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Advances in Environmental Biology, 6(5): 1769-1773, 2012
ISSN 1995-0756
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ORIGINAL ARTICLE
Assessment of Na+⁄ H+ Antiporters and H+-ATPase Pumps Transcriptional Changes in
Aeluropus littoralis Dealing with Salt Stress
1
Nasiri Najmeh, 1Shokri Ehsan, 2Nematzadeh Ghorbanali
1
M.Sc in Plant Breeding, Genetic & Agricultural Biotechnology Institute of Tabarestan (GABIT), University of
Agriculture Sciences and Natural Resources, Sari, Iran
2
Prof. in plant genetics, Genetic & Agricultural Biotechnology Institute of Tabarestan (GABIT), University of
Agriculture Sciences and Natural Resources, Sari, Iran
Nasiri Najmeh, Shokri Ehsan, Nematzadeh Ghorbanali; Assessment of Na+⁄ H+ Antiporters and H+ATPase Pumps Transcriptional Changes in Aeluropus littoralis Dealing with Salt Stress
ABSTRACT
NHX and SOS proteins are widely regarded as key players in the sequestration and efflux of sodium (Na+)
in plant cells to avert ion toxicity in the cytosol under salinity stress. These proton-coupled sodium transporters
use the proton-motive force created by the vacuolar (V-type) and plasma membrane (PM-type) H+-ATPase
pumps. Here, we use Aeluropus littoralis as a halophytic monocot model to study salt-related transcriptional
changes in NHX1, SOS1, and subunit c genes. The study provides evidence for induced expression of the Na+⁄H+
antiporters and PM-type H+-ATPase in response to salt stress. In addition it was found that the subunit c
mRNAs of V-type pump was transcripted constitutively in A.littoralis shoots that maybe a halophytic trait.
Finally the results suggest that a precise coordination at transcriptional levels between antiporters and H+ pumps
was crucial during plant facing to high salinity.
Key words: A.littoralis, NHX1, SOS1, H+-ATPase pumps, subunit c
Introduction
The C4 grass, Aeluropus littoralis, grows in the
salt marshes and coastal zones. It can tolerate
seawater salinity and the salinity fluctuation from
water
evaporation
and
tidal
inundation,
approximately from 300 mM to 600 mM. The
salinity tolerance mechanisms of Aeluropus species
include cellular, organizational, and whole plant
adaptations, such as ion compartmentation, presence
of salt glands on the leaves, ion exclusion at the root,
and ion partitioning in divergent organs. Under salt
stress, the maintenance of K+ and Na+ homeostasis is
crucial [16]. Thus, understanding how plants cope
with excessive Na+ in the environment is of great
agricultural importance as soil salinity accounts for
large yield losses in crops worldwide. Na+ stress
disrupts K+ uptake by root cells [4]. When Na+ enters
cells and accumulates to high levels, it becomes toxic
to enzymes. To prevent growth cessation or cell
death, excessive Na+ has to be extruded or
compartmentalized in the vacuole. Unlike animal
cells, plant cells do not have Na+-ATPases or
Na+/K+-ATPases, and they rely on H+-ATPases and
H+-pyrophosphatases to create a proton-motive force
that drives the transport of all other ions and
metabolites. Many of the transporters of H+, K+ and
Na+ have now been identified. The regulatory
mechanisms that control the expression and activity
of the transporters are beginning to be elucidated.
Vacuolar sequestration of Na+ not only lowers Na+
concentration in the cytoplasm but also contributes to
osmotic adjustment to maintain water uptake from
saline solutions. In plant, the NHX family of Na+/H+
anti-porter function in Na+ compartmentation [9].
NHX1 are localized in the tonoplast membrane, and
their transcript levels are up-regulated by salinity
stress [18]. In addition to NHX, The plasma
membrane Na+/H+ antiporter SOS1 is essential for the
salt tolerance of various model plants, including
Arabidopsis thaliana [1] and its halophytic relative
Thellungiella salsuginea [11], Tomato [12], and the
moss Physcomitrella patens [3]. SOS1 is thought to
mediate Na+ efflux at the root epidermis and longdistance transport from roots to shoots [17,12] while
protecting individual cells from Na+ toxicity [3-20].
SOS1 is also indirectly required for the uptake of
potassium (K+) in the presence of Na+, although the
mechanistic basis is not fully understood [3,20,21].
The transcript level of SOS1 is up-regulated by NaCl
stress but not by drought, cold, or abscisic acid [17].
Salt up-regulation of SOS1 expression is partially
under the control of the SOS2/SOS3 regulatory
pathway [17,22,7,5]. The activity of NHX and SOS
Corresponding Author
Shokri Ehsan, M.Sc in Plant Breeding, Genetic & Agricultural Biotechnology Institute of
Tabarestan (GABIT), University of Agriculture Sciences and Natural Resources, Sari, Iran
E-mail: [email protected]
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Adv. Environ. Biol., 6(5): 1769-1773, 2012
transport system is driven by the proton-motive force
generated by the vacuolar membrane H+-ATPase,
H+-pyrophosphatase and plasma membrane H+ATPase respectively. The direct stimulation of the
vacuolar and plasma membrane Na+/H+ antiport
system may be coordinated with the increased
activity of the v-type and pm-type H+ pumps, which
provide the driving force for the operation of the
cation exchanger. It has been shown that transcript
amount of V-type H+-ATPase different subunits
shows endogenous oscillations in response to salinity
[14]. More ever accumulation of mRNAs of PM H+ATPase under NaCl stress and the positive
correlation with salt tolerance are well documented
[10,13,6,15].
Materiels and Methods
Plant Samples Preparation:
Seedlings of halophyte plant A.littoralis were
irrigated daily with half-strength Hoagland nutrient
solution according to evaporation demand. Six-weekold seedlings were treated with 0 (control), 200 and
400 mmol/L NaCl. The final salinity level was
achieved by raising 100 mmol/L NaCl every 24 h in
order to avoid osmotic shock. Plant samples (shoot)
were taken for analysis 7 days after salt exposure.
The shoot Na+ and K+ concentrations were
determined with a Shimadzu AA-680 atomic
absorption / flame spectrophotometer.
Semi-quantitative RT-PCR:
Oligonucleotide primers for amplification of
PCR products of between 350 and 600 bp from A.
littoralis were designed using Oligo 7 software
according to the highly conserved amino acid
sequence of diverse genes (NHX, SOS1, PMATPase,VHA-c) obtained from GenBank. Total
RNA extracted using TRIZOL reagents (Invitrogen,
Inc.,CA,USA) and was exposured to Rnase free
DnaseI treatment according to Fermentas procedure
to eliminate residual genomic DNA. Single strand
cDNA was then obtained using 1µg of total RNA
and oligo (dT) primers, with the RevertAid Reverse
Transcriptase System (Fermentas) for RT-PCR. After
RT, cDNA was amplified for 19-21 cycles (94ºC 45s, 50.3ºC -45s, and 72ºC -90 s), which was
determined to be within the linear range of
amplification. For equal loading of the first standard
cDNA in RT-PCR reaction, the concentration must
be adjusted according to an internal control gene, so
the housekeeping gene β-actin was used as a control
for variations in the input of RNA.
Result:
Quantification of NHX1 transcripts in A.litorallis
shoot parts via semi-quantitative RT-PCR showed
that salinization was affected NHX1 levels positively,
as with increasing salinity NHX1 levels increased. In
comparison with control, the mRNA level of NHX
was 2.9 and 3.6 fold higher in 200 mM and 400 mM
stressed plants respectively (Fig. 2). In the presence
of NaCl, SOS1 transcripts were also increased in
shoot parts of A.litorallis when compared with
normal condition. SOS1 mRNA abundance higher in
200 mM and 400 mM stressed plants while in nonstress condition no visible band was amplified for
SOS1 gene one week after salt exposure (Fig. 2).
More ever NHX1 and SOS1 mRNA levels in 400 mM
stressed plant was higher than 200 mM treated plants
in shoot parts. The transcript level for V-H+-ATPase
subunit c was found to be constitutively expressed in
shoot tissues of A.litorallis plant and there wasn’t a
significant difference between non-stress and treated
plants (Fig. 2). Finally the transcripts of plasma
membrane H+-ATPase in shoot of A.litorallis plants
was up-regulated in response to salinity levels but
there wasn’t a significant difference between 200 and
400 mM treated plants. Also the PMA transcripts in
non-stress plants were not visibly amplified (Fig. 2).
Shoot K+ to Na+ ratio drastically decreased with
increase in salinity levels in A.litorallis. Sodium
increased considerably while K+ slightly decreased.
In comparison to control, Na+ accumulation was
greatly enhanced in shoot parts of salt treated plants,
but when salinity severity increased no significant
difference was observed between 200 and 400 mM
treated plants for accumulated sodium ions. In
contrast, shoot potassium content reduced in salt
treated plants in comparison to control (Fig. 1A &
B).
Discussion:
Transcriptional changes of subunits of the
vacuolar H+-ATPase in response to salinity stress
have been reported from a number of plants. Saltinduced transcriptional activation of V-ATPase
subunits A, B, E, and c have been shown in common
ice plant [2, 6, 19]. In this work we have shown that
the expression of V-ATPase subunit c was not
changed at transcriptional level in A.littoralis shoots
one week after salt stress. The constant V-H+ATPase transcript amounts maybe had a close
relationship with the activity of V-H+-ATPase, which
provided the energy for the compartmentalization of
sodium in response to salinity. It is apparent that
salinity affects vacuolar V-ATPase subunits gene
expression differently and keeps up high mRNA
levels of V-ATPase subunit c constantly in
A.littoralis shoots maybe halophytic trait. In addition
the mRNA level of NHX was about 3.5 fold higher
in salt treated plants indicated that Na+ sequestration
in vacuoles play a critical role in establishing salt
tolerance. This suggests that the salt sequestration
capacity of shoot parts including leaves, stem, nodes
and leaf sheaths allows the plant to accumulated
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higher amounts of sodium chloride in the early days
of dealing with salt stress. Moreover, it has been well
documented that salt treatment of plants induces the
activities of the plasma membrane proton pumps
both in Halophytes and Glycophytes. Accumulation
of mRNAs of PM H+-ATPase under NaCl stress and
the positive correlation with salt tolerance are well
documented [10, 13, 6, 15]. In Suaeda maritima (a
natural halophyte) and Oryza sativa salt-tolerant
cultivar accumulation of PM H+-ATPase gene
transcript was greater than that in a non-tolerant
cultivar of rice treated with NaCl [15]. In this study
when Aeluropus plants were treated with NaCl,
plasma membrane H+-ATPase transcripts increased.
Parallel to this increase in PM H+-ATPase
transcripts, the SOS1 mRNAs also increased in
treated plants and such an increase in SOS1 levels
was more evident at 400 mM treatment. This saltinducible transcription in both PM H+-ATPase and
SOS1 levels could be attributed to relatively high
Na+/H+ exchange activity in plasma membrane site
that efficiently extruded Na+ from cells.
Fig. 1: Effect of salinity on Sodium and Potassium concentrations (A) and the ratio of K+ to Na+ in shoot parts
of A.littoralis (B). Six-week-old seedlings were treated with 0 (control), 200 and 400 mM NaCl.
Fig. 2: Transcripts amount of NHX1, SOS1,V- type H+ATPase (subunit c) and PM- H+ATPase genes in the
shoot part of A.littoralis under different concentration of NaCl. A. littoralis β-actin gene was used as an
internal control for the same quantity of total RNAs. Experiments were repeated at least three times to
obtain similar results.
Conclusion:
Salinity tolerance of plants is a complex trait
involving adaptation at the level of cells, organs and
the whole plant. The key factor of salinity tolerance,
beside osmotic adjustment, is the control of
intracellular ion homeostasis.This work provides
some molecular evidence that underlie salinity
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Adv. Environ. Biol., 6(5): 1769-1773, 2012
tolerance in A.littoralis and provides supporting
information for transcriptional regulation of vacuolar
and plasma membrane type H+-ATPase by under
salinity and their coordination with transport proteins
involved in the sequestraion of Na+ into the vacuole,
or the removal of Na+ across the PM, including the
Na+/H+
exchanger.
Such
coordination
at
transcriptional level appears to be key mechanisms
for salinity tolerance in A.littoralis as they have been
shown to be in other halophytes.
9.
10.
11.
12.
Acknowledgments
This study was financial supported with GABIT
(Genetic & Agricultural Biotechnology Institute of
Tabarestan).
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