Plant Physiology and Biochemistry 43 (2005) 347–354

www.elsevier.com/locate/plaphy

Cloning and characterization of a wheat vacuolar cation/proton antiporter

and pyrophosphatase proton pump
Faïçal Brini a, Roberto A. Gaxiola b, Gerald A. Berkowitz b, Khaled Masmoudi a,*
a
Plant Molecular Genetics Unit, Center of Biotechnology of Sfax, B.P’K’, 3038 Sfax, Tunisia
b
Department of Plant Science, Agricultural Biotechnology Laboratory, University of Connecticut, 1390 Storrs Road, Storrs, CT 06269-4163, USA
Received 12 July 2004; accepted 16 February 2005

Available online 17 March 2005

Abstract

Sodium at high millimolar levels in the cytoplasm is toxic to plant and yeast cells. Sequestration of Na+ ions into the vacuole through the
action of tonoplast proton pumps (an H+-ATPase in the case of yeast, and either a H+-pyrophosphatase (H+-PPase) or H+-ATPase in the case
of plants) and a Na+/H+ antiporter is one mechanism that confers salt tolerance to these organisms. The cloning and characterization of genes
encoding these tonoplast transport proteins from crop plants may contribute to our understanding of how to enhance crop plant response to
saline stress. We cloned wheat orthologs of the Arabidopsis genes AtNHX1 and AVP1 using the polymerase chain reaction and primers
corresponding to conserved regions of the respective coding sequences, and a wheat cDNA library as template. The wheat NHX cDNA cloned
by this approach was a variant of the previously reported TNHX1 gene. The vacuolar H+-PPase pump we cloned (TVP1) is the first member of
this gene family cloned from wheat; it is deduced translation product is homologous to proteins encoded by genes in barley, rice, and Arabi-
dopsis. Function of TNHX1 as a cation/proton antiporter was demonstrated using the nhx1 yeast mutant. TNHX1 was capable of suppressing
the hyg sensitivity of nhx1. Functional characterization of the wheat H+-PPase TVP1 was demonstrated using the yeast ena1 (plasma mem-
brane Na+-efflux transporter) mutant. Expression of TVP1 in ena1 suppressed its Na+ hypersensitivity. Expression analysis of salt-stressed
wheat plants showed substantial up-regulation of TNHX1 transcript levels as compared to control plants, while transcript accumulation for
TVP1 was not greatly affected by exposure of plants to salt stress.
© 2005 Elsevier SAS. All rights reserved.

Keywords: Na+/H+ antiporter; H+-pyrophosphatase; Sodium sequestration; Salt tolerance; Transcript accumulation; Wheat

1. Introduction ion accumulation in the cytosol [3,12,15,25]. The capacity

Salinity is a major constraint to crop (including wheat [22])
productivity; reducing yields on saline soils and limiting
expansion of agriculture onto previously uncultivated land
[8]. Adaptation of plants to salt stress (i.e. resumption of
growth after exposure to high soil salinity) requires cellular
ion homeostasis involving net intracellular Na+ and Cl– uptake
and subsequent vacuolar compartmentalization without toxic
Abbreviations: H+-PPase, proton pumping pyrophosphatase; hyg, hygro-
mycin; ORF, open reading frame; UTR, untranslated region; YNB, yeast
nitrogen base; YPD, yeast extract/peptone/dextrose; YPGAL, yeast
extract/peptone/galactose.
* Corresponding author. Tel.: +216 74 440 816x1092; fax: +216 74 440
818.
E-mail address: khaled.masmoudi@cbs.rnrt.tn (K. Masmoudi).
0981-9428/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.plaphy.2005.02.010
for vacuolar compartmentalization of Na+ and Cl– is an adap-
tation mechanism conserved in halophytes and glycophytes
[3,15]; however the process is more efficient in halophytes.
Vacuolar partitioning of Na+ and Cl– contributes to the main-
tenance of cellular water status. Together with K+ and organic
solute accumulation in the cytosol and organelles, Na+ and
Cl– sequestration in the vacuole balances intracellular osmotic
status of cells in salt grown plants [27]. Cellular ion exclu-
sion cannot provide complete adaptation of plants to high soil
salinity, presumably because of the osmotic stress compo-
nent of salinity stress [3]. Continued cell growth would be
restricted under osmotic stress because of an unfavorable
water balance, limiting the water uptake necessary for cell
expansion. The integrated processes of cell division and
expansion (i.e. growth) require substantial water uptake; cells
increase their volume after division by as much as 100-fold
348 F. Brini et al. / Plant Physiology and Biochemistry 43 (2005) 347–354

[20]. Vacuolar expansion is a primary mechanism underlying
this massive cell enlargement.
Energy-dependant Na+ transport (i.e. against a concentra-
tion gradient) across plant cell membranes (plasma lemma
and tonoplast) is usually coupled to the proton (H+) electro-
chemical potential established by H+-translocating pumps
[3,13,15,31]. H+ transport across these membranes increases
with salt treatment and may be attributed both to pump acti-
vation and enhanced transcription [15]. Plasma membrane
and tonoplast transporters facilitate Na+ efflux from the cyto-
sol by coupling Na+ transport to the (energetically favorable)
transport of H+. Both plasma membrane and tonoplast Na+/H+
antiporter activities increase in response to salt treatment, at
least in halophytic species [3]. Overexpression of the vacu-
olar Na+/H+ antiporter and H+-pyrophosphatase pump (H+-
PPase) has resulted in enhanced plant tolerance to both salin-
ity [2,12,36], and drought stress [12].
In Saccharomyces cerevisiae the primary pathway for Na+
extrusion is mediated by Ena1 [14,28], the plasma mem-
brane Na+-ATPase. Ena1 yeast mutants are hypersensitive to
high growth medium Na+, and expression of the Arabidopsis
tonoplast H+-PPase suppresses this mutant phenotype pre-
sumably due to increased capacity for Na+ sequestration in
the yeast vacuole [11]. Sequestration of cytosolic Na+ into
the yeast vacuole occurs through the action of Nhx1 [23].
Recent analyses of the genes involved in cation detoxifica-
tion in yeast have led to a model in which the Nhx1 Na+/H+
exchanger acts in concert with the vacuolar ATPase and the
Gef1 anion channel to sequester cations in a prevacuolar com-
partment [10,24]. This model posits that sequestration of
sodium by Nhx1 depends on the vacuolar H+-ATPase and
Gef1, the chloride channel. Gef1-mediated anion influx allows
establishment by the vacuolar H+-ATPase of a proton gradi-
ent sufficient in magnitude to drive Na+ accumulation in the
vacuole (against a concentration gradient) via Na+/H+ ex-
change.
In this study, we describe the molecular cloning of two
wheat cDNAs encoding the tonoplast H+-PPase and Na+/H+
antiporter, their expression patterns in salt-stressed plants, and
their functional characterization using heterologous expres-
sion in Na+-sensitive yeast mutants.
2. Results
2.1. Molecular characterization of wheat
TNHX1 and TVP1
The full-length cDNAs of TNHX1 and TVP1 were cloned
and sequenced as described (see Section 4). Sequence analy-
sis of the TNHX1 cDNA revealed an open reading frame
(ORF) of 1641 bp with a 3′-untranslated region (UTR) of
207 bp. Alignment of this ORF (GenBank accession no.
AY296910) with that of the previously cloned TNHX1 cDNA
(GenBank accession no. AY040245) indicated differences at
six positions. We attribute this to varietal differences in the
genomes used to generate the corresponding libraries and con-
sider our clone to be a variant of TNHX1. The TVP1 cDNA
we cloned has an ORF of 2289 bp with a 5′-UTR of 30 bp
and a 3′ noncoding region of 352 bp. A clade analysis of plant
NHX vacuolar Na+/H+ antiporters, including the wheat cDNA
cloned here, is presented in the phylogenetic tree shown in
Fig. 1A. Wheat TNHX1 (shown in bold) forms a clade with
the most closely related plant NHX homolog, HvNHX2 from

Fig. 1. Phylogenetic relationship between deduced protein sequences of plant vacuolar Na+/H+ antiporters (A) and vacuolar H+-PPase pumps (B). The Arabi-
dopsis (’At’), wheat (’T’), barley (’Hv’), and rice (’Os’) Na+/H+ antiporters are included in the analysis shown in (A). The wheat (’T’), barley (’H’), rice (’O’),
Arabidopsis (’A’), and tobacco (’Nt’) H+-PPase pumps are included in the analysis shown in (B). The wheat Na+/H+ antiporters (TNHX1 and TNHX2) can be
paired into two phylogenetic subgroups, while the wheat H+-PPase forms a unique group with the barley H+-PPase HVP2. Multiple sequence alignment was
performed using the CLUSTALW computer program [16]. The accession numbers for the protein sequences are as follows: AtSOS1 (AAF76139), Arabidopsis
thaliana; six A. thaliana (AtNhx) isogenes, AtNhx1 (AF510074); AtNhx2 (AF490586); AtNhx3 (AC011623); AtNhx4 (AB015479); AtNhx5 (AC005287) and
AtNhx6 (AC010793); OsNhx1 (AB021878), Oryza sativa (rice); HvNhx1 (AB089197) and HvNhx2 (AY247791), Hordeum vulgare (barley); TNhx1(AY296910)
and TNhx2 (AY040246), T. aestivum (wheat); TVP-1 (AY296911), T. aestivum; HVP-1 (AB032839) and HVP-2 (D13472), H. vulgare; NtVP5 (X77915),
Nicotiana tabacum (tobacco); OVP-1 (D45383), OVP-2 (D45384), OVP-3 (AB126350) and OVP-5 (AB126351), O. sativa; AVP-1 (M81892), AVP-2 (AF182813)
and AVP-3 (AB015138), A. thaliana.
 F. Brini et al. / Plant Physiology and Biochemistry 43 (2005) 347–354
barley. Fig. 1B shows a phylogenetic analysis of plant vacu-
olar H+-PPases. TVP1 (shown in bold) forms a clade with the
barley vacuolar H+-PPase HVP2.
The deduced amino acid sequence of TNHX1, and se-
quences of representative homologs from other species are
shown in Fig. 2. Hydropathy analysis (not shown) of the
TNHX1 sequence confirms the presence of the 12 transmem-
brane domains present in members of this protein family;
these domains are identified in Fig. 2. Also shown (as a boxed
area within transmembrane domain III in Fig. 2) is the
amiloride binding motif that is common to these proteins [5];
this motif is present in TNHX1. The deduced amino acid
sequence of the variant of TNHX1 that we cloned (GenBank
accession no. AY296910) is 99% similar to the amino acid
sequence of the TNHX1 previously reported (GenBank acces-
sion no. AY040245). Sequence comparison between TNHX1
and TNHX2 showed 75% identity at the nucleotide level, while
no similarity was found between the 3′-UTR of TNHX2 and
the 3′-UTR of TNHX1.
Regions of the deduced amino acid sequence of TVP1 (and
corresponding sequences of some homologs) are shown in
Fig. 3. A region of the plant vacuolar H+-PPases that is highly
conserved (G240-M287 in TVP1) is shown in Fig. 3A. Within
this region, a putative pyrophosphatase catalytic site [20] is
present (boxed region in Fig. 3A); this motif can also be found
in the TVP1 sequence. Another functional domain present in
plant vacuolar H+-PPases is the ’EYYTS’ motif shown in
Fig. 3B that is involved in H+ transport [35]. It should be
noted that all the plant vacuolar H+-PPases, including TVP1
(see Fig. 3B), contain a conserved glutamate residue (E221 in
TVP1; E229 in AVP1) [37]. It has been suggested that this
residue plays a role in coupling pyrophosphate hydrolysis to
H+ translocation across the vacuolar membrane [7,35].
2.2. Functional characterization of the wheat antiporter
TNHX1 and vacuolar H+-PPase using yeast mutants
Heterologous expression in yeast has been used in several
studies to characterize the Arabidopsis vacuolar Na+/H+ anti-
porter AtNHX1 [6,11,34]. However, little functional analysis
work has been done with NHX1 homologs from crop plants.
Xia et al. [34] have recently expressed the sugar beet (Beta
vulgaris) BvNHX1 antiporter in yeast for functional charac-
terization. Here, we used a similar strategy to undertake the
first characterization of a Na+/H+ antiporter cDNA cloned
from the cereal crop wheat. Gaxiola et al. [11] and Darley et
al. [6] demonstrated function of the Arabidopsis AtNHX1
cDNA as a vacuolar Na+/H+ antiporter by partial suppression
of hygromycin (hyg) hypersensitivity of the nhx1 yeast
mutant. Hygromycin is a cationic antibiotic that is toxic to
cells upon accumulation in the cytosol. The basis for hyg
hypersensitivity of the yeast nhx1 mutant is likely a reduc-
tion in NHX-mediated sequestration of the toxic cation in the
yeast vacuole [1,35]. As shown in Fig. 4A, the expression of
the wheat cDNA (TNHX1) we have identified as a putative
vacuolar Na+/H+ antiporter does in fact partially complement
349
the nhx1 mutant phenotype, suppressing hyg hypersensitiv-
ity. In the absence of hyg, the nhx1 mutant transformed with
any of the plasmids (along with the wild type yeast) grew on
YPD medium, but in the presence of hyg, the nhx1 mutant
grew only when transformed with the plant TNHX1 cDNA
(Fig. 4A). Yokoi et al. [35] has demonstrated that several of
the six Arabidopsis NHX genes (AtNHX2, AtNHX5) in addi-
tion to AtNHX1 encode vacuolar Na+/H+ antiporters that
complement yeast nhx1 sensitivity to hyg, as we report here
for the wheat Na+/H+ antiporter TNHX1.
In previous studies, Gaxiola et al. [11] used the Na+ hyper-
sensitivity of the ena1 yeast mutant to demonstrate function
of the Arabidopsis vacuolar H+-PPase. Results of this work
are consistent with a model whereby expression of a plant
vacuolar H+-PPase would allow for greater growth of ena1 in
the presence of high external Na+ due to increased sequestra-
tion into the vacuole of Na+ that enters the yeast cytosol. In
this prior work with the Arabidopsis H+-PPase AVP1 [11] a
gain-of-function (E427D) mutant construct of AVP1 was used
in studies employing the yeast ena1 for functional character-
ization. Here, we studied the wild type TVP1. We find that
the TVP1 cDNA also suppresses the Na+ hypersensitivity of
ena1 (Fig. 4B). All ena1 mutants along with the wild type
yeast grew on YPGAL medium with no added NaCl, while
the ena1 mutant grew in the presence of 0.5 M NaCl only
when transformed with the plant TVP1 cDNA (Fig. 4B). Our
results with TVP1 are consistent with prior studies of AVP1
expression in yeast [11,17], in suggesting that TVP1 is also
an H+-PPase pump localized to the vacuolar and/or prevacu-
olar tonoplast membrane. In work with AVP1, Kim et al. [17]
localized the plant protein to the yeast vacuole membrane as
well as demonstrating PPase activity. However, we cannot
discount the possibility that TVP1 suppression of the ena1
Na+ hypersensitivity occurs by TVP1 H+-pump action at the
yeast plasma membrane. This could possibly suppress ena1
hypersensitivity to Na+ through the action of the yeast cell
membrane Na+/H+ antiporter NHA1 [26]. The results shown
in Fig. 4 suggest that the two wheat cDNAs TNHX1 and TVP1
can function in a model eukaryote (yeast) cell as a Na+/H+
antiporter and H+-PPase, respectively.
2.3. Southern blot analysis
Southern blot analyses of genomic DNA isolated from
wheat (Triticum durum) leaves were performed using probes
corresponding to 3′-UTR sequences of the TVP1 and TNHX1
cDNAs (Fig. 5). As shown in Fig. 5A, a single band was
detected in the case of TVP1 when DNA was subjected to
digestion with HindIII, EcoRI, SacI, or XhoI. We conclude
from this hybridization pattern that the wheat genome has a
single copy of the TVP1 gene. Southern analysis of HindIII,
EcoRI and XhoI-digested DNA probed with TNHX1 also iden-
tified one hybridizing band (Fig. 5B). This results shown in
Fig. 5B indicate that the wheat genome has a single copy of
the TNHX1 gene and further, that TNHX1 and TNHX2 are not
allelic; i.e. that they are encoded by two separate genes in the
350 F. Brini et al. / Plant Physiology and Biochemistry 43 (2005) 347–354

Fig. 2. Multiple alignment of the deduced amino acid sequence of TNHX1, the Na+/H+ antiporter we cloned from wheat, along with the barley (HvNhx1), rice
(OsNhx1), Arabidopsis (AtNhx1), human (HsNHE1), and yeast (ScNhx1) sequences. Identical residues are highlighted in black, residues with conservative
substitutions are shaded in gray, and dashes indicate gaps in a sequence. Putative transmembrane domains are indicated by Roman numerals. The amiloride
binding motif is framed and the consensus sequence is indicated by the boxed residues above the alignment in the transmembrane domain III region. Numbers
flanking the sequences correspond to positions of amino acids directly next to the number.
 F. Brini et al. / Plant Physiology and Biochemistry 43 (2005) 347–354
Fig. 3. Alignment of several conserved regions of the deduced amino acid
sequence of the wheat H+-PPase TVP1 with corresponding regions of a bar-
ley (HVP1) and an Arabidopsis (AVP1) H+-PPase. Sequences were aligned
using the Clustal X program. (A) A conserved region of the proteins flan-
king the putative H+-PPase catalytic site (shown within the box). (B) The
conserved motif found in plant H+-PPases that is critical for H+-pumping by
the protein. Numbers flanking the sequences correspond to positions of amino
acids directly next to the number.
Fig. 4. Functional characterization of the wheat Na+/H+ antiporter TNHX1
(A) and the wheat H+-PPase TVP1 (B) using yeast mutants. (A) Growth of
the nhx1 yeast mutant transformed with either the empty plasmid (Nhx1), or
the TNHX1 cDNA. The yeast strains were grown on YPD medium with (+),
or without (–) 0.05 mg ml–1 hyg. (B) Growth of the ena1 yeast mutant trans-
formed with either the empty plasmid (ena1), or the TVP1 cDNA. The yeast
strains were grown on YPGAL medium with (+) or without (–) 0.5 M NaCl.
Growth of the isogenic wild type yeast strain (WT) is shown for both expe-
riments. For both TNHX1 and TVP1, results are shown for two independent
yeast clones (TNHX1-2; TNHX1-3; TVP1-1; TVP1-2). In all cases, growth
of fourfold serial dilutions of each culture is shown. Experiments shown in
this figure were repeated, with similar results (data not shown).
wheat genome. The nucleotide sequences of TNHX1 and
TNHX2 have 75% identity.
2.4. Expression pattern of TVP1 and TNHX1 in wheat
under salt stress
In order to examine the effect of salt stress on the expres-
sion of the vacuolar Na+/H+ antiporter and H+-PPase genes,
total RNA isolated from roots and shoots of wheat seedlings
was subjected to Northern blot analyses. A Northern analysis
of RNA isolated from control and salt-stressed plants using
the 3′-UTR of TNHX1 as a probe is shown in Fig. 6A. Salt
stress significantly increased the transcript levels of TNHX1
in roots and, to a lesser extent, in leaves. As suggested by the
results of the Southern analysis shown in Fig. 5B, we can
discount the possibility that the 3′-UTR TNHX1 probe hybrid-
izes to TNHX2 message. In prior studies of Arabidopsis, Gaxi-
351
Fig. 5. Genomic Southern blot analysis of wheat (T. durum) vacuolar
H+-PPase (A) and Na+/H+ antiporter (B). Wheat genomic DNA (10 µg) was
digested with the restriction endonucleases HindIII (H), EcoRI (E), XhoI
(X) or SacI (S), and hybridized with either the 32P-labelled 3′-UTR specific
TVP1 (A) or 3′-UTR specific TNHX1 (B). DNA fragments of a 1 kb ladder
were used as molecular markers (indicated on the left of each panel).
Fig. 6. Analysis of TNHX1 (A) and TVP1 (B) expression under salt stress.
Expression in roots and leaves of control wheat seedlings (C) and seedlings
subjected to irrigation with 200 mM NaCl was monitored. Ribosomal RNA
(rRNA) was used as a control. Experiments shown in this figure were repea-
ted a second time with similar results (data not shown).
ola et al. [11] found increases in transcript level of the TNHX1
homolog AtNHX1 in salt-stressed Arabidopsis seedlings. Sub-
sequently, Yokoi et al. [35] found that the transcript levels of
several, but not all of the AtNHX genes increase in response
to salinity stress.
In contrast to the strong up-regulation of TNHX1 in roots
of salt-stressed wheat seedlings, we found only a modest (if
at all) effect of salinity stress on TVP1 expression in roots
(Fig. 6B). Results shown in Fig. 6B also show no effect of
salinity stress on the level of TVP1 transcript in leaves of salt-
stressed wheat seedlings.
3. Discussion
Work presented in this report focuses on two vacuolar tono-
plast membrane transport proteins of wheat, a Na+/H+ anti-
porter (TNHX1) and an H+-PPase (TVP1). It has been previ-
ously demonstrated that overexpression of the Arabidopsis
H+-pyrophosphatase (AVP1) confers salt tolerance to yeast
strains only when the yeast contains a functional vacuolar
Na+/H+ exchanger (Nhx1) [11]. Thus, the Na+/H+ exchanger
352 F. Brini et al. / Plant Physiology and Biochemistry 43 (2005) 347–354
acts in concert with the vacuolar H+-PPase (and ATPase) to
sequester cations in the vacuole (and prevacuolar compart-
ment). Heterologous expression in yeast mutants allowed for
an indirect characterization of their functions. The approach
we take here to identify the cDNAs as encoding tonoplast ion
transport proteins is consistent with the strategy used in prior
reports to demonstrate function of the Arabidopsis homologs
AVP1 and AtNHX1. Both genes were shown to be present in
the T. durum genome. This work represents the first cloning
and functional characterization of a vacuolar H+-PPase from
the important cereal crop wheat. It has been over a decade
since the first report of the cloning of a cereal crop (barley)
H+-PPase [32]; since this time no functional characterization
of a cereal crop vacuolar H+-PPase has been published. In
addition to the characterization of TVP1 from wheat included
in our work, we became aware during the preparation of this
manuscript of a recently published paper reporting the func-
tional characterization of the barley vacuolar H+-PPase [9].
The H+-PPases are considered to form a multigene family.
Two cDNA clones (OVP1 and OVP2) encoding vacuolar
H+-PPases isolated from rice were reported [29]. Indeed, there
are more than five isoforms in rice, three isoforms in barley
[32,21] and at least three isoforms in Arabidopsis. A highly
conserved colinearity of genes between rice and wheat is well
established [19]. Here, we isolate the first isoform from wheat.
Further screening of the wheat cDNA library may lead to the
isolation of other vacuolar H+-PPase isoforms.
Recently, it has been reported in barley that salt stress
increased the transcript level of a tonoplast H+-PPase (HVP1)
in roots for a short time (5 h), and subsequently increased
that of another isoform (HVP10) after treatment for 24 h [9].
In our case with TVP1, we find only a modest (if at all)
increase in the transcript level in roots of salt-stressed plants
as compared to TVP1 levels in roots of control plants. As was
the case in barley, we found no detectable effect of salinity
stress on expression of the vacuolar H+-PPase (TVP1) in
leaves. In contrast, we found that the transcript level of the
wheat Na+/H+ antiporter gene (TNHX1) increased greatly in
roots and to some extent in leaves of wheat seedlings treated
with 200 mM NaCl. It seems that the results of transcript
accumulation indicate that the expression of TVP1 is not coor-
dinated with that of TNHX1 in roots of wheat seedlings treated
with NaCl. The greater increase of TNHX1 expression in roots
treated with salt might be due to the accumulation of Na+ in
the vacuoles of root cells. Unlike the results with wheat
reported here, expression of the vacuolar H+-PPase and
Na+/H+ antiporter appear to be coordinated in barley [9] sub-
jected to salinity stress.
Members of these families of plant proteins, tonoplast
Na+/H+ antiporters and H+-PPases, are known to be impor-
tant regulators of plant response to salinity stress. Salinity
stress limits growth and yields of wheat in many regions of
the world; enhanced sequestration of Na+ taken up into wheat
leaves could be one strategy to engineer salinity tolerance.
The results included in this report, therefore, are relevant to
this important agricultural challenge. Further study of these
wheat ion transporter genes could lead to a better understand-
ing of the molecular basis for salinity tolerance in an impor-
tant cereal crop. In addition, over-expression of the Arabi-
dopsis homologs of these genes has led to salinity (for both
the antiporter and pyrophosphatase) and drought (in the case
of the pyrophosphatase) tolerance. Our finding that both wheat
genes, the Na+/H+ antiporter and H+-PPase, confer salt toler-
ance in yeast suggests it may be worthwhile to elucidate the
contribution of these proteins to salt tolerance in a staple crop
plant such as wheat. Engineering plants to increase func-
tional levels of these proteins may be one strategy to develop
tolerance to this important abiotic stress.
4. Methods
4.1. Yeast strains and plasmids
All strains used are isogenic to W303 (ura3-1 can1-
100 leu2-3, 112trp1-1 his3-11, 15). Plasmids pRS8 and
pYES2 (Invitrogen) were used to construct PMA1/TNHX1
and pGAL1/TVP1, respectively. The pRS8 plasmid was con-
structed (Mascorro-Gallardo and Gaxiola, unpublished data)
using the pRS424 plasmid [4] as a backbone with the high
expression level PMA1 (S. cerevisiae plasma membrane
H+-ATPase) promotor [30] driving expression. The wild type
W303, and the yeast mutant strains nhx1::HIS3 and
ena1::HIS3 (see [11] for construction and/or source) were
used for the work reported here. Cells were grown in YPD
(1% (w/v) yeast, 2% (w/v) peptone, and 2% (w/v) dextrose;
Difco; Franklin Lakes, NJ), or YPGAL (1% (w/v) yeast, 2%
(w/v) peptone, and 2% (w/v) galactose; Difco). Transforma-
tion was performed using the EZ-Yeast transformation kit
(BIO 101; Carlsbad, CA).
4.2. Plasmid constructs
PCR products were generated from a wheat (Triticum aes-
tivum) root tissue cDNA library. The full-length TNHX1 ORF
was amplified with PfuTurbo DNA polymerase (Stratagene,
La Jolla, CA) using wheat cDNA library as template and prim-
ers corresponding to the 5′ and 3′ ends of the wheat gene
TNHX1 (GenBank accession no. AY040245) with BamHI and
SphI, restriction sites added, respectively. These oligonucle-
otide primers were 5′-ATAAGAATGCGGCCGCGGCAT-
GGGGCTCGATTT-3′, and 5′-ACATGCATGCCTGGGCT-
TCGACTTAACTAC-3′, respectively. The PCR product was
cloned into the pCR2.1-Topo vector (Invitrogen, Carlsbad,
CA), generating plasmid pTNHX1. After sequencing, the
TNHX1 cDNA was digested with BamHI and SphI and cloned
into pRS8 digested with the same restriction enzymes.
The wheat vacuolar H+-PPase cDNA ’TVP1’ was cloned
as follows. The 5′ end of the cDNA was amplified from the
wheat library using a degenerate primer ’HvP2’ correspond-
ing to the 5′ end of the barley H+-PPase HVP1 (accession no.
AB032839) sequence (5′-TGGCAAGGYTGRCTCTTTG-
 F. Brini et al. / Plant Physiology and Biochemistry 43 (2005) 347–354
TGCTGAAYCC-3′) and the T7 primer (5′-TAATACGACT-
CACTATAGGG-3′). The missing 3′ end of the TVP1 cDNA
was amplified from the same wheat cDNA library using a 5′
primer corresponding to a conserved region of plant (barley,
rice, Arabidopsis) vacuolar H+-PPases, and a 3′ primer cor-
responding to the polylinker region of the pYES2 plasmid.
Forward and reverse primer sequences were 5′-ACCCGTA-
CAGCATTGTTC-3′ and 5′-TGAATGTAAGCGTGAC-
ATAA-3′, respectively. The two PCR products generated from
the above reactions (corresponding to the 5′ and 3′ regions of
the target TVP1 cDNA) were cloned into the pCR2.1-Topo
vector, generating plasmids pTVP5′.1 and pTVP3′.2. Se-
quencing of pTVP5′.1 and pTVP3′.2 allowed for the genera-
tion of a new set of oligonucleotide primers corresponding to
the 5′ end and 3′ end of the TVP1 ORF. The forward primer
(with an added BamHI restriction site) and reverse primer
sequence, respectively, were 5′-CGGGATCCGGCA-
TGGCGATCCTCGGG-3′ and 5′-CTTCTAGATGTA-
CTTGAACAG-3′. The PCR product was first cloned into the
pCR2.1-Topo vector, generating plasmid pTVP1. The insert,
corresponding to the full-length TVP1 ORF was isolated with
BamHI and EcoRI digestion of pTVP1, and then cloned into
pYES2 plasmid digested with the same restriction enzymes.
The reconstituted TVP1 ORF was sequenced (GenBank acces-
sion no. AY296911).
4.3. Functional assays using yeast mutants
The nhx1 yeast mutant was transformed with either the
empty pRS8 plasmid, or the pRS8 plasmid containing TNHX1.
The two nhx1 strains, along with the wild type (W303) strain
were grown at 28 °C for 2 days in selective yeast nitrogen
base (YNB) medium lacking tryptophan (Qbiogene/Bio 101,
Carlsbad, CA) (pH 6.5). Aliquots (5 µl) of the saturated cul-
tures, and fourfold serial dilutions of the cultures were spot-
ted onto YPD plates supplemented with (+) or without (–)
0.05 mg ml–1 hyg. The strains were cultured at 28 °C for
2 days on YPD medium before growth evaluation. The ena1
yeast mutant was transformed with either the empty
pYES2 plasmid, or the pYES2 plasmid containing TVP1. The
two ena1 strains, along with the wild type (W303) strain were
grown at 28 °C for 2 days in selective YNB-ura medium. Ali-
quots (5 µl) of the saturated cultures, and fourfold serial dilu-
tions of the cultures were spotted onto YPGAL plates supple-
mented with (+) or without (–) 0.5 M NaCl. The strains were
cultured at 28 °C for 2 days before growth evaluation. The
assays used here to functionally characterize both TNHX1
and TVP1 are based on work [11] done with the same yeast
mutants to functionally characterize the Arabidopsis ho-
mologs of TNHX1 and TVP1 (i.e. AtNHX1 and AVP1, respec-
tively).
4.4. Plant growth conditions
Seeds of T. durum cv. ’Oum Rabiaa’ were germinated on
wet filter paper, transferred to hydroponic culture with one
353
quarter-strength Murashige and Skoog (MS) medium, and
grown in a growth chamber (16 h photoperiod, 25 °C) for
1 week prior to initiation of salinity stress. A group of plants
were maintained on the same medium (control treatment),
and for a second group of plants, the growth medium was
replaced with one quarter-strength MS medium with 200 mM
NaCl added. Plants were grown for 3 days further under these
conditions.
4.5. Genomic blot hybridization
Genomic DNA was isolated from leaves of wheat as
described previously [18]. Samples (10 µg) of the DNA were
digested separately with HindIII, EcoRI, XhoI and SacI restric-
tion endonucleases. Digested DNA was electrophoretically
fractionated on a 1% (w/v) agarose gel and transferred to
Hybond N+ nylon membranes (Amersham) by capillary trans-
fer. After prehybridization with 200 µg ml–1 of salmon sperm
DNA, the immobilized DNAs were hybridized overnight with
probes that were 32P-labeled using the Rediprime random
primer labeling kit (Amersham). A PCR amplification prod-
uct corresponding to the 3′-UTR was used as a probe for
TNHX1 and TVP1. The primers used to generate the TNHX1
probe are 1481:5′-GCTCGGCTCATCGTGTA-3′ and
1840:5′-CTGAACGAGATTAATTTACAG-3′. The primers
used to generate the TVP1 probe are 2302:5′-CTGTTCAA-
GTACATCTAGAAG-3′ and 2620:5′-CAAGGGTTCAA-
ACATTATCG-3′. After hybridization and washing, mem-
branes were exposed to an imaging plate, and the radioimage
of the plate was analyzed using the Molecular Imager FX-Plus
(Bio-Rad, France).
4.6. Northern blot hybridization
Total RNA (10 µg) of each sample (roots and leaves from
control and salt-stressed wheat seedlings) was extracted by
phenol/LiCl precipitation [33]. The RNA was electrophore-
sed on 1% (w/v) formaldehyde-agarose gels, and transferred
to Hybond N+ membranes. Northern blot hybridizations
(using the 3′-UTR TNHX1 and TVP1 DNA probes men-
tioned above) were performed at 42 °C in 50% (v/v) forma-
mide, 0.12 M Na2HPO4, pH 7.2, 0.25 M NaCl, 7% (w/v)
SDS and 1 mM EDTA. After hybridization with the DNA
probes described above, membranes were exposed to an imag-
ing plate and the radioimage of the plate was analyzed using
the Molecular Imager FX-Plus.
Acknowledgements
We thank Dr. Daniel Schachtman (Donald Danforth Plant
Science Center, St Louis, MO) for the gift of the wheat cDNA
library. This work was supported jointly by grants from the
Secrétariat d’Etat à la Recherche Scientifique et à la Tech-
nologie (SERST), Tunisia, and the USDA-RSED awards
FG-TN102 and 5831483022. R.A.G. was supported by the
354 F. Brini et al. / Plant Physiology and Biochemistry 43 (2005) 347–354
National Research Initiative of the USDA Cooperative State
Research, Education, and Extension Service under grant no.
2001-35100-10772. G.A.B. was supported by NSF award no.
MCB-0344141.
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