plant H
+
pyrophosphatase, supposedly increasing the vacuolar/ prevacuolar pH gradient and
thus the driving force for ScNHX1 mediated cation accumulation, results in increased
salt tolerance, increased K+ levels, and reduced intracellular Na
+ levels in the presence
of endogenous ScNHX1 only. Based on these observations it can be stated that the
sequestration model, that suggests that NHX mediated salt tolerance is a consequence
of the accumulation of toxic Na+ inside the vacuole, away from the cytosol is too
simple, and that at least part of the tolerance is due to K+ accumulation or altered K
+
homeostasis, although the precise mechanism remains unclear.
However, the data on ion accumulation indicate that the role for NHX antiporters
in salt tolerance is above all related to osmotic adjustment by ion accumulation inside
the vacuole or endosomal compartments. Apart from a role in osmotic adjustment by
Na+ or K
+ accumulation in conditions of salt stress, NHX proteins were suggested to
fulfil a role in K+ homeostasis in normal growth conditions, based on their ion
specificity and affinity (Zhang and Blumwald 2001; Venema et al 2002). Most of the
cellular K+ is present in the vacuole where it has a biophysical function to maintain
turgor and drive cell expansion. The smaller cytoplasmic pool has both osmotic and
biochemical functions. Whilst K+ is actively included in the vacuole in normal growth
conditions, active export of K+ from the vacuole to the cytoplasm is necessary in
severe K+ depletion to maintain adequate cytosolic K
+ concentrations. An acidification
of the cytoplasm is observed in these conditions that could serve as a signal to induce
high affinity K+ uptake, or K
+ efflux from the vacuole (Walker et al 1996).
Published research has shown that NHX antiporters play roles in salt tolerance,
vacuolar pH regulation and K+ homeostasis. Contrary to general belief, NHX mediated
salt tolerance is not strictly related to Na+ accumulation, and reduced Na
+ content and
113
increased K+ content are equally often observed. This suggests that the genes function
in vacuolar osmotic adjustment via K+ or Na
+ accumulation, in accordance with their
ion specificity and gene induction pattern, or that salt tolerance is induced by other
mechanisms possibly by indirect effects on vesicle trafficking via endosomal pH
regulation. In this respect it has to be pointed out that plant salt tolerance mechanisms
appear to be plant species or variety specific, determined by differential responses at
the tissue, cell-type and subcellular level. Also vacuolar or cytoplasmic pH and K+
concentrations are variable between cell types, and differentially regulated in different
species. The differential expression of NHX genes in plants or varieties that
accumulate or exclude Na+ indicates that NHX proteins could play crucial roles in such
differences, but this has not been studied in much detail. Only in one case, the alkaline
pH in vacuoles of Ipomea nil, a species and cell-type specific role of NHX was clearly
demonstrated. Little more information can be gathered from general overexpression
experiments and crude experiments comparing species based on total shoot or root ion
content and antiporter expression levels. It indeed seems odd, that unregulated
overexpression of one gene, promoting vacuolar accumulation of ions in vacuoles
across the plant, can substantially improve such a complex trait as salt tolerance in
such a wide variety of plants. To describe function of the NHX genes in plants, genetic
studies using individual or multiple NHX knock-out mutants are required, as well as
detailed studies on tissue distribution and vacuolar or cytoplasmic ion content.
Function of plant NHX genes in endosomal vesicle trafficking has not been studied so
far and the search for regulatory mechanisms and NHX interacting partners is lacking
far behind compared to yeast and animal studies. The present study will be crucial to
provide more clues to the real function of the various NHX isoforms in plants.
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In conclusion, it can be explained that halophytes have efficient mechanisms to
sequester Na+ into vacuoles. Salicornia plants can grow in high salt and also
accumulate salt in the stem tissue therefore it is reasonable to isolate antiporter genes
from this plant and further utilize in developing transgenic plants, which might have
greater tolerance against salt stress. A full length (2,110 bp) Na+/H
+ antiporter
(SbNHX1) gene (accession number: EU448383) has been isolated from the halophyte
Salicornia brachiata, cloned and characterised. The putative SbNHX1 protein revealed
the presence of 11 strong transmembrane domains. The glycosylation sites seem to be
well conserved in SbNHX1. This is the first study to demonstrate glycosylation of
plant NHX. Detailed analysis confirms that the SbNHX1 is a vacuolartype Na?/H?
antiporter. SbNHX1 gets clustered with halophytes within the Class-I type of NHX
proteins. It has shown good expression (12 fold increase) under salt stress. The
overexpression of SbNHX1 gene showed enhanced salt tolerance in transgenic
tobacco. In future, SbNHX1 gene can be a potential candidate gene for enhancing
abiotic stress tolerance in crop plant.