Plant, Cell and Environment (2010) 33, 793–804
doi: 10.1111/j.1365-3040.2009.02105.x
Variation in salinity tolerance and shoot sodium
accumulation in Arabidopsis ecotypes linked to
differences in the natural expression levels of
transporters involved in sodium transport
pce_2105
793..804
D. JHA, N. SHIRLEY, M. TESTER & S. J. ROY
The Australian Centre for Plant Functional Genomics and the University of Adelaide, PMB1, Glen Osmond, SA 5064,
Australia
ABSTRACT
Salinity tolerance can be attributed to three different
mechanisms: Na+ exclusion from the shoot, Na+ tissue tolerance and osmotic tolerance. Although several key ion
channels and transporters involved in these processes are
known, the variation in expression profiles and the effects of
these proteins on Na+ transport in different accessions
of the same species are unknown. Here, expression profiles
of the genes AtHKT1;1, AtSOS1, AtNHX1 and AtAVP1
are determined in four ecotypes of Arabidopsis thaliana.
Not only are these genes differentially regulated between
ecotypes, the expression levels of the genes can be linked to
the concentration of Na+ in the plant. An inverse relationship was found between AtSOS1 expression in the root and
total plant Na+ accumulation, supporting a role for AtSOS1
in Na+ efflux from the plant. Similarly, ecotypes with high
expression levels of AtHKT1;1 in the root had lower
shoot Na+ concentrations, due to the hypothesized role of
AtHKT1;1 in retrieval of Na+ from the transpiration
stream. The inverse relationship between shoot Na+ concentration and salinity tolerance typical of most cereal crop
plants was not demonstrated, but a positive relationship was
found between salt tolerance and levels of AtAVP1 expression, which may be related to tissue tolerance.
Key-words: AtSOS1; AtHKT1;1; AtAVP1; AtNHX1; sodium
transport.
INTRODUCTION
Soil salinity is a major abiotic stress affecting agricultural
land which results in significant losses of crop yield. A large
contributor to salinity stress is the build-up of the sodium
ion (Na+) in the cytoplasm of leaf cells. High cytoplasmic
Na+ interferes with processes that require the binding of
potassium (K+), protein synthesis and the activation of key
metabolic enzymes (Bhandal & Malik 1988; Blaha et al.
Correspondence: M. Tester.
[email protected]
© 2010 Blackwell Publishing Ltd
Fax:
+61 8 8303 7102;
e-mail:
2000; Tester & Davenport 2003; Munns, James & Lauchli
2006; Munns & Tester 2008). One key mechanism of salinity
tolerance is the ability of a plant to control Na+ transport at
both the tissue and cellular level, either by secreting Na+
into tissues, cells and organelles where it can do little
damage, or by minimizing the amount of Na+ entering the
plant through its roots (Tester & Davenport 2003; Apse &
Blumwald 2007; Munns & Tester 2008). Exclusion of Na+
from the shoot has frequently been observed as a central
mechanism in salinity tolerance for cereal crops such as
durum wheat (Gorham 1990; Munns & James 2003), bread
wheat (Gorham 1990; Schachtman & Munns 1992; Poustini
& Siosemardeh 2004), barley (Forster 2001; Wei et al. 2003;
Pritchard et al. 2004; Garthwaite, von Bothmer & Colmer
2005) and rice (Zhu, Kinet & Lutts 2001); however, it has
become apparent in some crop varieties that tissue tolerance mechanisms are also important (El-hendawy, Hu &
Schmidhalter 2005; Genc, McDonald & Tester 2007). Therefore, an understanding of the mechanisms and control of
movement of Na+ through a plant and the genes involved is
crucial if the ability of our current crop varieties to grow in
saline soil is to be improved.
Na+ exclusion from the shoot has often been cited as an
important mechanism in plant salinity tolerance (Munns
et al. 2000; Tester & Davenport 2003; Garthwaite et al.
2005; Colmer, Flowers & Munns 2006). In Arabidopsis and
other species, genes belonging to the HKT or SOS1 families have been implicated in controlling Na+ movement
throughout the plant. The HKT gene family encodes proteins that are responsible for the influx of Na+ into a cell
(Uozumi et al. 2000). Members of the family can be
divided into two subgroups depending on their function,
as either a Na+ uniporter or a Na+ and K+ symporter
(Platten et al. 2006). Members of subgroup 1, the Na+ uniporters (Platten et al. 2006), have been shown to be important in salinity tolerance. In Arabidopsis, Athkt1;1 mutants
and the ecotypes Ts1 and Tsu1, which have no root
AtHKT1;1 expression, hyperaccumulate Na+ in the shoot
while showing reduced Na+ accumulation in the root
(Maser et al. 2002; Berthomieu et al. 2003; Rus et al. 2004;
Rus et al. 2006). Further characterization has revealed that
793
794 D. Jha et al.
AtHKT1;1 is important in retrieving Na+ from the xylem
in the root, thereby minimizing the amount of Na+ entering the shoot through the transpiration stream (Davenport
et al. 2007). A role of AtHKT1;1 in the recirculation of
Na+ from the shoot, through removal of Na+ from the
xylem and the facilitation of Na+ loading into the phloem,
has also been proposed by Sunarpi et al. (2005). AntiHKT1; 1 antibodies were found to bind to the plasma
membrane of shoot xylem parenchyma cells and Athk1-3
knockouts had higher concentrations of Na+ in the shoot
xylem and lower concentrations in the phloem than wildtype plants. Similar functions have been identified or proposed for members of HKT subgroup 1 in rice (Ren et al.
2005) and wheat (James, Davenport & Munns 2006; Byrt
et al. 2007).
Members of the plasma membrane Na+/H+
antiporter SOS1 family have also been implicated in
reducing the amount of Na+ transported to the shoot in
the transpiration stream; however, unlike members of the
HKT family, SOS1 proteins have been shown to efflux Na+
from cells back into the external medium (Qiu et al. 2002;
Shi et al. 2002). In Arabidopsis, Atsos1 mutants have been
shown to be hypersensitive to Na+ stress, showing severe
growth retardation and high shoot and xylem Na+ concentrations which ultimately leads to the death of the plant
(Wu, Ding & Zhu 1996; Shi et al. 2000, 2002). While initial
studies localized AtSOS1 gene expression to epidermal
cells at the root tip and in parenchyma cells at the xylem/
symplast boundary, therefore suggesting a role in regulating xylem loading (Shi et al. 2002), more recent results
suggest a role in Na+ efflux from the root (Shabala et al.
2005). Similar results for other SOS1 proteins have also
been observed in poplar (Wu et al. 2007), Thellungiella
halophila (Vera-Estrella et al. 2005), wheat (Mullan,
Colmer & Francki 2007) and rice (Martinez-Atienza et al.
2007). Interestingly, the salt sensitivity of Atsos1 mutants
can be reduced by mutation of ATHKT1;1 (Rus et al.
2004).
A key group of transporters involved in Na+ tissue tolerance mechanisms are the vacuolar Na+/H+ antiporters, such
as AtNHX1, which are responsible for detoxifying the cytoplasm by pumping Na+ into the vacuole (Gaxiola et al. 1999;
Pardo et al. 2006). There are numerous studies showing that
overexpression of AtNHX1, or its homologs from other
plant species, results in salt-tolerant plants (Apse et al. 1999;
Zhang & Blumwald 2001; Xue et al. 2004; He et al. 2005;
Chen et al. 2007). Similarly, overexpression of AtAVP1, or
its homologs, leads to increased salinity tolerance by
enhancing the accumulation of Na+ in the vacuole (Gaxiola
et al. 2001; Zhao et al. 2006). AtAVP1 encodes a vacuolar
H⫾translocating pyrophosphatase which increases the difference in the electrochemical potential of H+ between the
vacuole and cytoplasm, thereby energizing the movement
of Na+ into the vacuole through Na+/H+ antiporters such as
AtNHX1 (Gaxiola et al. 2001; Tester & Davenport 2003;
Munns & Tester 2008).
While much is known about the mechanism of action of
individual plant transporters under conditions of salt stress,
little work has been carried out on the characterization and
interaction of the genes encoding these transporters.
Furthermore, it is not known how widespread particular
mechanisms are to deal with salt stress, or whether different
plants regulate different genes to achieve a particular level
of tolerance.
The aim of this study was to investigate the salinity tolerance of four Arabidopsis ecotypes, Columbia (Col),
Landsberg erecta (Ler), Wassilewskija (Ws) and C24, commonly used in salinity studies (Zhu, 2000; Quesada et al.
2002; Gao et al. 2003; Sunarpi et al. 2005; Kim et al. 2007;
Kant et al. 2008; Møller et al. 2009), to determine whether
the tolerance of these ecotypes is correlated with the
expression of four important transporters, AtHKT1;1,
AtSOS1, AtNHX1 and AtAVP1, which are involved in regulating the movement of Na+. While the results reveal no
inverse relationship between shoot Na+ accumulation and
salinity tolerance in these Arabidopsis ecotypes, there is a
positive relationship between tolerance and levels of
AtAVP1 expression, which may be related to tissue tolerance that could be conferred by this vacuolar H+ pump.
Inverse relationships were demonstrated between the
expression levels of AtSOS1 in the root and total plant
Na+ accumulation and between the expression levels of
AtHKT1;1 in the root and shoot Na+ concentration. These
can be explained by the removal of Na+ from the root by
AtSOS1 and by retrieval of Na+ from the transpiration
stream in the roots by AtHKT1;1.
MATERIALS AND METHODS
Plant material and growth conditions
Seeds of Arabidopsis thaliana ecotypes Col, Ws, Ler and
C24 were obtained from the European Arabidopsis Stock
Centre (Nottingham, UK). Seeds were first surface sterilized, by soaking in 70% ethanol for 2 min followed by 3–4
rinses in sterile milli-Q water, before individual seeds were
planted in 1.5 mL microfuge tubes filled with half-strength
Arabidopsis nutrient solution (Arteca & Arteca 2000) and
0.8% Bacto agar. The seeds were vernalized for 2 d at 4 °C
and then transferred to a growth room with a 10 h light/14 h
dark photoperiod, an irradiance of 70 mmol m-2 s-1 and a
constant temperature of 21 °C. The bottom 0.5–0.7 cm of
the microfuge tubes were removed after emergence of the
cotyledon and the roots of the seedling had grown approximately two-thirds down the length of the tube. Upon emergence of the root from the agar, the plants were transferred
to a constantly aerated hydroponics tank containing fullstrength Arabidopsis nutrient solution. The pH of the
hydroponic solution was monitored and maintained at pH
5.7. Salt stress was applied 5 weeks after germination by the
addition of 100 mm NaCl in 12 hourly increments of 25 mm.
Calcium activity in the growth medium was maintained at
0.3 mm at each salt application by addition of the correct
amount of calcium, as calculated using Visual Minteq
Version 2.3 (KTH, Department of Land and Water
Resources Engineering, Stockholm, Sweden).
© 2010 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 793–804
Transporters important in salinity tolerance in Arabidopsis 795
Phenotyping of ecotypes
Plants were harvested after 5 d of salt treatment. Whole
roots and shoots of control and salt-treated plants were
excised separately and fresh weights were recorded. Plant
material was dried at 65 °C for 2 d and salinity tolerance
was determined by comparing differences between the dry
weights of control and salt-treated plants. Samples were
digested in 70% nitric acid and 30% hydrogen peroxide for
2.5 h at 120 °C in a Hot Block (Environmental Express, Mt.
Pleasant, SC, USA). Na+ and K+ concentrations were measured using inductively coupled plasma spectrometry–
atomic emission spectra. The results presented are the
average and standard error of the mean (SEM) for five
biological replicates.
Expression analysis of Na+ related transporters
Total RNA was extracted using TRIzol reagent (Invitrogen,
Carlsbad, CA, USA), following the protocol described by
Chomczynski 1993. Genomic DNA contamination was
removed using Ambion’s DNA-free kit (Promega,
Madison, WI, USA) and 200 ng of total RNA was used to
synthesize cDNA using Superscript III (Invitrogen).
Quantitative real-time PCR (Q-PCR) was performed on
the cDNA for the transporters AtHKT1;1 (At4g10310),
AtSOS1 (At2g01980), AtAVP1 (At1g78920) and AtNHX1
(At5g27150), following the protocol outlined in Burton
et al. 2008 using a RG6000 Rotor-Gene Real Time Thermal
Cycler (Corbett Research, Sydney, Australia). For primer
sequences, see Supporting Information Table S1.
A two-round normalization of Q-PCR data, by geometric
averaging of multiple control genes, was carried out as
described by Vandesompele et al. 2002. Cyclophilin
(At2g36130), tubulin alpha-2 chain (TUA2, At1g50010),
actin 2 (ACT2, At3g18780) and glyceraldehyde 3-phosphate
dehydrogenase A (GAPA, At3g26650) were used in this
study. Briefly, the expression data for each of the genes are
normalized with respect to the biological replicates from
the same treatment, using the three control genes which
show the lowest variation between samples. The geometric
mean of expression for all genes is then calculated from the
normalized replicates and SE determined. The second
round of normalization standardizes the mean of gene
expression from each individual treatment to each other
(Vandesompele et al. 2002). The SE derived from the first
round of normalization was scaled using the normalization
factors for the second round. The results presented are the
average ⫾ SEM for three biological replicates.
22
Na+ influx and translocation assays
Unidirectional sodium influx, root to shoot Na+ translocation and the amount of sodium retained in the upper part
of the root was measured using 22Na+ (GE Healthcare,
Rydalmere, Australia), following previously published protocols (Essah, Davenport & Tester 2003; Davenport et al.
2007). Unidirectional 22Na+ influx was determined in roots
of 4-week-old seedlings grown in square Petri dishes on
half-strength Murashige and Skoog medium (pH 5.7)
(Sigma-Aldrich, St. Louis, MO, USA), 1% w/v sucrose and
0.3% phytagel (Sigma-Aldrich). Excised roots, pooled from
three to four individual plants, were pre-treated for 10 min
in unlabelled influx solution, containing 50 mm NaCl and
0.5 mm CaCl2. Roots were blotted gently and transferred to
15 mL of labelled influx solution, containing 50 mm NaCl,
0.5 mm CaCl2 and 5.5 mCi 22Na+, and gently incubated at
room temperature (RT) on a rotating shaker for 2 min. The
roots were rinsed twice in ice-cold rinsing solution (50 mm
NaCl and 10 mm CaCl2) for 2 and 3 min, respectively.
Once blotted dry, the roots were weighed, mixed with 4 mL
of EcoLume liquid scintillation fluid (MP Biomedicals,
Sydney, Australia) and measured using a liquid scintillation
counter (Beckman Coulter LS6500, Gladesville, Australia).
For measurements of root to shoot sodium translocation,
as well as the amount of 22Na+ retained in the upper root,
plants were grown in hydroponics for 5 weeks, followed by
pre-treatment with 50 mm NaCl for 5 d, as described earlier.
Individual plants were removed from the hydroponics and
the lower half of their roots suspended in 50 mL polypropylene tubes, containing 10 mL of labelled influx solution
(50 mm NaCl, 0.5 mm CaCl2 and 5 mCi 22Na+). The plants
were gently incubated at RT on a gently rotating shaker for
1 h before being separated into upper unlabelled root,
lower labelled root and shoot. The samples were then
rinsed, blotted, weighed, mixed with scintillation fluid and
measured as previously described.
RESULTS
Arabidopsis salinity tolerance not related to
shoot Na+ concentration
The Arabidopsis ecotypes Col, Ler, Ws and C24 were
grown in hydroponics for 5 weeks before being incubated
in either 0 or 100 mm NaCl for a further 5 d. As expected,
salt-stressed plants showed a significant reduction in the
amount of dry mass produced over the 5 d period;
however, the level of reduction varied between ecotypes
(Fig. 1 and Table 1). Ler and Ws are the more tolerant
ecotypes, showing a minimal reduction of dry matter production and a high plant tolerance index when grown in
100 mm NaCl, while the growth rate of the two saltsensitive ecotypes, Col and C24, was significantly reduced.
Although there are large variations in dry weight production between ecotypes under salt stress, there appears to
be little variation in terms of percentage water content of
their roots and shoots, or in their root dry weight ratio
under salt stress (Table 1).
Variation can also be observed in the concentrations of
Na+ and K+ accumulating in the shoots and roots of the four
different ecotypes (Table 2). Col and Ws accumulate low
concentrations of Na+ in the shoot and higher concentrations in the root when compared with C24 and Ler. C24,
however, appears better able to maintain higher concentrations of K+ in its roots when compared with the other
© 2010 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 793–804
796 D. Jha et al.
Figure 1. Representative photographs of
Arabidopsis ecotypes Columbia (Col),
Wassilewskija (Ws), Landsberg erecta
(Ler) and C24 grown for 5 weeks in
hydroponics before treatment with either
0 or 100 mm NaCl.
Table 1. Differences in salinity tolerance of four ecotypes of Arabidopsis
Ecotype
Col
Ws
Ler
C24
Treatment
(NaCl)
Total dry
mass (mg)
0 mm
100 mm
0 mm NaCl
100 mm
0 mm NaCl
100 mm
0 mm NaCl
100 mm
59.9 ⫾ 5.9
35.1 ⫾ 2.9
38.6 ⫾ 1.6
32.6 ⫾ 1.7
22.4 ⫾ 0.8
20.8 ⫾ 2.0
42.5 ⫾ 4.6
27.4 ⫾ 1.7
Plant tolerance
index (Total DWsalt/
Total DWcontrol)
Root DW ratio
(rootDW/root +
shootDW)
0.59
0.137
0.175
0.134
0.155
0.094
0.091
0.085
0.072
0.84
0.93
0.65
% Water content
(FW-DW)/FW
Root
Shoot
95.2
94.6
95.4
95.2
93.9
95.4
94.5
94.9
87.7
86.9
86.9
86.6
84.2
85.7
85.5
85.7
Plants were grown in hydroponics for 5 weeks before treated with either 0 or 100 mm NaCl for 5 d. Fresh (FW) and dry weights (DW) of both
roots and shoots were measured and used to calculate total plant dry mass, root DW ratio, % tissue water content and plant salinity tolerance.
Results for total dry mass are the mean ⫾ SEM of five biological replicates.
Col, Columbia; Ws, Wassilewskija; Ler, Landsberg erecta.
Root
Shoot
Ecotype
Treatment
[Na+] (mm)
[K+] (mm)
[Na+] (mm)
[K+] (mm)
Col
0 mm NaCl
100 mm NaCl
0 mm NaCl
100 mm NaCl
0 mm NaCl
100 mm NaCl
0 mm NaCl
100 mm NaCl
1.5 ⫾ 0.1
57 ⫾ 3
2 ⫾ 0.4
55 ⫾ 9
2 ⫾ 0.2
35 ⫾ 1
1.5 ⫾ 0.1
36 ⫾ 2
77 ⫾ 2
38 ⫾ 1
74 ⫾ 3
50 ⫾ 6
93 ⫾ 5
61 ⫾ 3
80 ⫾ 2
71 ⫾ 3
3.6 ⫾ 0.4
87 ⫾ 10
5.3 ⫾ 0.7
138 ⫾ 18
10 ⫾ 0.4
193 ⫾ 17
9.3 ⫾ 1.1
230 ⫾ 6
113 ⫾ 2
93 ⫾ 11
135 ⫾ 5
115 ⫾ 7
105 ⫾ 2
91 ⫾ 3
130 ⫾ 2
97 ⫾ 3
Ws
Ler
C24
Table 2. Na+ and K+ concentrations in the
roots and shoots of four ecotypes of
Arabidopsis grown hydroponically for 5
weeks before treated to either 0 or 100 mm
NaCl for 5 d
Results are the mean ⫾ SEM for five biological replicates.
Col, Columbia; Ws, Wassilewskija; Ler, Landsberg erecta.
© 2010 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 793–804
Transporters important in salinity tolerance in Arabidopsis 797
vital signalling mechanism as there is no change in the
expression of the transporters studied in either the root or
the shoot of C24, while the other ecotypes show considerable changes (Table 3). Using gas chromatography-mass
spectrometry, and protocols previously established in the
laboratory (Jacobs et al. 2007), we have also observed that
there are no changes in the profile of 93 metabolites
(including sugars, organic acids, amino acids and fatty acids)
in the roots of salt-stressed C24 when, under similar circumstances, there are changes in the metabolite profile in the
roots of Col-0 (data not shown).
Plant tolerance
(Total DWsalt / Total DWcontrol)
(a)
1.0
Ler
0.9
Ws
0.8
0.7
C24
Col
0.6
0.5
0
50
100
150
200
250
The variation observed between the growth and cation
accumulation characteristics of the four ecotypes is also
seen at the expression level. In the roots, Col, Ws and Ler
show an up-regulation of the genes AtSOS1, AtAVP1 and
AtNHX1 under salt-stressed conditions which is not
observed in C24, while only Col and Ws show an increase in
AtHKT1;1 expression (Table 3). Similar observations are
apparent in the shoot of salt-stressed Col, Ws and Ler,
where significant increases in AtAVP1 are observed which
are not seen in C24; it appears that AtNHX1 is up-regulated
under saline conditions only in the shoot of Ler (Table 3).
In the shoots, AtSOS1 is up-regulated only in salt-treated
Col and Ler (Table 3).
Shoot Na+ concentration (mM)
Plant tolerance index
(Total DWsalt/Total DWcon)
(b)
1.0
Ler
0.9
Ws
0.8
0.7
C24
Col
0.6
0.5
0
10
20
30
40
50
60
Expression profiling of Na+-related transporters
70
Total dry mass in control plants (mg)
Figure 2. (a) Relationship between shoot Na+ concentration
and plant salinity tolerance, as measured by total dry weight
(DW) in salt stressed ⫼ total DW in control, in four ecotypes of
Arabidopsis. (b) Relationship between total dry mass in control
plants at the end of the experimental period and their salinity
tolerance index. Plants were grown for 5 weeks in hydroponics
before treatment with either 0 or 100 mm NaCl for 5 d. Results
are the mean ⫾ SEM of five biological replicates. Col, Columbia;
Ws, Wassilewskija; Ler, Landsberg erecta.
ecotypes. All ecotypes show a reduction in shoot K+ concentrations under salt stress.
Interestingly, the plant salinity tolerance index is not
inversely related to the amount of shoot Na+. Col, the
ecotype accumulating the lowest shoot Na+, exhibits the
lowest salinity tolerance, whereas Ler accumulates significantly higher concentrations of shoot Na+ while still maintaining near-normal levels of growth in 100 mm NaCl
(Tables 1 & 2, Fig. 2a). Indeed, it appears that salinity tolerance in Arabidopsis may be related to a tissue tolerance
mechanism (Fig. 2a), or to plant growth rate, with those
ecotypes exhibiting slower growth and biomass accumulation in control conditions being more salt-tolerant plants
(Fig. 2b). C24 is an exception to this observation, having
high shoot Na+ accumulation and low tolerance. Indeed,
C24 may not detect the salinity stress or might be missing a
Plant tissue tolerance related to
AtAVP1 expression
In both root and shoot, plant salinity tolerance is positively
correlated with AtAVP1 expression (Fig. 3a,b). Ler and Ws,
which exhibit the highest expression levels of AtAVP1 in
both roots and shoots, demonstrate the highest salinity tolerance when compared with ecotypes Col and C24, which
have low levels of both AtAVP1 expression and salinity
tolerance. The expression of AtAVP1 is also correlated with
the levels of AtNHX1 expression under salt stress, in both
roots and shoots, with the salt-tolerant Ler showing significantly higher levels of both AtAVP1 and AtNHX1 than the
salt-sensitive C24 (Fig. 3c,d). Interestingly, this relationship
between the genes is not observed in non-stressed conditions. Overall, though, there is no strong relationship
between root or shoot AtNHX1 expression and salinity
tolerance (data not shown). There was also no correlation
observed between tissue Na+ concentrations or amounts
and expression of either AtAVP1 or AtNHX1.
Plant Na+ content related to AtSOS1 and
AtHKT1;1 expression in roots
The amount of total plant Na+ was negatively correlated
with root AtSOS1 expression. Those ecotypes with high
root AtSOS1 expression under salt-stressed conditions, such
as Ler and Col, showed significantly lower total plant Na+
than C24 which has low root AtSOS1 expression and high
© 2010 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 793–804
798 D. Jha et al.
Ecotype
Treatment
AtSOS1
AtHKT1;1
AtAVP1
AtNHX1
3
Gene expression in roots (Normalized mRNA copies/mL cDNA (¥10 ) )
Col
0 mm NaCl
7 ⫾ 2.1
13 ⫾ 5.3
81 ⫾ 21
100 mm NaCl
20 ⫾ 0.7
35 ⫾ 5.4
188 ⫾ 7.0
Ws
0 mm NaCl
3 ⫾ 2.9
9 ⫾ 4.1
84 ⫾ 44
100 mm NaCl
21 ⫾ 0.4
34 ⫾ 1.8
248 ⫾ 4.0
Ler
0 mm NaCl
8 ⫾ 1.4
5 ⫾ 1.4
199 ⫾ 10
100 mm NaCl
25 ⫾ 2.4
6 ⫾ 4.7
479 ⫾ 56
C24
0 mm NaCl
6 ⫾ 1.5
1 ⫾ 0.4
140 ⫾ 18
100 mm NaCl
8 ⫾ 1.3
2 ⫾ 0.8
130 ⫾ 3.0
63 ⫾ 23
101 ⫾ 3.6
69 ⫾ 16
84 ⫾ 16
58 ⫾ 4.1
126 ⫾ 12
72 ⫾ 26
45 ⫾ 4.4
Gene expression in shoots (Normalized mRNA copies/mL cDNA (¥103))
Col
0 mm NaCl
3 ⫾ 0.9
1.5 ⫾ 0.2
202 ⫾ 25
100 mm NaCl
13 ⫾ 0.6
0.7 ⫾ 0.2
353 ⫾ 8
Ws
0 mm NaCl
4 ⫾ 0.6
0.6 ⫾ 0.03
178 ⫾ 9
100 mm NaCl
6 ⫾ 2.1
0.3 ⫾ 0.3
347 ⫾ 49
Ler
0 mm NaCl
1⫾2
0.3 ⫾ 0.2
224 ⫾ 60
100 mm NaCl
18 ⫾ 4
0.1 ⫾ 0.5
566 ⫾ 160
C24
0 mm NaCl
8 ⫾ 3.5
0.3 ⫾ 0.08
238 ⫾ 27
100 mm NaCl
10 ⫾ 2.3
0.4 ⫾ 0.07
267 ⫾ 42
166 ⫾ 13
122 ⫾ 9
119 ⫾ 3
101 ⫾ 15
106 ⫾ 17
156 ⫾ 31
129 ⫾ 32
80 ⫾ 12
Table 3. Expression profile of AtSOS1,
AtHKT1;1, AtAVP1 and AtNHX1 in the
roots and in the shoots of Col, Ws, Ler and
C24 grown for 5 weeks in hydroponics
before treatment with 0 or 100 mm NaCl
for 5 d
Results are the mean ⫾ SEM of three biological replicates.
Col, Columbia; Ws, Wassilewskija; Ler, Landsberg erecta.
total plant Na+ (Fig. 4a). A similar inverse relationship
could be observed between shoot Na+ concentrations and
root AtHKT1;1 expression. Those ecotypes, such as Col and
WS, which have high root expression of AtHKT1;1 show
lower shoot Na+ accumulation under salt-stressed conditions when compared with Ler and C24 which do not significantly up-regulate AtHKT1;1 (Fig. 4b). No correlation
was observed between shoot Na+ levels and shoot expression of either AtSOS1 or AtHKT1;1.
Higher root to shoot transfer of Na+ in C24
than in Col
Due to the significant differences in expression of
AtHKT1;1 between Col and C24, the effect of this gene on
initial Na+ entry and transportation from the root to the
shoot was investigated further in the two ecotypes using
22
Na+. While the unidirectional influx of Na+ is comparable
between Col and C24, there are significant differences
between the amount of 22Na+ retained in the roots and that
translocated to the shoots between the two ecotypes
(Table 4). Although C24 translocates almost three-quarters
of the 22Na+ entering its roots to the shoot, Col translocates
less than half. Indeed, Col is able to retain more Na+ in the
upper half of the root than C24 suggesting the presence of
a mechanism for removing Na+ from the transpiration
stream that is present in Col but not in C24 (Table 4).
DISCUSSION
Arabidopsis, a good model for understanding
salinity tolerance?
For many crop species, salinity tolerance is linked to shoot
Na+ exclusion (Gorham 1990; Schachtman & Munns 1992;
Forster 2001; Zhu et al. 2001; Munns & James 2003; Poustini
& Siosemardeh 2004; Garthwaite et al. 2005); however, in the
four ecotypes of Arabidopsis used in this study, no such
relationship was found. Instead, with the exception of C24, a
positive relationship was found between Na+ accumulation
and plant salinity tolerance, suggesting that Arabidopsis may
use mechanisms involved with Na+ tissue tolerance, such as
intracellular compartmentation and increased accumulation
of compatible solutes (Munns & Tester 2008), more than Na+
exclusion. Interestingly, there appears to be an inverse correlation between growth rate and tolerance, with those
ecotypes that are slow growing under control conditions
appearing to be more salt tolerant under stressed conditions
(Table 1 and Fig. 2b). Both Ler and Ws grew significantly
slower than Col and C24 but showed greater salinity tolerance, perhaps because the slower growth rate results in
slower water uptake, thereby enabling these plants to better
partition Na+ entering the shoot from the transpiration
stream. Rus et al. (2006) also observed no relationship
between elevated shoot Na+ concentrations resulting in
increased salt sensitivity in the ecotype Tsu1 which exhibited
reduced AtHKT1;1 expression, increased shoot Na+ and a
greater salt tolerance when compared to the low shoot Na+
accumulating Col-0. This suggests that other mechanisms of
tolerance, such as osmotic tolerance or tissue tolerance, may
be more important in enabling Arabidopsis plants to grow in
saline conditions than Na+ exclusion.
Salinity tolerance in wheat has been shown to rely on
Na+ exclusion from the shoot (Munns & James 2003). Our
results suggest that Na+ exclusion in Arabidopsis is not
linked to salinity tolerance as strongly as it is in the cereals,
consistent with previous concerns that Arabidopsis is not a
good model system for studying overall salinity tolerance in
cereals (Møller & Tester 2007); the use of other more relevant model species, such as rice, may be preferable.
However, there is increasing evidence that shoot Na+ exclusion is not the only mechanism of salinity tolerance in
© 2010 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 793–804
Transporters important in salinity tolerance in Arabidopsis 799
(b)
Plant tolerance
(Total DWsalt / Total DWcontrol)
Plant tolerance
(Total DWsalt / Total DWcontrol)
(a)
1.0
Ler
0.9
Ws
0.8
0.7
C24
Col
0.6
1.0
Ler
0.9
Ws
0.8
0.7
C24
Col
0.6
0.5
0.5
0
200
400
600
0
800
600
800
(d)
200
150
100
50
0
0
200
400
600
800
Root AtAVP expression
Normalized copy number / m L cDNA (×103)
Shoot AtNHX1 expression
3
Normalized copy number / m l cDNA (×10 )
Root AtNHX1 expression
3
Normalized copy number / m L cDNA (×10 )
400
Shoot AtAVP expression
Normalized copy number / m L cDNA (×103)
Root AtAVP expression
Normalized copy number / m L cDNA (×103)
(c)
200
200
150
100
50
0
0
200
400
600
800
Shoot AtAVP expression
Normalized copy number / m L cDNA (×103)
Figure 3. (a,b) Relationship between root or shoot AtAVP expression, respectively, and plant salinity tolerance in four ecotypes of
Arabidopsis. (c,d) Relationship between root or shoot AtAVP and AtNHX1 expression, respectively, for Col (䊊), Ws (䉭), Ler (䉮) and
C24 (䊐). Plants were grown for 5 weeks in hydroponics before treatment with either 0 (open symbols) or 100 mm (closed symbols) NaCl
for 5 d. The solid lines are linear regressions plotted between AtAVP1 and AtNHX1 gene expression at 100 mm NaCl. Results are the
mean ⫾ SEM of three biological replicates. Col, Columbia; DW, dry weight; Ws, Wassilewskija; Ler, Landsberg erecta.
cereals; some studies, such as that of Australian bread wheat
varieties (Genc et al. 2007) and of near-wild relatives of
wheat, such as Triticum monococcum (Rajendran, Tester &
Roy 2009), reveal a lack of correlation between shoot Na+
and tolerance. Arabidopsis may serve as a model for studying the other salinity tolerance mechanisms which may be
present in cereals, although further work will be required to
establish whether or not this is the case.
Arabidopsis is nevertheless a good system for understanding Na+ transport in to and through a plant and for
investigating the effects of manipulation of the expression
of transporters on shoot Na+ concentrations. Indeed, many
of the genes identified as important in the transport of Na+,
including some of those used in this study, were first discovered in Arabidopsis. Expression of these Arabidopsis genes
in other species have been shown to increase the salt tolerance of these species (Zhang & Blumwald 2001; Xue et al.
2004; He et al. 2005; Zhao et al. 2006; Bao et al. 2009). In
addition, the ability to complement knockout mutants of
Na+ transporters in Arabidopsis, using orthologous genes
from cereals, provides important information regarding the
function of these cereal genes, such as the complementation
of Atsos1 knockouts with OsSOS1 (Martinez-Atienza et al.
2007). Furthermore, Arabidopsis has been used for the
development of systems for manipulating the temporal and
spatial expression of genes to control the transport of ions
through a plant. Cell type-specific expression of AtHKT1;1
in root stelar cells resulted in reduced shoot Na+ and
increased salinity tolerance (Møller et al. 2009). Such
knowledge is invaluable to research in crops.
© 2010 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 793–804
800 D. Jha et al.
(a)
50
C24
40
Ws
30
Ler
+
Total Na m moles / plant
60
Col
20
10
0
0
5
10
15
20
25
30
Root AtSOS1 expression
3
Normalized copy number / m L cDNA (×10 )
+
Shoot Na concentration (mM)
(b)
250
200
150
(Møller et al. 2009) confirm that the gene is important in
reducing the amount of Na+ translocated to the shoot. In this
study of wild-type ecotypes, an inverse relationship between
levels of root AtHKT1;1 expression and shoot Na+ accumulation (Fig. 4b) was also found, as well as a positive relationship between root Na+ accumulation and root expression
(Tables 2 & 3, respectively). Ws and Col, which both significantly up-regulate AtHKT1;1 under salt stress conditions,
show lower shoot and higher root Na+ concentrations than
the high shoot and low root accumulators, Ler and C24,
which show no increase in gene expression. Similar observations have been reported in other Arabidopsis populations,
such as the ecotypes Ts-1 and Tsu-1 which show low
AtHKT1;1 expression and high shoot Na+ accumulation
(Rus et al. 2006). Interestingly, it was also found in this study
that low shoot Na+ accumulation was not linked to salinity
tolerance, with the Tsu1 plants having low root AtHKT1;1
expression, high shoot Na+ accumulation and lower leaf
death observed than Col-0. By selection of high and low
AtHKT1;1 expressing ecotypes (Col and C24, respectively),
it can be demonstrated using 22Na+ that this difference in
gene expression is related to the amount of Na+ being translocated to the shoot and not the initial influx of Na+ from the
growth medium (Table 4). It is hypothesized that Col uses
AtHKT1;1 to remove Na+ from the transpiration stream for
compartmentation in the root.
100
AtSOS1
50
0
0
10
20
30
40
50
Root AtHKT1;1 expression
3
Normalized copy number / m L cDNA (×10 )
Figure 4. (a) Relationship between root AtSOS1 expression
level and total plant Na+ concentration. (b) Relationship between
root AtHKT1;1 expression level and shoot Na+ concentration for
Col (䊊), Ws (䉭), Ler (䉮) and C24 (䊐). Plants were grown for 5
weeks in hydroponics before treatment with either 0 (open
symbols) or 100 mm (closed symbols) NaCl for 5 d. Results are
the mean ⫾ SEM of three biological replicates. Col, Columbia;
DW, dry weight; Ws, Wassilewskija; Ler, Landsberg erecta.
The roles of some key genes involved in
Na+ transport
AtHKT1;1
Previous work has highlighted the importance of AtHKT1;1
in reducing the amount of shoot Na+ accumulation through
the retrieval of Na+ from root xylem cells (Davenport et al.
2007) as well as recirculation from shoot to root (Berthomieu et al. 2003; Sunarpi et al. 2005), rather than through
reducing the initial entry of Na+ into the roots (Berthomieu
et al. 2003;Essah et al. 2003).Studies using AtHKT1;1 knockouts (Maser et al. 2002; Berthomieu et al. 2003; Rus et al.
2004) and root stellar-specific AtHKT1;1 overexpression
While increased root expression of AtHKT1;1 leads to
lower shoot Na+ accumulation, increased expression of
AtSOS1 in roots results in lower total plant Na+ (Fig. 4a),
consistent with a role for AtSOS1 in the efflux of Na+ from
the plant roots back to the growth solution. While the
results of this study reveal differences in AtSOS1 root
expression in natural populations leading to reduced plant
Na+, similar observations have been made in transgenic
Arabidopsis. In high-salt conditions, constitutive overexpression of AtSOS1 in Arabidopsis has been shown to
reduce plant Na+ accumulation by over 50%, with transgenic plants showing greater survival and growth rates than
wild-type controls (Shi et al. 2003). Furthermore, Atsos1
knockout plants hyperaccumulate Na+ and show severe
reductions in plant survival (Shi et al. 2002). AtSOS1 has
been shown to be a plasma membrane-bound Na+/H+ antiporter which transports Na+ out of a cell (Shi et al. 2000,
2002; Qiu et al. 2003; Mahajan, Pandey & Tuteja 2008); this
would suggest that the primary function of AtSOS1 involves
the movement of Na+ from the root back into the growth
medium.
Such an observation would seem to be at odds with
gene::GUS and gene::GFP fusions which suggest that
AtSOS1 is expressed predominantly around vascular tissue,
the only epidermal tissue expression being at the root tip
(Shi et al. 2002). In this case, the expectation is that salt
would be pumped into the transpiration stream, resulting in
an increase in shoot Na+, unless the protein were specifically
targeted towards the plasma membrane on the opposite
© 2010 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 793–804
Transporters important in salinity tolerance in Arabidopsis 801
Table 4. Measurements of unidirectional 22Na+ influx in excised roots, % translocation of 22Na+ to shoot and % 22Na+ retained in the
unlabelled upper root, in the ecotypes Col and C24
Unidirectional 22Na+ influx
Retrieval of 22Na+ from xylem
22
Ecotype
Na+ retained in
upper root (% relative
to total in plant)
22
Na+ influx (mmoles g-1
root FW min-1)
Na+ translocated to
shoot (% relative
to total in plant)
22
22
Na+ retained in upper
root (% relative to total
in upper root + shoot)
Col
C24
1.92 ⫾ 0.14 (n = 23)
1.55 ⫾ 0.09 (n = 39)
47.5 (n = 11)
72.5 (n = 9)
23.7 (n = 11)
14.2 (n = 9)
32.2 (n = 11)
16.3 (n = 9)
Results for unidirectional influx are presented as the mean ⫾ SEM of 9 to 39 biological replicates.
Col, Columbia; Ws, Wassilewskija; Ler, Landsberg erecta.
side of the cell from the xylem conducting elements. Clearly,
further work is required to investigate this discrepancy.
However, Shabala et al. have demonstrated more recently
that epidermal AtSOS1 activity could be found along the
whole root and not just at the root tip (Shabala et al. 2005),
which is consistent with the results presented here.
AtAVP1
Ecotypes which have increased salinity tolerance were
found to have higher AtAVP1 expression in either or both
the root and shoot (Fig. 3a,b). While AtAVP1 is not directly
involved in Na+ transport, it is involved in establishing
and maintaining an electrochemical potential difference
for protons between the cytosol and the vacuole, to enable
Na+/H+ antiporters, such as AtNHX1, to pump Na+ into the
vacuole (Gaxiola et al. 2001). Previous overexpression
studies have shown that when AtAVP1, or its homologs, are
constitutively overexpressed, plants such as tobacco (Gao
et al. 2006; Duan et al. 2007), rice (Zhao et al. 2006) and
Arabidopsis (Gaxiola et al. 2001; Brini et al. 2007) have significantly higher salinity tolerance. The results presented
here are similar to those from the overexpression studies
but are a consequence of the natural variation in gene
expression levels in ecotypes of the same species. It is shown
that those ecotypes with high AtAVP1 expression also have
greater salinity tolerance (Fig. 3a,b).
As might be expected, the level of expression of AtAVP1
was positively correlated with the expression of AtNHX1 in
all tissues, but this was seen only in salt-stressed conditions
(Fig. 3c,d). Overexpression of NHX genes has often been
described to increase the salinity tolerance of Arabidopsis
(Apse et al. 1999; Brini et al. 2007), rice (Chen et al. 2007),
cotton (He et al. 2005), wheat (Xue et al. 2004) and tomato
(Zhang & Blumwald 2001); nevertheless, the activity of the
transporter depends upon a difference in electrochemical
potential for protons, such as could be established and
maintained by AtAVP1 (Munns & Tester 2008). However, it
is interesting to note that in the present work, there was no
relationship observed between tolerance or tissue Na+ concentrations and AtNHX1 expression.This may reflect either
the greater importance of AtAVP1 in determining salinity
tolerance or a greater role for post-translational control of
AtNHX1 activity.
Limitations of study
It should be noted, however, that changes in gene expression are not necessary linked with changes in protein abundance, nor can changes in gene expression elucidate the
activity of the individual transporters. It may well be the
case that one ecotype possesses a more effective version of
a Na+ transporting protein or there are differences between
the ecotypes in the effectiveness of the regulators of these
proteins, such as differences within the CBL/CIPK Ca+ signalling pathways. This study has only established correlations between the expression of commonly studied genes
involved in Na+ transport through a plant and is not
intended to establish causal relationships.
Does C24 lack a Na+ sensor?
Of particular interest in this study is the lack of response of
C24 to salinity. While it is clear that the plant is salt stressed
(Table 2, Fig. 1), there is no substantial increase in expression of AtHKT1;1, AtSOS1, AtNHX1 or AtAVP1 in either
root or shoot tissue (Table 3). Similar observations have
been made in C24 of a lack of increase in expression of
other salt-responsive genes (S.J. Roy et al. unpublished
data) and in the metabolic profile of roots (D. Jha et al.
unpublished data). By contrast, Col, which is similarly a
salt-sensitive ecotype, responds to salinity stress by
up-regulating genes in the root and the shoot. Indeed, all
the genes studied were up-regulated in the root of Col
plants (Table 3), and both AtSOS1 and AtAVP1 were
up-regulated in the shoot (Table 3). The results here suggest
either that C24 up-regulates other, perhaps novel, transporters not examined in this study, or that the ecotype lacks
a key protein involved in the detection of salt stress or of a
component at a high level in the pathway signalling salt
stress to the cell. If the latter is the case, identification of
this detection and/or signalling mechanism could provide
insights into how a plant perceives salinity stress.
CONCLUSIONS
While it is demonstrated that there is no inverse relationship between shoot Na+ accumulation and salinity tolerance
in Arabidopsis, shoot Na+ accumulation was found to vary
© 2010 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 793–804
802 D. Jha et al.
significantly between Arabidopsis ecotypes and this
appears to be affected by the expression levels of
AtHKT1;1, AtAVP1 and AtSOS1 (and not AtNHX1). An
inverse relationship is observed between the expression
levels of AtSOS1 in the root and whole plant Na+ accumulation, as well as between the expression levels of
AtHKT1;1 in the root and Na+ concentration in the shoot.
The effects of increased AtSOS1 expression are proposed to
be due to increased Na+ extrusion from the root; the effects
of AtHKT1;1 expression are likely to be due to enhanced
retrieval of Na+ from the transpiration stream in the roots.
The results also indicate that the expression of AtAVP1
and AtNHX1 is co-regulated, and that increased levels of
AtAVP1 expression are correlated with increased plant
salinity tolerance.
ACKNOWLEDGMENTS
The authors would like to thank the Australian Research
Council for funding, Robin Hosking for help with the
hydroponics set-up and the Waite Analytical Services (University of Adelaide).
REFERENCES
Apse M.P. & Blumwald E. (2007) Na+ transport in plants. FEBS
Letters 581, 2247–2254.
Apse M.P., Aharon G.S., Snedden W.A. & Blumwald E. (1999) Salt
tolerance conferred by overexpression of a vacuolar Na+/H+ antiport in Arabidopsis. Science 285, 1256–1258.
Arteca R.N. & Arteca J.M. (2000) A novel method for growing
Arabidopsis thaliana plants hydroponically. Physiologia Plantarum 108, 188–193.
Bao A.-K., Wang S.-M., Wu G.-Q., Xi J.-J., Zhang J.-L. & Wang
C.-M. (2009) Overexpression of the Arabidopsis H+-PPase
enhanced resistance to salt and drought stress in transgenic
alfalfa (Medicago sativa L.). Plant Science 176, 232–240.
Berthomieu P., Conéjéro G., Nublat A., et al. (2003) Functional
analysis of AtHKT1 in Arabidopsis shows that Na+ recirculation
by the phloem is crucial for salt tolerance. The EMBO Journal
22, 2004–2014.
Bhandal I.S. & Malik C.P. (1988) Potassium estimation, uptake, and
its role in the physiology and metabolism of flowering plants.
International Review of Cytology 110, 205–254.
Blaha G., Stelzl U., Spahn C.M.T., Agrawal R.K., Frank J. &
Nierhaus K.H. (2000) Preparation of functional ribosomal complexes and effect of buffer conditions on tRNA positions
observed by cryoelectron microscopy. Methods in Enzymology
317, 292–306.
Brini F., Hanin M., Mezghani I., Berkowitz G.A. & Masmoudi K.
(2007) Overexpression of wheat Na+/H+ antiporter TNHX1 and
H+-pyrophosphatase TVP1 improve salt- and drought-stress tolerance in Arabidopsis thaliana plants. Journal of Experimental
Botany 58, 301–308.
Burton R.A., Jobling S.A., Harvey A.J., Shirley N.J., Mather D.E.,
Bacic A. & Fincher G.B. (2008) The genetics and transcriptional
profiles of the cellulose synthase-like HvCslF gene family in
barley. Plant Physiology 146, 1821–1833.
Byrt C.S., Platten J.D., Spielmeyer W., James R.A., Lagudah E.S.,
Dennis E.S., Tester M. & Munns R. (2007) HKT1; 5-like cation
transporters linked to Na+ exclusion loci in wheat, Nax2 and
Kna1. Plant Physiology 143, 1918–1928.
Chen H., An R., Tang J.-H., Cui X.-H., Hao F.-S., Chen J. & Wang
X.-C. (2007) Over-expression of a vacuolar Na+/H+ antiporter
gene improves salt tolerance in an upland rice. Molecular Breeding 19, 215–225.
Chomczynski P. (1993) A reagent for the single-step simultaneous
isolation of RNA, DNA and proteins from cell and tissue
samples. BioTechniques 15, 532–537.
Colmer T.D., Flowers T.J. & Munns R. (2006) Use of wild relatives
to improve salt tolerance in wheat. Journal of Experimental
Botany 57, 1059–1078.
Davenport R.J., Munoz-Mayor A., Jha D., Essah P.A., Rus A.N.A.
& Tester M. (2007) The Na+ transporter AtHKT1;1 controls
retrieval of Na+ from the xylem in Arabidopsis. Plant, Cell &
Environment 30, 497–507.
Duan X.-G., Yang A.-F., Gao F., Zhang S.-L. & Zhang J.-R.
(2007) Heterologous expression of vacuolar H+-PPase
enhances the electrochemical gradient across the vacuolar membrane and improves tobacco cell salt tolerance. Protoplasma 232,
87–95.
El-Hendawy S.E., Hu Y. & Schmidhalter U. (2005) Growth, ion
content, gas exchange, and water relations of wheat genotypes
differing in salt tolerances. Australian Journal of Agricultural
Research 56, 123–134.
Essah P.A., Davenport R. & Tester M. (2003) Sodium influx
and accumulation in Arabidopsis. Plant Physiology 133, 307–
318.
Forster B. (2001) Mutation genetics of salt tolerance in barley: an
assessment of Golden Promise and other semi-dwarf mutants.
Euphytica 120, 317–328.
Gao F., Gao Q., Duan X., Yue G., Yang A. & Zhang J. (2006)
Cloning of an H+-PPase gene from Thellungiella halophila and
its heterologous expression to improve tobacco salt tolerance.
Journal of Experimental Botany 57, 3259–3270.
Gao X., Ren Z., Zhao Y. & Zhang H. (2003) Overexpression of
SOD2 increases salt tolerance of Arabidopsis. Plant Physiology
133, 1873–1881.
Garthwaite A.J., von Bothmer R. & Colmer T.D. (2005) Salt tolerance in wild Hordeum species is associated with restricted entry
of Na+ and Cl- into the shoots. Journal of Experimental Botany
56, 2365–2378.
Gaxiola R.A., Rao R., Sherman A., Grisafi P., Alper S.L. & Fink
G.R. (1999) The Arabidopsis thaliana proton transporters,
AtNHX1 and AVP1, can function in cation detoxification in
yeast. Proceedings of the National Academy of Sciences of the
United States of America 96, 1480–1485.
Gaxiola R.A., Li J., Undurraga S., Dang L.M., Allen G.J., Alper S.L.
& Fink G.R. (2001) Drought- and salt-tolerant plants result from
overexpression of the AVP1 H+-pump. Proceedings of the
National Academy of Sciences of the United States of America 98,
11444–11449.
Genc Y., McDonald G.K. & Tester M. (2007) Reassessment of
tissue Na+ concentration as a criterion for salinity tolerance in
bread wheat. Plant, Cell & Environment 30, 1486–1498.
Gorham J. (1990) Salt tolerance in the Triticeae: K/Na discrimination in synthetic hexaploid wheats. Journal of Experimental
Botany 41, 623–627.
He C., Yan J., Shen G., Fu L., Holaday A.S., Auld D., Blumwald
E. & Zhang H. (2005) Expression of an Arabidopsis
vacuolar sodium/proton antiporter gene in cotton improves
photosynthetic performance under salt conditions and
increases fiber yield in the field. Plant Cell Physiology 46, 1848–
1854.
Jacobs A., Lunde C., Bacic A., Tester M. & Roessner U. (2007) The
impact of constitutive heterologous expression of a moss Na+
transporter on the metabolomes of rice and barley. Metabolomics 3, 307–317.
© 2010 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 793–804
Transporters important in salinity tolerance in Arabidopsis 803
James R.A., Davenport R.J. & Munns R. (2006) Physiological characterization of two genes for Na+ exclusion in durum wheat,
Nax1 and Nax2. Plant Physiology 142, 1537–1547.
Kant P., Gordon M., Kant S., Zolla G., Davydov O., Heimer Y.M.,
Chalifa-Caspi V., Shaked R. & Barak S. (2008) Functionalgenomics-based identification of genes that regulate Arabidopsis
responses to multiple abiotic stresses. Plant, Cell & Environment
31, 697–714.
Kim B.G., Waadt R., Cheong Y.H., Pandey G.K., Dominguez-Solis
J.R., Schultke S., Lee S.C., Kudla J. & Luan S. (2007) The calcium
sensor CBL10 mediates salt tolerance by regulating ion homeostasis in Arabidopsis. The Plant Journal 52, 473–484.
Mahajan S., Pandey G.K. & Tuteja N. (2008) Calcium- and saltstress signaling in plants: shedding light on SOS pathway.
Archives of Biochemistry and Biophysics 471, 146–158.
Martinez-Atienza J., Jiang X., Garciadeblas B., Mendoza I., Zhu
J.-K., Pardo J.M. & Quintero F.J. (2007) Conservation of the salt
overly sensitive pathway in rice. Plant Physiology 143, 1001–
1012.
Maser P., Eckelman B., Vaidyanathan R., Horie T., Fairbairn D.J.,
Kubo M., Yamagami M., Yamaguchi K., Nishimura M. &
Uozumi N. (2002) Altered shoot/root Na+ distribution and bifurcating salt sensitivity in Arabidopsis by genetic disruption of the
Na+ transporter AtHKT1. FEBS Letters 531, 157–161.
Møller I., Gilliham M., Jha D., Mayo G., Roy S.J., Coates J., Haseloff
J. & Tester M. (2009) Salinity tolerance engineered by cell typespecific over-expression of a Na+ transporter in the Arabidopsis
root. The Plant Cell 21, 2163–2178.
Møller I.S. & Tester M. (2007) Salinity tolerance of Arabidopsis: a
good model for cereals? Trends in Plant Science 12, 534–540.
Mullan D., Colmer T. & Francki M. (2007) Arabidopsis–rice–wheat
gene orthologues for Na+ transport and transcript analysis in
wheat–L. elongatum aneuploids under salt stress. Molecular
Genetics and Genomics 277, 199–212.
Munns R. & James R.A. (2003) Screening methods for salinity
tolerance: a case study with tetraploid wheat. Plant and Soil 253,
201–218.
Munns R. & Tester M. (2008) Mechanisms of salinity tolerance.
Annual Review of Plant Biology 59, 651–681.
Munns R., Hare R.A., James R.A. & Rebetzke G.J. (2000) Genetic
variation for improving the salt tolerance of durum wheat. Australian Journal of Agricultural Research 51, 69–74.
Munns R., James R.A. & Lauchli A. (2006) Approaches to increasing the salt tolerance of wheat and other cereals. Journal of
Experimental Botany 57, 1025–1043.
Pardo J.M., Cubero B., Leidi E.O. & Quintero F.J. (2006) Alkali
cation exchangers: roles in cellular homeostasis and stress tolerance. Journal of Experimental Botany 57, 1181–1199.
Platten J.D., Cotsaftis O., Berthomieu P., et al. (2006) Nomenclature
for HKT transporters, key determinants of plant salinity tolerance. Trends in Plant Science 11, 372–374.
Poustini K. & Siosemardeh A. (2004) Ion distribution in wheat
cultivars in response to salinity stress. Field Crops Research 85,
125–133.
Pritchard J., Tomos A.D., Farrar J.F., Minchin P.E.H., Gould N., Paul
M.J., MacRae E.A., Ferrieri R.A., Gray D.W. & Thorpe M.R.
(2004) Turgor, solute import and growth in maize roots treated
with galactose. Functional Plant Biology 31, 1095–1103.
Qiu Q.-S., Barkla B.J., Vera-Estrella R., Zhu J.-K. & Schumaker
K.S. (2003) Na+/H+ exchange activity in the plasma membrane of
Arabidopsis. Plant Physiology 132, 1041–1052.
Qiu Q.-S., Guo Y., Dietrich M.A., Schumaker K.S. & Zhu J.-K.
(2002) Regulation of SOS1, a plasma membrane Na+ /H+
exchanger in Arabidopsis thaliana, by SOS2 and SOS3. Proceedings of the National Academy of Sciences of the United States of
America 99, 8436–8441.
Quesada V., Garcia-Martinez S., Piqueras P., Ponce M.R. & Micol
J.L. (2002) Genetic architecture of NaCl tolerance in Arabidopsis. Plant Physiology 130, 951–963.
Rajendran K., Tester M. & Roy S.J. (2009) Quantifying the three
main components of salinity tolerance in cereals. Plant, Cell &
Environment 32, 237–249.
Ren Z.-H., Gao J.-P., Li L.-G., Cai X.-L., Huang W., Chao D.-Y.,
Zhu M.-Z., Wang Z.-Y., Luan S. & Lin H.-X. (2005) A rice
quantitative trait locus for salt tolerance encodes a sodium transporter. Nature Genetics 37, 1141–1146.
Rus A., Lee B.-h., Munoz-Mayor A., Sharkhuu A., Miura K., Zhu
J.-K., Bressan R.A. & Hasegawa P.M. (2004) AtHKT1 facilitates
Na+ homeostasis and K+ nutrition in Planta. Plant Physiology
136, 2500–2511.
Rus A., Baxter I., Muthukumar B., Gustin J., Lahner B., Yakubova
E. & Salt D.E. (2006) Natural variants of AtHKT1 enhance Na+
accumulation in two wild populations of Arabidopsis. PLoS
Genetics 2, 1964–1973.
Schachtman D.P. & Munns R. (1992) Sodium accumulation in
leaves of Triticum species that differ in salt tolerance. Functional
Plant Biology 19, 331–340.
Shabala L., Cuin T., Newman I. & Shabala S. (2005) Salinityinduced ion flux patterns from the excised roots of Arabidopsis
SOS mutants. Planta 222, 1041–1050.
Shi H., Ishitani M., Kim C. & Zhu J.-K. (2000) The Arabidopsis
thaliana salt tolerance gene SOS1 encodes a putative Na+/H+
antiporter. Proceedings of the National Academy of Sciences of
the United States of America 97, 6896–6901.
Shi H., Quintero F.J., Pardo J.M. & Zhu J.-K. (2002) The putative
plasma membrane Na+/H+ antiporter SOS1 controls longdistance Na+ transport in plants. The Plant Cell 14, 465–477.
Shi H., Lee B.-h., Wu S.-J. & Zhu J.-K. (2003) Overexpression
of a plasma membrane Na+/H+ antiporter gene improves salt
tolerance in Arabidopsis thaliana. Nature Biotechnology 21,
81–85.
Sunarpi H.T., Horie T., Motoda J., et al. (2005) Enhanced salt tolerance mediated by AtHKT1 transporter-induced Na+ unloading
from xylem vessels to xylem parenchyma cells. The Plant Journal
44, 928–938.
Tester M. & Davenport R. (2003) Na+ tolerance and Na+ transport
in higher plants. Annals of Botany 91, 503–527.
Uozumi N., Kim E.J., Rubio F., Yamaguchi T., Muto S., Tsuboi A.,
Bakker E.P., Nakamura T. & Schroeder J.I. (2000) The Arabidopsis HKT1 gene homolog mediates inward Na+ currents in
Xenopus laevis oocytes and Na+ uptake in Saccharomyces cerevisiae. Plant Physiology 122, 1249–1260.
Vandesompele J., De Preter K., Pattyn F., Poppe B., Van Roy N., De
Paepe A. & Speleman F. (2002) Accurate normalization of realtime quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology 3, 1–11.
Vera-Estrella R., Barkla B.J., Garcia-Ramirez L. & Pantoja O.
(2005) Salt stress in Thellungiella halophila activates Na+ transport mechanisms required for salinity tolerance. Plant Physiology 139, 1507–1517.
Wei W., Bilsborrow P.E., Hooley P., Fincham D.A., Lombi E. &
Forster B.P. (2003) Salinity induced differences in growth, ion
distribution and partitioning in barley between the cultivar Maythorpe and its derived mutant Golden Promise. Plant and Soil
250, 183–191.
Wu S.J., Ding L. & Zhu J.K. (1996) SOS1, a genetic locus essential
for salt tolerance and potassium acquisition. The Plant Cell 8,
617–627.
Wu Y., Ding N., Zhao X., Zhao M., Chang Z., Liu J. & Zhang L.
(2007) Molecular characterization of PeSOS1: the putative
Na+/H+ antiporter of Populus euphratica. Plant Molecular
Biology 65, 1–11.
© 2010 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 793–804
804 D. Jha et al.
Xue Z.-Y., Zhi D.-Y., Xue G.-P., Zhang H., Zhao Y.-X. & Xia G.-M.
(2004) Enhanced salt tolerance of transgenic wheat (Tritivum
aestivum L.) expressing a vacuolar Na+/H+ antiporter gene with
improved grain yields in saline soils in the field and a reduced
level of leaf Na+. Plant Science 167, 849–859.
Zhang H.-X. & Blumwald E. (2001) Transgenic salt-tolerant
tomato plants accumulate salt in foliage but not in fruit. Nature
Biotechnology 19, 765–768.
Zhao F.-Y., Zhang X.-J., Li P.-H., Zhao Y.-X. & Zhang H. (2006)
Co-expression of the Suaeda salsa SsNHX1 and Arabidopsis
AVP1 confer greater salt tolerance to transgenic rice than the
single SsNHX1. Molecular Breeding 17, 341–353.
Zhu G.Y., Kinet J.M. & Lutts S. (2001) Characterization of rice
(Oryza sativa L.) F3 populations selected for salt resistance. I.
Physiological behaviour during vegetative growth. Euphytica
121, 251–263.
Zhu J.-K. (2000) Genetic analysis of plant salt tolerance using
Arabidopsis. Plant Physiology 124, 941–948.
SUPPORTING INFORMATION
Additional Supporting Information may be found in the
online version of this article:
Table S1. Sequences of gene-specific primer pairs used in
QRT-PCR experiments, including Temperature of acquisition (Taq) for Q-PCR.
Please note: Wiley-Blackwell are not responsible for the
content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the
article.
Received 15 September 2009; received in revised form 4 December
2009; accepted for publication 4 December 2009
© 2010 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 793–804