Download Alkali cation exchangers: roles in cellular homeostasis and stress

Journal of Experimental Botany, Vol. 57, No. 5, pp. 1181–1199, 2006
Plants and Salinity Special Issue
doi:10.1093/jxb/erj114 Advance Access publication 2 March, 2006
Alkali cation exchangers: roles in cellular homeostasis
and stress tolerance
José M. Pardo*, Beatriz Cubero, Eduardo O. Leidi and Francisco J. Quintero
Instituto de Recursos Naturales y Agrobiologı´a, Consejo Superior de Investigaciones Cientı´ficas,
Reina Mercedes 10, Sevilla 41012, Spain
Received 24 October 2005; Accepted 11 January 2006
Abstract
Introduction
Uptake and translocation of cations play essential
roles in plant nutrition, signal transduction, growth,
and development. Among them, potassium (K1) and
sodium (Na1) have been the focus of numerous
physiological studies because K1 is an essential
macronutrient and the most abundant inorganic cation
in plant cells, whereas Na1 toxicity is a principal
component of the deleterious effects associated with
salinity stress. Although the homeostasis of these two
ions was long surmised to be fine tuned and under
complex regulation, the myriad of candidate membrane
transporters mediating their uptake, intracellular distribution, and long-distance transport is nevertheless
perplexing. Recent advances have shown that, in
addition to their function in vacuolar accumulation of
Na1, proteins of the NHX family are endosomal transporters that also play critical roles in K1 homeostasis,
luminal pH control, and vesicle trafficking. The plasma
membrane SOS1 protein from Arabidopsis thaliana,
a highly specific Na1/H1 exchanger that catalyses Na1
efflux and that regulates its root/shoot distribution, has
also revealed surprising interactions with K1 uptake
mechanisms by roots. Finally, the function of individual members of the large CHX family remains largely
unknown but two CHX isoforms, AtCHX17 and AtCH23,
have been shown to affect K1 homeostasis and the
control of chloroplast pH, respectively. Recent advances on the understanding of the physiological processes that are governed by these three families of
cation exchangers are reviewed and discussed.
Plant nutrition depends on the activity of membrane
transporters that translocate minerals from the soil into
the plant and mediate their intra- and intercellular distribution. The genome of Arabidopsis thaliana appears to
encode >800 membrane transport proteins, 65% of which
are secondary active transporters. Classification of these cotransporters has been based on both phylogeny and their
known or predicted functions as transporters for cations,
anions, and organic compounds, including sugars and
amino acids (Saier, 2000). In plants, most co-transporters
are energized by the proton electrochemical gradient
generated by primary proton pumps working in all cell
membranes, but alternative couplings also exist. Multigene
families, whose members often exhibit overlapping expression patterns and a high degree of sequence homology,
encode most types of plant membrane transporters. Furthermore, more than one transporter family transports many
inorganic nutrients. Their extensive array of membrane
transporters may provide plants with flexible strategies to
cope with fluctuations in their environment and to minimize
the adverse effects of nutrient deficiency and an excess of
toxic ions.
Computer-assisted phylogenetic analyses of the completed Arabidopsis genome sequence have revealed a large
family of putative cation/H+ antiporters (Mäser et al.,
2001). Based on the relative electrochemical gradients of
their substrates, cations, and protons, most if not all of these
exchangers are thought to extrude cations from the cytosol
to the outside across the plasma membrane or into intracellular compartments, including the vacuole. The best
examples of these exchangers are the Ca2+/H+ and Na+/
H+ exchangers at the vacuole membrane that extrude Ca2+
and Na+, respectively, from the cytosol to maintain low
cytosolic concentrations (Blumwald, 2000; Hepler, 2005).
Key words: Endosomes, intracellular localization, ion exchangers, membrane transport, mineral nutrition, pH regulation,
potassium, salinity, sodium, vacuole, vesicle trafficking.
* To whom correspondence should be addressed. E-mail: [email protected]
ª The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
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1182 Pardo et al.
In addition, similar transport activities are present in the
plasma membrane and organelles (Pardo and Quintero,
2002; Hepler, 2005). Up to four phylogenetic subfamilies
of cation/H+ exchangers have been identified within the
Arabidopsis genome that may exchange Ca2+, Na+, and K+
with H+ (Mäser et al., 2001). The CaCA gene subfamily of
Ca2+/H+ exchangers contains 11 members that have been
named CAX1 to CAX11. CAX1 and CAX2, but not CAX3
or CAX4, suppressed defective vacuolar Ca2+ transport in
a yeast vcx1 mutant (Cheng et al., 2002). Accumulated
evidence indicates CAX1, CAX2, and CAX3 may play
a central role in Ca2+ and metal (Mn2+, Cd2+) sequestration
into the vacuole, but the other family members remain to be
characterized. AtMHX, a protein related phylogenetically
to the CAX family, facilitates H+-coupled transport of Mg2+
and Zn2+ into plant vacuoles (Shaul et al., 1999). There are
two members of the NhaD subfamily (NHD1 and NHD2)
that have similarity to Na+/H+ antiporters previously found
in bacteria, both of which remain uncharacterized. In
Arabidopsis, there are eight members of the CPA1 family,
including the best characterized proteins, AtNHX1 and
SOS1, that catalyse Na+/H+ exchange at the tonoplast and
plasma membrane, respectively (Blumwald, 2000; Qiu
et al., 2002). The large CPA2 subfamily has 33 members
that include 28 CHX proteins thought to mediate cation/H+
exchange and five homologues of the K+/H+ antiporter
AtKEA1. Members of this family are just beginning to be
characterized and the progress made so far will be discussed
in this review.
The focus of this review is on the major alkali cation
exchanger families for which initial characterizations have
been achieved for individual members with respect to K+
and Na+ homeostasis. K+ is an indispensable macronutrient
and the most abundant cation in plants. K+ is essential for
many metabolic processes and a major contributor to cell
turgor. In Arabidopsis, K+ transport can occur through
members of the HAK/KUP/KT transporter family, KAT
and AKT channel families, NHX exchangers, and possibly
also via other non-characterized transporters such as those
belonging to the CHX and KEA families. In spite of these
apparent redundancies, individual transporters are thought
to achieve diverse and specialized functions, both at the
subcellular and organismic levels. By contrast, Na+ is
normally a non-essential element that is toxic to many plant
species when present in high concentrations. Na+ stress
often results in deficiency of K+ because of the physicochemical similarities between Na+ and K+ (Maathuis and
Amtmann, 1999). For instance, K+ uptake by HAK/KUP/
KT transporters is inhibited by Na+ (Santa-Maria et al.,
1997), and inhibition of the Arabidopsis AKT1 K+ channel
by intracellular Na+ has also been reported (Qi and
Spalding, 2004). By contrast, at low K+ availability,
moderate levels of Na+ actually promote plant growth by
replacing K+ in its role as provider of turgor (RodriguezNavarro, 2000). In low K+ conditions, Na+ can stimulate
high-affinity uptake of K+ in Arabidopsis (Spalding et al.,
1999). In barley, high-affinity Na+ uptake was detected in
roots of plants exposed to a K+-free medium. This Na+
uniport activity was insensitive to external K+ at the
beginning of K+ starvation and inhibited by K+ several
hours later (Haro et al., 2005). Besides their roles in
regulating the uptake and distribution of K+ and Na+, plant
alkali cation exchangers are emerging as critical regulators
of basic cellular processes such as the regulation of luminal
pH regulation in endosomes, vesicle trafficking, and protein sorting. Moreover, genetic studies with knockout mutants and transgenic lines with ectopic overexpression of
these transporters are revealing unexpected implications on
ion nutrition. In this review, recent developments on the
current understanding of the function of alkali cation exchangers are summarized and discussed.
Endosomal exchangers of the NHX family
Phylogeny and subcellular localization
The Arabidopsis AtNHX1 protein was the first Na+/H+
exchanger identified in plants (Gaxiola et al., 1999). Since
then, the number of homologous NHX transporters identified has grown dramatically. DNA sequences encoding
NHX proteins from >60 plant species, including gymnosperms and dicotyledonous and monocotyledonous angiosperms, have been deposited in the different datasets of
GenBank. Following the phylogenetic classification of
Saier (2000), plant NHXs are members of the NHE/NHX
subfamily of Na+/H+ exchangers, a subgroup of the cation
antiporter CPA1 family that is present across all taxonomic
groups of eukaryotes. With the sole known exception of
yeast, where a single NHX1 gene exists, NHE/NHX exchangers are present as multiple isoforms in all genomes
sequenced to date. Plant NHX exchangers are proteins of
about 550 residues in length and have the typical transporter topology of 10–12 transmembrane domains with
a hydrophilic C-terminal tail that is thought to be cytosolic.
Recently, however, luminal localization of the C-terminal
domain of the Arabidopsis vacuolar exchanger AtNHX1
has been reported (Yamaguchi et al., 2003). The highest
sequence homology among NHXs occurs in the N-terminal
part that forms the membrane pore, whereas C-terminal
domains are more dissimilar. In animals, where the NHE
family has been studied in great detail, the C-terminal
domain regulates ion translocation by the membranous
region of the molecule. Variation in amino acid sequences
of the C-terminus probably reflects differential regulation
of each isoform (Putney et al., 2002). In addition to the
conserved domain of the CPA superfamily (Pfam00999),
NHE/NHX exchangers have a small conserved stretch of 14
residues in the fourth transmembrane segment, with the
consensus FF(I/L) (Y/F)LFLLPPI thought to be the binding
site of the drug amiloride and its derivatives, which are
inhibitors of this family of exchangers (Putney et al., 2002).
Alkali cation exchangers 1183
On the basis of protein sequence similarity, the NHE/
NHX subfamily can be classified in two major groups that
have been named plasma membrane (PM) and intra-cellular
(IC) according to their subcellular localization (Brett et al.,
2005a). The PM group is exclusively present in animal
cells, whereas members of the IC group can be found in
animals, plants, and fungi, with the exception of NHE8-like
exchangers that are found only in animals and would have
appeared later in evolution to fulfil specific physiological
needs of animal cells (Brett et al., 2005a) (Table 1). All
plant NHXs characterized to date are assigned to the IC
group and can be further divided into two main groups
that will be denoted herein as class I and class II. In
Arabidopsis, members of the class-I category (AtNHX1–
4) are 56–87% similar to each other, whereas AtNHX5 and
6 (class II) are 79% similar but only 21–23% similar to
class-I isoforms (Yokoi et al., 2002). All NHX proteins of
class I characterized to date are localized in the vacuolar
membrane and form a separate clade within the IC group
that is composed exclusively of plant exchangers (Fig. 1).
By contrast, class-II members are found in endosomal
vesicles of plants (Table 1) and homologous proteins with
various endosomal localizations are also present in animals
and fungi (Table 1). In Arabidopsis and rice, where the
whole genomic sequence is known, the size of the NHX
gene family is almost identical. Arabidopsis contains six
NHX genes and rice has five, which are distributed in
a similar way: AtNHX1–4 and OsNHX1–4 constitute class
I, and AtNHX5–6 and OsNHX5 constitute class II (Fig. 1).
Members of the class-I category of Arabidopsis and
rice show 54–87% similarity. Similarity among class-II
members is 72–79%, but they are only 21–23% similar to
class-I isoforms. These data indicate that divergence
between class-I and class-II exchangers in plants occurred
before the separation of dicotyledons and monocotyledons.
The selectivity for ion substrate seems to be an additional
distinctive feature of each subgroup. Vacuolar exchangers
of class-I catalyse Na+/H+ or K+/H+ exchange with equal
affinity (Venema et al., 2002; Apse et al., 2003), whereas
the endosomal class II shows a preference for K+ over
Na+ as substrate (Venema et al., 2003). All these differential characteristics suggest that vacuolar and endosomal
exchangers play distinct roles in planta as discussed below.
Ion specificity
Plant antiporters were originally described exchanging
Na+/H+ at the tonoplast of beetroots and they were consequently related to Na+ sequestration into the vacuole, a critical feature for salt tolerance (Blumwald and Poole, 1985;
Niemietz and Willenbrink, 1985). The Na+/H+ exchange
was electroneutral and driven by the vacuolar proton
gradient established by the activity of the proton pumps
V-ATPase and V-PPase (Blumwald, 1987). Tonoplast
vesicles from beetroot were able to exchange Na+ and K+
by H+, but amiloride inhibited Na+ transport activity and
not K+ exchange, suggesting that separate entities mediated
Na+/H+ and K+/H+ exchanges (Blumwald and Poole,
1985). In tonoplast vesicles from salt-treated barley roots,
a Na+/H+ antiporter was induced showing no significant
activity with other monovalent cations (K+, Li+, Cs+, Rb+)
(Garbarino and DuPont, 1988). By contrast, other studies
performed with cotton or sunflower roots found antiporter
activity exchanging both K+ and Na+ at similar rates
(Hassidim et al., 1990; Ballesteros et al., 1997). In the
halophytes Atriplex numularia, Plantago maritima, and
Table 1. NHX/NHE homologues for which the subcellular localization has been experimentally determined
Organism
Gene
Saccharomyces cerevisiae
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Atriplex gmelini
Ipomoea nil
Ipomoea nil
Ipomoea tricolor
Oryza sativa
Hordeum vulgare
Beta vulgaris
Arabidopsis thaliana
Lycopersicon esculentum
Homo sapiens
Homo sapiens
Homo sapiens
Caenorhabditis elegans
Homo sapiens
Caenorhabditis elegans
Caenorhabditis elegans
Caenorhabditis elegans
NHX1
NHX1
NHX2
NHX4
NHX1
NHX1
NHX2
NHX1
NHX1
NHX2
NHX1
NHX5
NHX2
NHE6
NHE7
NHE9
NHX5
NHE8
NHX3
NHX8
NHX9
Type
Class
Class
Class
Class
Class
Class
Class
Class
Class
Class
Class
Class
Class
Class
Class
Class
I
I
I
I
I
I
I
I
I
I
II
II
II
II
II
II
Subcellular localization
Reference
Prevacuolar compartment
Vacuole
Vacuole
Vacuole
Vacuole
Vacuole
Vacuole
Vacuole
Vacuole
Vacuole
Vacuole
Endosomal compartment
Endosomal compartment
Endosomal compartment
Trans-Golgi network
Endosomal compartment
Intracellular
Mid- to trans-Golgi
Intracellular
Intracellular
Intracellular
Nass and Rao, 1998
Yokoi et al., 2002
Yokoi et al., 2002
B Cubero and JM Pardo, unpublished results
Hamada et al., 2001
Yamaguchi et al., 2001
Ohnishi et al., 2005
Yoshida et al., 2005
Fukuda et al., 2004
Vasekina et al., 2004
Xia et al., 2002
B Cubero and JM Pardo, unpublished results
B Cubero and K Venema, unpublished results
Brett et al., 2002
Numata and Orlowski, 2001
Nakamura et al., 2005
Nehrke and Melvin, 2002
Nakamura et al., 2005
Nehrke and Melvin, 2002
Nehrke and Melvin, 2002
Nehrke and Melvin, 2002
1184 Pardo et al.
Fig. 1. Phylogenetic tree of intracellular NHE/NHX exchangers.
Sequence alignment was performed using Clustal X with the Arabidopsis
and rice NHXs families and members of the intracellular (IC) group
whose subcellular localization has been experimentally demonstrated.
The IC group is clearly divided in two different clades corresponding to
class I and class II, as described in the text. GenBank accession numbers
are: AtNHX1, AAF21755; AtNHX2, AAM08403; AtNHX3,
AAM08404; AtNHX4, AAM08405; AtNHX5, AAM08406; AtNHX6,
AAM08407; OsNHX1, AAQ63678.1; OsNHX2, NP_910977.1;
OsNHX3, AK072026; OsNHX4, AP008212; OsNHX5, AK072114;
AgNHX1, BAB11940.1; InNHX1, BAB16380.1; InNHX2, BAD91201;
ItNHX1,
BAB60901.1;
HvNHX2,
BAC56698.1;
LeNHX2,
CAC83608.1; HsNHE6, NP_006350.1; HsNHE7, NP_115980.1;
HsNHE9, NP_775924.1; CeNHX5, NP_741891.1.
Mesembryanthemum crystallinum, the Na+/H+ antiport
activity was greater than the K+/H+ exchange (Hassidim
et al., 1990; Staal et al., 1991; Barkla et al., 1995).
Monitoring by NMR of luminal pH in vacuoles of
Catharanthus roseus showed that Na+, K+, and Ca2+
produced vacuolar alkalinization, but also that Na+ was
more effective than equimolar K+ concentrations to induce
luminal alkalinization (Guern et al., 1989). These studies
using biochemical approaches with whole vacuoles or
tonoplast vesicles are of limited use for the assignment
of discrete ion exchanges to specific transport entities
because an array on different exchangers may contribute
additively to the recorded ion fluxes and, consequently, the
substrate specificity of individual transporters cannot be
discerned with confidence.
The molecular cloning of genes encoding ion transporters has permitted a systematic, case-by-case approach
to the problem of substrate specificity. The first plant Na+/
H+ antiporter of the NHX family was cloned in Arabidopsis
(Gaxiola et al., 1999). Expression of AtNHX1 in an nhx1
yeast mutant suppressed its NaCl sensitivity and enhanced
intracellular compartmentalization of Na+ (Gaxiola et al.,
1999; Quintero et al., 2000). Experiments with vacuolar
vesicles isolated from yeast expressing AtNHX1 showed
low-affinity electroneutral Na+/H+ exchange (Darley et al.,
2000). Although initial reports with leaf vacuoles from
transgenic Arabidopsis overexpressing AtNHX1 indicated
that Na+/H+ exchange activity was much higher than in
control plants without affecting K+/H+ exchange (Apse
et al., 1999), subsequent work with transgenic tomato
overexpressing AtNHX1 showed enhanced antiport activity with both Na+ and K+, albeit with lower specificity for
K+ (Zhang and Blumwald, 2001). To study further its
substrate selectivity, a histidine-tagged AtNHX1 protein
was purified to homogeneity and reconstituted into artificial
lipid vesicles where AtNHX1 was the only transport protein present (Venema et al., 2002). The AtNHX1 protein
catalysed Na+ and K+ transport with similar affinity in the
presence of an imposed pH gradient. Li+ and Cs+ ions were
also transported with lower affinity, but organic cations
were not. This is in agreement with the ability of AtNHX1
to increase the Na+ and Li+ tolerance of yeast (Quintero
et al., 2000; Yokoi et al., 2002). Indirect evidence from
restored tolerance of yeast nhx1 mutants to NaCl and KCl
suggests that vacuolar proteins OsNHX1 from rice, and
InNHX1 and InNHX2 from Ipomoea nil (morning glory),
also have dual Na+ and K+ specificity (Fukuda et al., 2004;
Ohnishi et al., 2005). Collectively, the evidence gathered
suggests that NHX proteins of class I, which are resident
in the tonoplast, function as (Na+,K+)/H+ exchangers that
transport alkali cations with relatively low selectivity.
Interestingly, topological studies with AtNHX1 showed
that deletion of the C-terminus modified the cation specificity of the antiport when expressed in yeast, increasing the transport rate of Na+ over K+ in exchange for
vacuolar H+ (Yamaguchi et al., 2003). These results imply
that Na+/K+ selectivity may be under physiological control
through a regulatory C-terminal domain.
The only class-II NHX antiporter of plants whose
activity has been analysed in any detail is LeNHX2 from
tomato (Venema et al., 2003), which by amino acid
sequence comparison is most similar to AtNHX5 of
Arabidopsis (Fig. 1). The LeNHX2 protein co-localizes
with prevacuolar and Golgi markers in both yeast and
plants. LeNHX2 complemented the salt- and hygromycinsensitive phenotype of nhx1 yeast mutant and affected
the accumulation of K+, but not Na+, in intracellular compartments. Purified LeNHX2 reconstituted in proteoliposomes catalysed K+/H+ exchange but only minor
Na+/H+ exchange in vitro. Indirect evidence suggests that
AtNHX5 shares with LeNHX2 the same K+ preference.
The enhanced K+ and Na+ accumulation in nhx1 yeast
mutant expressing the vacuolar class-I isoforms AtNHX1
and AtNHX2 indicates that both were able to transport K+
and Na+ (Yokoi et al., 2002). By contrast, an nhx1 yeast
mutant transformed with AtNHX5 (class II) accumulated
Alkali cation exchangers 1185
+
comparatively less Na than AtNHX1 and AtNHX2, and
conveyed negligible tolerance to Li+ (Yokoi et al., 2002).
Moreover, a translational AtNHX5:GFP fusion localized
to the prevacuolar compartment of onion cells (B Cubero
and JM Pardo, unpublished results). Similar results were
reported for the human NHE7 exchanger, a mammalian
homologue of class-II exchangers. NHE7 localizes in transGolgi vesicles and catalyses K+ or Na+ exchange (Numata
and Orlowski, 2001).
Taken together, these data suggest that diverse subcellular localizations of class-I and class-II NHX proteins
in plants (Table 1) correlate with distinct substrate-ion
specificities and physiological roles. Vacuolar isoforms
(class I) are low-selectivity transporters exchanging alkali
cations and protons. In physiological terms, this dual
affinity implies that under normal growth conditions
class-I NHX proteins would mediate the accumulation of
K+ and Na+ into vacuoles, thereby contributing to osmotic
regulation and the generation of turgor essential for cell
expansion. Indeed, Arabidopsis plants bearing a knockout
in gene NHX1 show impaired leaf expansion, although this
phenotype has not been unequivocally linked to vacuolar
dysfunction (Apse et al., 2003). Under salinity stress, Na+
transport would be preferred simply because of the greater
abundance of this ion within cells or by modulation of ion
specificity through a regulatory mechanism involving the
C-terminal part of the protein (Yamaguchi et al., 2003). In
this regard, it would be of interest to test the ion selectivity
of NHX proteins from salt-tolerant species. The capability
for alkali cation transport in exchange for protons also
poses NHX proteins as prime candidates for pH regulation,
like their animal NHE counterparts. Presumably, the
localization of class-II NHX proteins in endosomal compartments other than the vacuole imposes restrictions on ion
selectivity since proper functioning of the endomembrane
system must rely on K+/H+ exchange for pH regulation to
prevent undue accumulation of potentially toxic Na+ into
the endosomal lumen. For instance, K+ and, to a much
lesser extent, Na+ and Li+ act as modulators of the activity of the Kex2/furin family of endoproteases required for
the maturation of proteins along the secretory pathway in
yeast and animal cells (Rockwell and Fuller, 2002).
Roles in stress responses
Selective ion uptake and differential ion compartmentalization are main features that explain disparities in tolerance
to salinity between glycophytes and halophytes (Flowers
et al., 1977; Greenway and Munns, 1980; Jeschke, 1984).
Salinity affects nutrient acquisition by interfering with K+
uptake by carriers and channels (Maathuis and Amtmann,
1999). At the cellular level, intracellular ion sequestration
into vacuoles for osmotic adjustment, strong ion selectivity
in the cytosol (preference of K+ over Na+), and accumulation of compatible (non-toxic) organic solutes in the
cytosol to equilibrate water potential across the tonoplast
are widely accepted mechanisms contributing to salt
tolerance (Greenway and Munns, 1980; Gorham et al.,
1985). The sequestration of ions that are potentially
damaging to cellular metabolism (e.g. Clÿ, Na+) into the
vacuole while maintaining high K+/Na+ ratios in the cytosol
would provide the osmotic driving force required for water
uptake in saline environments and, at the same time,
provide plants with an efficient instrument for ion detoxification (Greenway and Munns, 1980; Yeo, 1983).
The biochemical characterization of Na+/H+ antiporters
in the tonoplast supplied the evidence for a mechanism of
Na+ accumulation in the vacuole (Blumwald and Poole,
1985). Salt treatment of sugar beet suspension cells led to
an increase in the Vmax of Na+/H+ antiport activity
(Blumwald and Poole, 1987). The induction of vacuolar
ATPase (protonmotive force for the cation exchanger) and
Na+/H+ antiport activity by salt stress has been recorded in
glycophytes and halophytes (Garbarino and DuPont, 1988;
Barkla et al., 1995, 1999; Ballesteros et al., 1997; Parks
et al., 2002). In some salt-sensitive species (Plantago
media), no antiport activity could be recorded after salt
treatment (50 mM NaCl) (Staal et al., 1991). However, in
glycophytes bearing limited salt tolerance, like cotton
and sunflower, a detectable (Na+,K+)/H+ antiport activity
was shown in the absence of salt (Hassidim et al.,
1990; Ballesteros et al., 1997). Thus, biochemical and
physiological data support the role of Na+/H+ antiporters
regulating cytoplasmic Na+ concentration by vacuolar
sequestration when Na+ trespasses on the plasma membrane selectivity barrier. Differences between glycophytes
and halophytes in the basal and inducible antiport activity and ion selectivity might be related to their degree of
salt tolerance.
Osmotic stress rather than ion-specific stress seems to
activate NHX activity at the vacuole since mannitol and
KCl induced AtNHX1, AtNHX2, and OsNHX1 mRNA
abundance as much as NaCl (Gaxiola et al., 1999; Shi
and Zhu, 2002; Yokoi et al., 2002; Fukuda et al., 2004). In
yeast, ScNHX1 provided resistance to hyperosmotic shock
(Nass and Rao, 1999). After exposure to sudden hyperosmotic stress, wild-type cells showed rapid shrinkage of
their vacuoles but resumed growth earlier than nhx1 mutant
cells. Abscisic acid (ABA), a drought- and osmotic stressrelated hormone, enhanced expression of AtNHX1 and
AtNHX2 in Arabidopsis (Quintero et al., 2000; Yokoi et al.,
2002), LeNHX2 in tomato (Venema et al., 2003), and
GhNHX1 in cotton (Wu et al., 2004), but it failed to modify
antiport activity in Mesembryanthemum crystallinum
(Barkla et al., 1999). Differences in the ABA responsiveness of NHX genes may provide further clues to elucidate
the different mechanisms of regulation of antiport activity
in salt-sensitive and -tolerant species. Gene up-regulation
of AtNHX1 and AtNHX2 in Arabidopsis was dependent
on both ABA biosynthesis and signalling, since the stress
1186 Pardo et al.
response was greatly reduced in aba2-1, aba3-1, and
abi1-1 mutants but not in abi2-1 (Shi and Zhu, 2002;
Yokoi et al., 2002). Transcript abundance of AtNHX1 and
AtNHX2 was greater in sos1, sos2, or sos3 mutants,
presumably as a result of their exacerbated Na+ uptake
(Yokoi et al., 2002).
Overexpression of NHX antiporters has been used to
improve salt tolerance in several plant species (Apse et al.,
1999; Zhang and Blumwald, 2001; Zhang et al., 2001;
Ohta et al., 2002; Fukuda et al., 2004; Xue et al., 2004; Wu
et al., 2004, 2005). Transformed plants showed improved
plant survival and increased shoot growth over the control
lines under salt stress. Arabidopsis overexpressing AtNHX1
grew better than the wild type and accumulated Na+ in the
shoot, but the extent to which ectopic expression of
AtNHX1 increased Na+ concentration in tissues relative to
controls was not determined (Apse et al., 1999). Rice has
been transformed to overexpress NXH antiporters from
a halophyte (AgNHX1, Atriplex gmelini) (Ohta et al., 2002)
or its own (OsNHX1) (Fukuda et al., 2004). The AgNHX1plants showed greater survival rates after an osmotic shock
(300 mM NaCl, 3 d) (Ohta et al., 2002). Unfortunately,
only leaf Na+ contents on a dry-matter basis were reported,
offering no relevance for physiological interpretation as
K+ content and/or plant water status parameters were not
presented. Cells derived from OsNHX1-overexpressing
plants and growing at 100–200 mM NaCl showed greater
growth and Na+ concentration but lower K+ content than
controls (Fukuda et al., 2004). Surprisingly, K+ and Na+
concentrations in leaves of transformed plants did not show
significant differences (Fukuda et al., 2004). Ryegrass
plants transformed with OsNHX1 showed greater leaf Na+,
K+, and proline contents than control plants (Wu et al.,
2005). By contrast, leaves from transgenic wheat expressing AtNHX1 accumulated lower levels of Na+ but higher
levels of K+ than non-transgenic plants (Wu et al., 2005).
These conflicting reports call for a cautionary note.
Heterologous expression of proteins could modify the ion
selectivity of NHX antiporters, particularly if ion selectivity
by individual NHX proteins is under physiological control
that could be distorted by ectopic expression. Heterologous
expression of other ion exchangers has been reported to
result in dominant phenotypes of distorted ion homeostasis (Hirschi, 1999). Furthermore, relevant information is
still missing about the way NHX expression affects ion
compartmentalization within the cell, overall ion homeostasis, and osmotic adjustment, in order to fulfil the basic
physiological premises that sustain the increase in salt
tolerance.
Transient shifts of intracellular and apoplastic pH are
essential steps in several signal transduction processes.
Cytoplasmic acidification, combined with alkalinization
of the apoplast, loss of cellular K+, influx of external Ca2+,
and an oxidative burst are rapidly induced by microbial
elicitors. The increase in external pH originates from an
influx of protons, which contributes to cytoplasmic acidification. In addition, lysophosphatidylcoline (LPC) generated by phospholipase A2 at the plasma membrane triggers
the release of vacuolar protons via the activation of a Na+dependent proton efflux that was inhibited by amiloride
(Viehweger et al., 2002). In the presence of 1 lM LPC, the
proton efflux rate saturates at 10 mM Na+, a physiological
concentration of Na+ in the cytosol. These data suggest the
involvement of an NHX-like exchanger at the vacuolar
membrane that is stimulated by LPC, and have broader
implications for pH-controlled signal relay by this family
of proteins. The current availability of Arabidopsis knockout mutants in genes encoding vacuolar NHX isoforms
should allow the unequivocal assignment of function in
biotic-stress signalling to specific NHX exchangers.
Endosomal pH regulation
Cellular functioning requires the maintenance of pH optima
for the enzymes that participate in the different metabolic
pathways through mechanisms of pH stabilization often
called pH-stat. Plant metabolism generates H+ or OHÿ ions
that should be balanced to preserve intracellular pH (Raven,
1985). Biotic or abiotic stresses also induce cytoplasmic pH
changes triggering adaptive metabolic reactions (Sakano,
2001). The concerted transport of ions across membranes
may compose a biophysical pH-stat. Electrophysiological
studies suggest that tonoplast antiporters are among the
mechanisms responsible for the regulation of cytoplasmic
and vacuolar pH and of ion gradients induced by salt stress
(Carden et al., 2003).
The critical role of NHX exchangers in regulating
vacuolar pH is best illustrated by the flower colour
transition in Ipomoea spp. The petal colour of Ipomoea
changes from red-purple in flower buds to blue in the fully
open flower, and the same anthocyanin pigment accumulated in vacuoles is responsible for both colours. Anthocyanins give a blue colour in alkaline solution and a red
colour in an acidic environment. The flower colour
transition from red to blue is accompanied by an increase
in the pH of petal vacuoles from 6.6 to 7.7 in one of the few
instances in which vacuolar pH is alkaline relative to the
cytosol (Yoshida et al., 1995). Also during the colour
transition there is a simultaneous increase in protein levels
and activities of V-PPase, V-ATPase, and PM-ATPase, and
the de novo appearance of NHX1, whose polypeptide
and activity (measured as Na+/H+ exchange) was still
absent 6 h before the fully open stage was reached (Yoshida
et al., 2005). Insertion of a transposable element in the
InNHX1 gene of Ipomoea nil partly abrogated the vacuolar
pH shift and flower colour transition (Fukuda-Tanaka
et al., 2000; Yamaguchi et al., 2001). InNHX2, another
isoform that is also expressed in vegetative tissues may
co-operate with InNHX1 to control the alkaline pH shift
in petal vacuoles (Ohnishi et al., 2005).
Alkali cation exchangers 1187
Vesicle trafficking
Eukaryotic cells are spatially structured in a complex and
functionally co-ordinated system of internal membranes
that serve to carry proteins to their site of action. These
internal membranes subdivide the cell into a series of
membrane-bound compartments, defined by their lipid and
protein composition, and comprise the organelles of the
secretory and endocytic pathways: the endoplasmic reticulum (ER), the Golgi apparatus, the trans-Golgi network
(TGN), prevacuolar compartments (PVC), lytic compartments (vacuoles or lysosomes), and endosomes (Fig. 2).
Exchange of material between them is thought to be
achieved by small vesicles which bud from one membrane
and fuse with another in a highly controlled fashion. The
basic mechanisms involved in membrane-exchange reactions appear to be conserved in eukaryotes, from microorganisms to mammals. Newly synthesized proteins are
transported through the secretory pathway to the ER lumen
or membrane, pass from the ER through the Golgi
apparatus, and in late Golgi enter the secretory pathway
or are diverted to other cellular compartments: the vacuole
in yeast and plant cells, or the lysosome in animal cells. In
yeast and animals the material destined for the vacuole is
sorted away from that destined for the plasma membrane in
the TGN, whereas in plants the sorting can start as early as
at the cis-Golgi stacks (Hillmer et al., 2001) or bypass the
Golgi and be directed from the ER to storage vacuoles
(Hara-Nishimura et al., 1998). The vacuole also receives
material from the cell surface delivered by the process of
endocytosis. These two pathways overlap at a set of
membrane vesicles called the multivesicular body (MVB)
in animal cells and prevacuolar compartment (PVC) in
yeast and plants (Fig. 2). Besides the anterograde transport
to the vacuole, retrograde transport of proteins exists between the vacuole and earlier compartments: the MVB,
Golgi, or ER (Gotte and Lazar, 1999).
Fusion of transport vesicles with the target membranes
depends on SNARE (soluble N-ethylmaleimide-sensitive
factor adaptor protein receptor) molecules in the vesicle (vSNARE) and target (t-SNARE) membranes (Sollner et al.,
1993; Ungermann et al., 1998; Weber et al., 1998). The
interaction of these proteins is regulated by small GTPases
(RAB/YPT), belonging to the Ras superfamily. RAB/YPT
proteins cycle between active GTP-bound and inactive
GDP-bound states. The guanine nucleotide exchange factor
catalyses GDP/GTP exchange and RAB-GTP remains
anchored in the donor membrane where it intervenes in
the v- and t-SNARE interaction. A new cycle starts with the
hydrolysis of GTP enhanced by a GTPase-activating protein, GAP (Strom et al., 1993). ScNHX1, the endosomal
yeast Na+/H+ exchanger, was originally defined as an ion
homeostasis determinant mediating vacuolar sequestration
of Na+, coupling Na+ movement to the proton gradient
established by the vacuolar H+-ATPase (Nass et al., 1997;
Nass and Rao, 1998). Besides sodium sensitivity, nhx1
mutants show hygromycin sensitivity (Gaxiola et al., 1999;
Quintero et al., 2000), acidification of the vacuolar lumen
and impaired growth at low pH (Ali et al., 2004; Brett et al.,
2005b), and a ‘class E’ vacuolar protein-sorting phenotype
characterized by enlargement of the prevacuolar compartment and missorting of vacuolar carboxypeptidase Y to
the cell surface (Bowers et al., 2000). Ali et al. (2004)
Fig. 2. The plant endomembrane system. The connecting membrane exchange routes are represented by arrows. ER, Endoplasmic reticulum; GA,
Golgi apparatus; TGN, trans-Golgi network; Vac, Vacuole; EE, early endosome; PVC, prevacuolar compartment; PM, plasma membrane.
1188 Pardo et al.
demonstrated that the carboxy-terminal tail of ScNHX1
binds to GYP6, a GTPase-activating protein that also
regulates the YPT6-mediated vesicle fusion in the retrograde PVC to Golgi pathway (Fig. 3). It has been proposed
that NHX1 regulates the luminal pH of PVC and that GYP6
acts as a negative effector on ScNHX1 activity. When
ScNHX1 is inhibited by GYP6, a relative acidification of
the compartment would lead to relatively more acidic
vesicles that would be targeted to the vacuole. The relief
of ScNHX1 inhibition by competitive binding of YPT6 to
GYP6 would result in less-acidic vesicles that could be
directed to retrograde delivery to the TGN/Golgi (Fig. 3).
Recently, Brett et al. (2005b) have shown that the
trafficking defects of nhx1 strains, but not their salt sensitivity, can be mimicked by the addition of weak acids
or ameliorated by the addition of weak bases, providing
further evidence that pH regulation by ScNHX1 is critical
for trafficking pathways out of the late endosome.
There is also a critical requirement for a specific luminal
ionic concentration and pH in vesicular trafficking of
proteins in animal cells. Several pH-dependent processes
are: ligand–receptor dissociation (Dautry-Varsat et al.,
1983), endocytosis (Aniento et al., 1996; Chapman and
Munro, 1994; Maranda et al., 2001), post-translational
processing and sorting of newly synthesized proteins
(Halban and Irminger, 1994), binding of coat proteins to
early endosomes (reviewed in Stevens and Forgac, 1997),
and the association of vesicles with an ARF GTPase and its
GTP/GDP exchanger in pancreatic cells (Zeuzem et al.,
1992; Maranda et al., 2001). The abnormal lysosomal/
endosomal morphologies and associated defective trafficking observed in a class of lysosomal storage disorders are
associated with abnormal changes in luminal pH (Futerman
and van Meer, 2004). Changes in cellular pH alone severely
alter morphology and movement of organelles (Heuser,
1989). The plasma membrane Na+/H+ exchangers of the
NHE family are clearly associated with cellular pH
regulation in mammalian cells (Orlowski and Grinstein,
2004), but much less is known about the properties of the
intracellular subgroup despite the recent discovery of
numerous candidate genes from model animal organisms
and higher vertebrates, including human NHE6–NHE9
(reviewed by Brett et al., 2005a). Their intracellular
location within the endomembrane system and the remarkable conservation of protein trafficking mechanisms in
eukaryotes brings up the possibility that each may regulate
pH-mediated trafficking to or from different organelles.
NHE6 is found in endosomes (Brett et al., 2002; Nakamura
et al., 2005), NHE7 is located in the trans-Golgi network
of brain cells where it is thought to play a significant role
in organelle pH and volume homeostasis (Numata and
Orlowski, 2001), and NHE9 is found in the late recycling
endosomes (Nakamura et al., 2005) when expressed in
mammalian cell culture models. By regulating the pH
within these compartments, they are likely to influence
numerous pH-mediated trafficking events in the endocytic
Fig. 3. Model for endosomal traffic regulation in yeast. GYP6 binds to NHX1 at the PVC, inhibiting its activity and promoting anterograde transit (left
side) of relatively acidic vesicles to the vacuole (Vac). In retrograde traffic (right side) YPT6:GTP competes with NHX1 for GYP6 binding, relieving its
inhibition. The relative alkalinization of the endosome due to NHX1 activity promotes vesicular retrograde traffic to the trans-Golgi network and the
hydrolysis of GTP. YPT6:GDP returns to the TGN and is reactivated by a guanine nucleotide exchange factor protein.
Alkali cation exchangers 1189
pathway of cells such as linking ligand–receptor dissociation
to membrane recycling or lysosomal protein degradation.
The above precedents in yeast and animal cells, together
with endosomal localization and a strong preference for
K+/H+ exchange (Yokoi et al., 2002; Venema et al., 2003)
suggest that class-II NHX proteins are likely to fulfil
essential roles in the control of endosomal pH and vesicle
trafficking in plants (Fig. 2). By the criteria of cation
specificity and subcellular localization, class-II NHX transporters are the ScNHX1 counterparts in the endosomal
compartment of plant cells, and they are likely candidates to
intervene in vesicular trafficking by mechanisms equivalent
to those described by Ali et al. (2004) for ScNHX1. It is
tempting to hypothesize that class-II NHX transporters
would thus play a role in the regulation of vesicle trafficking by contributing to the maintenance of the required
pH and K+ gradients in the endosomal compartment. ClassI NHX transporters could also be involved in protein
sorting and vesicular trafficking processes since a T-DNA
insertional nhx1 mutant of Arabidopsis shows, among other
phenotypic traits, reduced leaf area which correlates with
the overall reduction of epidermal cell size. This phenotype
could be attributed not only to the loss of ability of the cells
to maintain a proper ion homeostasis in the vacuole, leading
to loss of vacuolar turgor, but also to a defective vacuole
biogenesis as it has been described for nhx1 mutants of
yeast (Bowers et al., 2000; Apse et al., 2003). Although
neither vacuole biogenesis nor vesicle trafficking has been
experimentally analysed in nhx mutants of plants, the transcriptome of wild-type and nhx1 mutant lines of Arabidopsis
does in fact show significant differences in the expression
of transcripts associated with protein processing and vesicular trafficking (Sottosanto et al., 2004). Whether vacuolar
NHX isoforms have a specific role in vesicle trafficking
or transcriptome alterations result indirectly from a putative
defect in vacuole formation is presently unknown.
Mineral nutrition
The vacuole occupies as much as 80–90% of the cell
volume in mature plant cells and quantitatively locates most
of the cell K+ (Leigh and Wyn Jones, 1984). Functionally,
vacuoles are K+ reservoirs that ensure adequate supply
to the cytosol. K+ concentration in vacuoles of barley
root varied proportionally with the overall tissue K+ concentration, whereas cytosolic K+ remained constant over a
wide range of tissue K+ concentrations. At diminishing K+
availability, the cytosolic K+ concentration was higher than
the vacuolar concentration (Walker et al., 1996). Cytosolic
K+ concentrations declined only when vacuolar K+ concentrations decreased to values around 20 mM (Leigh and
Wyn Jones, 1984).
In several studies performed on salt tolerance provided
by the overexpression of NHX genes, a greater leaf K+
concentration was found associated with the tolerant
phenotype (Zhang and Blumwald, 2001; Xue et al., 2004;
Wu et al., 2005). Considering that NHX antiporters of
class I may exchange K+ as well as Na+ for vacuolar H+
(see above), overexpression might affect cytosolic K+
by directing the active transport of K+ into the vacuole
when K+ availability is high (Walker et al., 1996). In
fact, electrophysiological studies suggest the suitability of
tonoplast antiporters for the homeostatic regulation of
cytosolic K+ when cells are provided with surplus concentrations of K+ (Walker et al., 1996). Thus, NHX antiporters
may play a significant role in K+ nutrition together with ion
channels by capturing K+ into vacuoles for cellular storage
and turgor generation at physiological growth conditions
(Leigh and Wyn Jones, 1984; Hsiao and Läuchli, 1986)
besides their protective function under salt or osmotic stress
(Nass and Rao, 1999; Blumwald, 2000). Under K+ deficiency, cytoplasmic acidification occurs and it may act as
a signal affecting K+ transport activity at the plasma
membrane and the tonoplast (Walker et al., 1996). Animal
NHE homologues are controlled by cytosolic pH through
C-terminal regulatory domains (Putney et al., 2002), and
a similar mechanism may exist for plant NHX proteins.
Potassium uptake seems affected in Arabidopsis nhx1
mutants as both a lower shoot K+ content and a lower
root K+ (Rb+) uptake rate than in the wild type have been
observed (Leidi et al., 2005). Thus, the reduced leaf
expansion in nhx1 mutants (Apse et al., 2003) may be
linked to a role of AtNHX1 in net K+ uptake and the gain in
cell turgor required for growth (Mengel, 1996). By contrast,
AtNHX1-overexpressing tomato showed greater K+ (Rb+)
uptake than control lines but were nevertheless prone to
showing K+ deficiency symptoms at leaf K+ concentrations
greater than the control line (Leidi et al., 2005). This
paradoxical phenotype is likely to be due to exacerbated
activity of the AtNHX1 antiporter in the transgenic lines
which could increase, unduly, the vacuolar pool at the expense of the cytoplasmic K+ pool, thereby inducing a K+
starvation signal and eliciting greater K+ uptake by roots.
Plasma membrane exchangers
SOS1 phylogeny and function
The SOS1 antiporter belongs to the NhaP subfamily of the
CPA1 family, which is related but is clearly different from
the NHE/NHX subfamily. In a previous phylogenetic
classification of Arabidopsis membrane transporters,
SOS1 was renamed NHX7 and included in the NHX
family (Mäser et al., 2001). This was probably due to the
fact that plant NhaP is a very small family, with only two
members, and the analysis method may have failed to
differentiate between NHX/NHE and SOS1/NhaP clusters.
When the phylogenetic analysis was performed with a
larger number of NhaP proteins from several organisms,
1190 Pardo et al.
its sensitivity was increased and the two different clades
could be easily distinguished (Fig. 4) (Brett et al., 2005a).
For this reason and to avoid confusion, it is suggested
that the NHX7 name should be discontinued in favour of
the original name SOS1.
The NhaP group of exchangers is present in bacteria,
protozoa, and plants (Brett et al., 2005a). The predicted
structure of NhaP-like exchangers consists of an N-terminal
transmembrane region followed by a hydrophilic Cterminal extension that, in eukaryotic NhaP homologues,
is remarkably long, with >600 residues. Since C-terminal
regions in many other transporters have been shown to
interact with regulatory proteins (Putney et al., 2002),
a complex regulation of eukaryotic NhaP should be
expected. Subcellular localization and biochemical characterization of NhaP in eukaryotic organisms is not available,
with the sole exception of Arabidopsis SOS1. By contrast
to NHX proteins, SOS1 has been shown to be localized at
the plasma membrane (Shi et al., 2002), where it catalyses
an electroneutral Na+/H+ exchange (Qiu et al., 2002; Shi
et al., 2002). In addition, SOS1 is highly specific for Na+
and cannot transport other monovalent cations, such as K+
or Li+. Since biochemical data supporting the presence of
plasma membrane Na+(K+)/H+ antiport activity were found
in only a limited number of plant species (Mennen et al.,
1990), the very existence of Na+/H+ exchangers in plasma
membranes was questioned. The cloning of the Arabidopsis
SOS1 gene and the presence of >120 ESTs (expressed
Fig. 4. Phylogenetic tree of plant NHE/NHXs and NhaP/SOS1 exchangers. Sequence alignment of the Arabidopsis and rice NHXs families
and all the plant full-length SOS1-like proteins was performed using
Clustal X. GenBank accession numbers: AtSOS1, AAF76139; AtSOL1,
NP_172918; OsSOS1, AAW33875; CnSOS1, CAD20320; Pp,
CAD91921.
sequence tags) encoding SOS1-like proteins from 31
different species demonstrates not only the existence of
this family of transporters but also its wide distribution
in plants. The size of plant NhaP gene families within
individual species is not clear because very few full-length
sequences are available. Only one SOS1-like gene has
been reported in Physcomitrella patens and in Cymodocea
nodosa (Benito and Rodriguez-Navarro, 2003). In the
fully sequenced genomes of rice and Arabidopsis, one
(Os12g44360) and two (SOS1 and At1g14660) genes,
respectively, have been found. The protein encoded by
locus At1g14660 is very similar to SOS1 (72% identity) but
is about 400 residues shorter than SOS1 at the C-terminus.
The function of the protein encoded by locus At1g14660
is still unknown. The strong phenotype of sos1 plants
suggests that protein At1g14660 probably plays a nonoverlapping role with SOS1 despite their high similarity
at the sequence level. By contrast, the rice SOS1 protein has been shown to be the functional equivalent of the
Arabidopsis SOS1 since it shows Na+/H+ antiport activity
and partially complements the Arabidopsis sos1 mutation
(J Martı́nez-Atienza and JM Pardo, unpublished results).
Based on the expression pattern of SOS1 and the
physiological characterization of sos1 mutant plants, it
has been suggested that SOS1 controls long-distance Na+
transport in Arabidopsis (Shi et al., 2002). The expression
pattern of SOS1 was examined using a SOS1 promoter:GUS
fusion. GUS activity was mainly detected in the inner
tissues surrounding the vasculature throughout the plant.
In the root, cross-sectional analysis revealed that GUS activity was localized primarily in the pericycle and in parenchyma cells bordering xylem vessels. In stem and petiole
cross-sections, GUS activity was also restricted to parenchyma cells at the xylem/symplast boundary. Besides its
association with the vasculature, SOS1 expression was
observed at the epidermal cells of the root tip. As undifferentiated cells at the root tip lack vacuoles large enough for
significant Na+ compartmentalization, SOS1 might be critical to prevent Na+ from accumulating in these cells by
actively extruding Na+ back to the soil solution.
The preferential expression of SOS1 in cells surrounding
the vasculature suggests a role of this transporter in longdistance Na+ transport in plants, since Na+ is transported
from the root to the shoot via the xylem. When grown in the
presence of a high Na+ concentration (100 mM NaCl), the
roots and aerial parts of sos1-mutant plants accumulated
more Na+ than wild-type plants. The Na+ concentration in
the xylem sap of both sos1-mutant and wild-type plants
increased substantially over time, but the Na+ concentration
was always higher for sos1. By contrast, sos1 mutant plants
accumulated less Na+ than the wild type in response to low
levels (25 mM NaCl) of salt stress (Ding and Zhu, 1997;
Shi et al., 2002). These contradictory results have been
explained by suggesting that SOS1 might function in
loading Na+ into the xylem for controlled delivery to the
Alkali cation exchangers 1191
shoot and storage in leaf mesophyll cells under moderate
salt stress. However, under extreme salinity stress and
catastrophic breakdown of Na+ exclusion mechanisms at
the root, a strong electrochemical gradient of Na+ across the
xylem/symplast boundary, which can be of greater magnitude than the pH gradient, would result in a reversal of
SOS1 activity and a retrieval of Na+ from the xylem to
prevent overaccumulation in the transpiration stream
(Shi et al., 2002). Fine-tuning of net ion uptake by roots,
xylem transport for long-distance distribution, and vacuolar
compartmentalization along the plant axis and in older
leaves, would be required to facilitate osmotic adjustment and plant growth in a saline environment.
Regulation of the SOS system
Significant progress has been made towards the understanding of SOS1 regulation. Na+/H+ exchange by SOS1 is
detected in plasma membrane vesicles from salt-stressed
Arabidopsis plants but not in control plants, indicating that
SOS1 activity is induced by stress (Qiu et al., 2002). The
up-regulation of SOS1 occurs at two levels at least: mRNA
stability and ion exchange rate. Key intermediaries in the
regulation of SOS1 are the protein kinase SOS2 and its
associated Ca2+-sensor protein SOS3. Mutant plants deficient in either SOS2 or SOS3 share the salt-sensitive and
K+-deficiency phenotype of sos1 plants (Zhu, 2000, and
references therein). SOS3 is a calcium-binding protein
capable of sensing calcium transients elicited by salt stress.
SOS2 is a serine/threonine protein kinase whose catalytic
domain is evolutionarily related to that of the yeast protein
SNF1 and the animal AMP-activated kinases (Liu et al.,
2000). The C-terminal part of SOS2 is unique to plants and
physically interacts with SOS3 in a Ca2+-dependent manner
through the FISL domain located at the boundary between
the regulatory C-terminus and the catalytic domain of SOS2
to relieve the kinase from auto-inhibition (Guo et al., 2001).
The FISL domain is itself inhibitory and its deletion results in constitutive activation of SOS2 (Guo et al., 2004).
The crystal structure of SOS3 has been resolved recently
(Sánchez-Barrena et al., 2005). Upon Ca2+ binding,
SOS3 undergoes dimerization and hydrophobic domains
are exposed. This change in surface properties may
facilitate interaction with SOS2 and the transmission of
the Ca2+ signal elicited by stress. Adjacent to the SOS3binding domain of SOS2 is the PPI domain for interaction
with ABI2, a type-2C protein phosphatase that is a negative intermediary in ABA signalling (Ohta et al., 2003).
Although this interaction may be a cross-talk point between
ABA signalling and the SOS pathway for ion homeostasis
under salinity stress, there is no direct evidence of the
regulation of SOS1 by ABA. Moreover, sos2 and sos3
mutants are specifically defective in salt tolerance but not in
ABA responses (Ohta et al., 2003). Conversely, mutants
deficient in the SOS2-like kinase PKS3 or the SOS3-like
protein SCaBP5, whose complex also interacts with ABI2,
are hypersensitive to ABA but not significantly affected in
salt tolerance (Guo et al., 2002). The dominant negative
protein phosphatase encoded by the abi2-1 allele, which
brings about ABA insensitivity rather than hypersensitivity,
cannot interact with SOS2 or PKS3 and permits greater
tolerance to salt shock, suggesting that dissociation of the
ABI2/SOS2 complex is a requisite for the progression of
the salt-stress signal. Whether or not activation of SOS1 is
a signalling output of the stress phenotype associated with
the abi2-1 allele is presently unknown.
Biochemical activation of Na+/H+ exchange of SOS1 by
salt stress is controlled through protein phosphorylation by
the SOS2/SOS3 kinase complex. Co-expression of the three
proteins in a yeast strain lacking endogenous Na+ transporters restored salt tolerance to a much greater extent than
SOS1 alone, whereas SOS2 or SOS3 individually failed to
stimulate SOS1 activity (Quintero et al., 2002). SOS2 and
SOS3 had no effect per se in the absence of SOS1. The
SOS2/SOS3 kinase complex promoted the phosphorylation
of SOS1 in yeast plasma membrane fractions and purified
SOS2 phosphorylated SOS1 in vitro, demonstrating that
SOS1 is a genuine substrate for SOS2 kinase. Consistent
with these interactions, SOS1 activity in the plasma membrane was not induced by salt stress in sos2 or sos3 mutant plants (Qiu et al., 2002). However, the addition of a
constitutively active, SOS3-independent form of SOS2 to
plasma membrane vesicles isolated from sos2 or sos3
plants, but not from sos1, restored SOS1 activity. Besides
kinase activation, SOS3 was also shown to recruit SOS2 to
the plasma membrane to achieve efficient interaction with
SOS1 (Quintero et al., 2002). N-myristoylation of SOS3 is
necessary for its functionality in planta (Ishitani et al.,
2000) and for the targeting of the SOS2/SOS3 complex to
the plasma membrane (Pardo et al., 2005). Nonetheless, the
evaluation in planta of an array of mutant alleles of SOS2
has revealed that the kinase may reach the plant plasma
membrane in the absence of a functional SOS3 protein,
perhaps as a result of interactions with other proteins such
as alternative SCaBP proteins or even ABI2 (Guo et al.,
2004). The SOS2/SOS3 kinase phosphorylates SOS1 at
the C-terminus and stimulates its Na+ extrusion activity
in yeast, and C-terminally truncated SOS1 mutants become
hyperactive and independent of SOS2/SOS3 (Pardo
et al., 2005). These results indicate that SOS1 is released
from auto-inhibition by its C-terminal through protein
phosphorylation.
The transcript level of SOS1 is up-regulated by salt stress
by a mechanism that is partly dependent on SOS2 and
SOS3 (Shi et al., 2000). Regulation of mRNA abundance
seems to be post-transcriptional because it was observed
even when SOS1 transcription was driven by the constitutive CaMV 35S promoter (Shi et al., 2003). The low level
of transcript accumulated in transgenic lines under standard
growth conditions suggests that the SOS1 transcript is
1192 Pardo et al.
unstable in the absence of salt and that stress causes a posttranscriptional stabilization of the transcript. Nothing is
known about the factor(s) that regulate SOS1 mRNA
stability.
Interaction of the SOS pathway with K+ nutrition
Arabidopsis mutants lacking the SOS1 transporter or one of
its regulatory proteins, SOS2 and SOS3, manifest symptoms of K+ deficiency on culture medium with low K+
(<200 lM K+) implying that the Ca2+-regulated SOS
pathway has a positive regulatory effect on K+ acquisition.
SOS1 is the only known target of the SOS2/SOS3 kinase
complex and it has been shown to mediate Na+/H+ exchange but to lack any detectable K+ transport capacity,
both in its basal state and after activation by the SOS2/
SOS3 kinase (Qiu et al., 2002; Quintero et al., 2002; Shi
et al., 2002). High Na+ concentration in the soil solution is
thought to disturb K+ acquisition by competing with
binding sites in transport systems that mediate K+ uptake
(Hasegawa et al., 2000), but the K+-deficiency phenotype of sos mutants is perplexing and raises the question
of how a disturbed Na+ homeostasis impinges on K+ nutrition beyond the competition for common transporters.
The K+-deficiency phenotype of sos mutants is conditional as it is exacerbated by low Ca2+ (0.15 mM) and
suppressed by high Ca2+ (3 mM). Ca2+ has long been
known to improve K+/Na+ selectivity, which effectively
reduces Na+ uptake and increases salinity tolerance. Based
on biochemical and electrophysiological studies, three coexisting pathways for Na+ influx have been proposed
(Tester and Davenport, 2003). Two pathways are transporter-mediated components that can be distinguished by
their relative sensitivity to extracellular Ca2+. The third
pathway (bypass flow) is attributed to leakage through
the apoplast and to discontinuities in the endodermis, for
instance at the points of emergence of lateral roots. The
relative contribution of each pathway varies with species
and growth conditions. Bypass flow is particularly apparent
in rice (Yeo et al., 1987). In excised Arabidopsis roots
incubated with 50 mM NaCl at low Ca2+ activity (0.2 mM),
the three components of Na+ influx were of similar
magnitude (;0.6 lmol gÿ1 minÿ1; Essah et al., 2003),
although bypass flow might have been underestimated by
the use of detached roots lacking transpirational water flux.
Electrophysiological work suggests that non-selective
cation channels (NSCCs) at the plasma membrane account
for at least part of the transporter-mediated Na+ uptake that
is Ca2+-sensitive. NSCCs are characterized by their poor
discrimination among inorganic monovalent cations. In
addition, some NSCCs are also permeable to divalent
cations like Ca2+ and Mg2+, but many are, instead, inhibited
by Ca2+ (reviewed by Demidchik et al., 2002). The
Arabidopsis genome has revealed the existence of two
large families of NSCCs with sequence similarity to animal
counterparts, namely the cyclic nucleotide-gated and
calmodulin-regulated channels (CNGCs) and the glutamate
receptor-like channels (GLRs), some of which may correspond to the NSCC activities characterized electrophysiologically. Maathuis and Sanders (2001) described in the
plasma membrane of Arabidopsis root protoplasts the
presence of CNGC activity that was deactivated by
cAMP and cGMP, by contrast to animal CNGCs and other
plant CNGCs which are activated by ligand (cAMP or
cGMP) binding to the channel protein (Demidchik et al.,
2002; Talke et al., 2003). Moreover, when supplemented
into the growth medium, both cyclic nucleotides effectively restricted Na+ uptake by seedlings and increased salt
tolerance (Maathuis and Sanders, 2001). At the molecular
level, the inward-rectifier and non-inactivating channel
AtCNGC1, which is expressed in plant roots and conducts
Na+ equally as well as K+, may provide a physiologically
significant Na+ entry from the soil solution in the Ca2+sensitive pathway (Sunkar et al., 2000; Leng et al., 2002;
Hua et al., 2003). Presumably, the inhibition of NSCC
channels (AtCNGC1 and others) by Ca2+ diminishes the
Na+ load of sos mutants and alleviates the K+-deficiency
phenotype in low K+.
Remarkably, hkt1 mutations that were initially isolated
as extragenic suppressors of the Na+ hypersensitivity of the
sos3 mutant also suppressed the K+-deficiency phenotype
of sos mutants completely, whereas moderate overexpression of AtHKT1 exacerbated K+ deficiency in wild-type
and sos3 seedlings (Rus et al., 2001, 2004). Heterologous
expression in yeast and Xenopus oocytes has shown that
plant HKT transporters are all permeable to Na+ and, in
some cases, to K+ as well. The wheat transporter TaHKT1
functions as a high-affinity Na+/K+ symporter at low
external K+ or Na+ concentrations but, in the presence of
sufficient amounts of both ions, K+ transport by TaHKT1
was blocked and only low-affinity Na+ uptake occurred
(Rubio et al., 1995; Gassman et al., 1996). In rice, OsHKT1
and OsHKT4 isoforms are specific high- and low-affinity
Na+ transporters, respectively, whereas OsHKT2 showed
Na+/K+-coupled transport (Horie et al., 2001; Garciadeblas
et al., 2003). High-affinity Na+ transport by OsHKT1 was
inhibited by 1 mM K+ but K+ transport itself was marginal
(Garciadeblas et al., 2003). In Arabidopsis, genetic and
electrophysiological analyses indicate that its single
AtHKT1 protein performs several physiological functions
all pertaining to specific Na+ transport. Mutants lacking
AtHKT1 failed to show significant reduction in short-term
Na+ uptake by roots (Essah et al., 2003) but manifested
a consistent increment of Na+ in aerial parts with a concomitant reduction in Na+ root content (Mäser et al., 2002;
Berthomieu et al., 2003; Rus et al., 2004). AtHKT1 gene
expression has been localized to the root stele and leaf
vasculature, with preferential accumulation in phloem
tissues (Mäser et al., 2002; Berthomieu et al., 2003).
Na+ content in the phloem sap of hkt1 mutants was reduced
Alkali cation exchangers 1193
+
7-fold, whereas Na content was slightly greater in the
xylem sap (Berthomieu et al., 2003). These results indicate
that AtHKT1 controls root/shoot Na+ partition, reducing
leaf Na+ accumulation by Na+ re-circulation from shoots to
roots. The current model posits that AtHKT1 mediates Na+
loading into the phloem sap in leaves and unloading in
roots, and that influx or efflux through AtHKT1 would be
determined by the local electrochemical gradient of Na+
(Berthomieu et al., 2003). Although export from leaves
could help to maintain low salt concentrations in the shoot, it
should be kept in mind, however, that there appears to be
comparatively little retranslocation of salt from leaves
relative to the amount imported in the transpiration stream
(Munns, 2005). Plants transpire about 50 times more water
that they retain in their leaves. Estimates of xylem and
phloem fluxes of Na+ and Clÿ indicated that, in barley,
phloem export from a leaf was only 10% of the import from
the xylem. As exemplified by rice (Ren et al., 2005),
retrieval of ions from the xylem sap could conceivably be
a more practical strategy to ward off excessive accumulation
of salts in the shoot (see below) than export in the phloem.
The evidence gathered from the genetic analysis of the
Na+ transporters SOS1 and AtHKT1 establishes that K+
nutrition is linked to Na+ homeostasis in more complex
ways than the widely accepted model that Na+ competes
with K+ for uptake through transport systems mediating
K+ acquisition. In the sos mutants extrusion of Na+ to the
soil solution is impaired due to insufficient SOS1 activity
at the root epidermis (Qiu et al., 2002; Shi et al., 2002).
SOS1 is also preferentially expressed in xylem parenchyma cells where it contributes to Na+ loading of the xylem
and subsequent translocation to the shoot along the transpiration stream (Shi et al., 2002). Consequently, Na+ is
accumulated in the root of sos mutants (Shi et al., 2002;
Rus et al., 2004). In this scenario of compromised xylem
loading, incremental Na+ deposition in the root stele by Na+
re-circulation and phloem unloading would be detrimental
and averted by inactivation of AtHKT1. Indeed, single sos3
and hkt1 mutations promote the undue accumulation of Na+
in roots and shoots, respectively, whereas a double sos3
hkt1 mutant achieves a more balanced partition of Na+ that
is closer to the profile of wild-type plants (Rus et al., 2004).
Excessive retention of Na+ in cells of the root stele of sos
mutants in a AtHKT1 wild-type background may impede
transverse flux of K+ to the stele and/or K+ loading of the
xylem sap under low K+ conditions. Reduction of stelar
Na+ by mutation of AtHKT1 may restore sufficient K+ flux
from the root cortex towards the xylem. It appears that
the transport function of the SOS system and AtHKT1 are
co-ordinated tightly and together they achieve Na+ (and
K+) homeostasis. Dysfunction of either system alters
long-distance transport and adequate partition of Na+,
thereby resulting in salt-sensitive phenotypes.
Recent evidence implicates the K+ inward-rectifying
channel AKT1 as a target for Na+-induced failure of K+
uptake (Qi and Spalding, 2004). In growth medium with
millimolar NH4+ concentrations, which block non-channel
K+ uptake systems, Arabidopsis seedlings rely on the
AKT1 channel for physiologically relevant K+ uptake. In
Na+-free media with high NH4+ , the AKT1-dependent K+uptake ability of the sos mutant root cells, measured
electrophysiologically, was normal and the growth rates
of these mutants displayed wild-type K+ dependence.
Addition of 50 mM NaCl strongly inhibited root-cell K+
permeability and growth rate in K+-limiting conditions of
sos1 but not wild-type plants. Moreover, addition of 10 mM
NaCl into the cytoplasmic side of patch-clamped wild-type
root cells inhibited AKT1 K+ channel activity completely.
Therefore, it appears that elevated cytoplasmic Na+ levels
resulting from loss of SOS1 function impaired K+ permeability in root cells and compromised K+ nutrition. It is also
conceivable that excessive retention of Na+ in the root stele
of sos mutants due to reduced xylem loading (Shi et al.,
2002; Rus et al., 2004) may result in the inhibition of
additional K+ transporters involved in long-distance transport of this nutrient, such as SKOR (shaker-like K+
outward-rectifying channel), a channel expressed in the
stele of the root that mediates K+ efflux into the xylem for
distribution from root to shoot (Gaymard et al., 1998).
However, growth measurements showed that akt1 seedlings were salt sensitive during early seedling development, but skor seedlings were normal, suggesting that the
detrimental effect of Na+ on K+ transport through channels
is probably more important at the uptake stage than at the
xylem-loading stage (Qi and Spalding, 2004).
Non-channel targets for Na+ inhibition in the longdistance transport pathway for K+ may nevertheless exist.
Lacan and Durand (1996) have shown that excised soybean
roots treated with NaCl reabsorbed Na+ from the xylem
vessels in exchange for K+. This net Na+/K+ exchange at
the xylem/symplast interface was strongly linked because
Na+ in the xylem sap enhanced K+ release, whereas
increased xylematic K+ prompted Na+ reabsorption. Na+
removed from the xylem sap was subsequently excreted to
the external medium. Lacan and Durand (1996) suggested
that reabsorption of Na+ from the acidic xylem sap by
a Na+/H+ antiporter was energetically enabled through H+
cycling by K+/H+ antiport and H+/anion symport. Based on
the expression pattern of SOS1 and the ion profile of sos1
plants at various salinity regimes, Shi et al. (2002) have
suggested that SOS1 has a dual role in loading and
reabsorption of Na+ from the xylem stream. When salinity
is moderate, SOS1 functions to load Na+ into the xylem
for controlled delivery to the shoot and storage in leaf
mesophyll cells. Under severe salt stress, Na+ might
quickly accumulate in the shoot, exceeding the capacity
of vacuoles in mesophyll cells to compartmentalize ions.
Under these conditions, SOS1 would function to restrict net
Na+ uptake at the root tip and to retrieve Na+ from the
xylem in the mature root, both processes helping to limit
1194 Pardo et al.
rapid accumulation of Na+ in the shoot. If Na+ and K+
fluxes at the xylem/symplast boundary of Arabidopsis were
coupled to produce the net Na+/K+ exchange as proposed
by Lacan and Durand (1996), a functional SOS1 protein
might be required to enable K+ loading to the xylem when
plants were growing in the presence of Na+ in a culture
medium with low availability of K+, conditions in which
sos mutants show symptoms of K+ deficiency.
Control of long-distance transport of Na+ may function
differently in rice plants. Fine mapping of a major QTL
for shoot K+ content and salt tolerance in rice identified
SKC1 (Ren et al., 2005), an HKT-type protein corresponding to gene OsHKT8 (Garciadeblas et al., 2003), as a
Na+-selective transporter preferentially expressed in the
parenchyma cells surrounding xylem vessels. SCK1/HKT8
transcripts were more abundant in roots and up-regulated
by salt stress (Ren et al., 2005). In electrophysiological
tests, the SCK1/HKT8 isoform from the relatively salttolerant variety Nona Bokra was more active in the
facilitation of Na+ transport across the plasma membrane
than its counterpart from the salt-sensitive Koshihiraki
variety, suggesting that greater capacity for Na+ retrieval
from the xylem by SCK1/HKT8 in Nona Bokra plants was
the basis for their salt-tolerance. Conversely, increased Na+
concentration in the xylem sap and leaves was associated
with the weaker SKC1 allele of the salt-sensitive variety.
Whether SCK1/HKT8 functions co-ordinately with the rice
SOS1 homologue to regulate the net Na+ load of the xylem
remains to be determined. Also unknown is whether or
not in rice there is a functional homologue of AtHKT1
from Arabidopsis permitting the recirculation of Na+
via the phloem and contributing further to the restriction
of Na+ accumulation in the leaves.
CHX family
Phylogeny and classification
The CHX transporters, along with the related KEA subfamily, constitute the CPA2 family of cation/proton antiporters. This is probably the largest group of cation
exchangers in plants. According to Sze et al. (2001) there
are 28 CHX isoforms in Arabidopsis and 17 in rice.
Arabidopsis CHXs are subdivided into five groups (I–V).
Rice CHXs can be assigned to groups I, IV, and V; there are
no rice orthologues of Arabidopsis groups II and III. The
functional meaning of this classification is unknown, but it
could be related to substrate specificity or subcellular
targeting as illustrated by the NHX family. All CHXs
proteins have a similar size of about 800 amino acids and,
so far, there are no biochemical data demonstrating the
transport mechanism or substrate specificity of any of the
CHX family members. In other organisms, transporters of
the CPA2 family are well characterized and they show very
different catalytic properties. KefB and KefC mediate K+
efflux in Escherichia coli, and they seem to act as ligandgated channels (Miller et al., 1997). NapA from Enterococcus hirae and NhaS3 from Synechocystis sp. are Na+/H+
antiporters (Waser et al., 1992; Inaba et al., 2001), and
GerN from Bacillus cereus has a dual exchange capacity as
an Na+/H+ or Na+/K+ antiporter (Southworth et al., 2001).
Finally, the yeast KHA1 antiporter, the only reported
eukaryotic CPA2 member, localizes in an intracellular
compartment (Maresova and Sychrova, 2005), probably
the mitochondria (Sickmann et al., 2003), and indirect
evidence suggests that it could transport K+. Any or several
of the transport modes described for homologous proteins
can be expected in the plant CHX family.
Function of CHX exchangers
Numerous transcriptome analyses have been carried out
in Arabidopsis but, unfortunately, the CHX gene family
seem to be expressed at a very low level and no functional
hint for any member of this family can be drawn from its
expression pattern. Changes in transcript levels were detected only for AtCHX10 and AtCHX15, which in both
cases were down-regulated by salt stress (Maathuis et al.,
2003). Recently, a detailed gene expression analysis of the
complete Arabidopsis CHX family was performed using
sensitive techniques (Sze et al., 2001). Most CHX genes
were specifically expressed during male gametophyte development. Important changes in K+ and H+ concentrations
have been reported to occur throughout the gametophyte
differentiation process in tobacco, and these oscillations
have been associated with the regulation of biosynthetic
reactions. Moreover, the whole anther is an important
K+ sink, having almost twice the concentration of apical
leaves (Andreyuk et al., 2001). Potassium transport is also
essential during pollen tube elongation, and disruption of
the Arabidopsis SPIK K+ channel results in impaired pollen
tube growth (Mouline et al., 2002). CHX exchangers with
K+ transport capacity may play different roles in any of
those complex processes taking place during gametophyte differentiation, but the large number of individual
CHX genes apparently expressed in these organs is nevertheless surprising.
Only two CHX isoforms, AtCHX17 and AtCH23, have
been studied to date following a genetic approach (Cellier
et al., 2004; Song et al., 2004). AtCHX17 encodes a protein, 820 residues long, that is most similar to the nhaS4
protein in Synechocystis sp. Expression of nhaS4 in an
E. coli mutant strain, deficient in several endogenous antiporters, could not restore Na+ tolerance but it allowed
growth in medium with low K+ concentration. Transcripts
of AtCHX17 were detected in roots only when the plants
were salt stressed, K+ starved, or upon treatment with acidic
pH or the hormone ABA. In two transcriptome analysis
reports, AtCHX17 was also up-regulated by Ca2+ deprivation and by osmotic- and cold-stress (Kreps et al., 2002;
Alkali cation exchangers 1195
Maathuis et al., 2003). A more detailed analysis using GUS
reporter activity and in-situ RT-PCR showed that AtCHX17
transcription was restricted to epidermal and cortical cells
of roots. GUS activity was also detected in anthers of
flowering plants. Transgenic plants containing the
AtCHX17 promoter-GUS gene did not show a significant
change when they were K+ starved or challenged with
acidic pH, and the up-regulation caused by salt stress was
much lower. This discrepancy was explained as the result of
post-transcriptional regulation or the absence of regulatory
sequences in the AtCHX17 promoter fragment used in
the GUS fusion construct. Attempts to express AtCHX17
in Saccharomyces cerevisiae were unsuccessful. To gain
information about AtCHX17 function, two independent
homozygous T-DNA insertion lines within the gene were
phenotypically characterized. No anatomical differences
with the wild type were found under all conditions tested,
including salt stress and K+ starvation, although saltstressed or K+-starved roots of chx17 plants contained
;20% less K+ than wild type. No significant changes were
observed in the shoot K+ content. More research data will
be necessary to confirm the putative function of AtCHX17
in K+ homeostasis and elucidate its substrate specificity.
The AtCHX23 exchanger localizes to the chloroplast
envelope and plays an essential role in chloroplast development (Song et al., 2004). Plants with reduced
expression of AtCHX23 by RNAi showed leaf chlorosis
and were smaller in size. Chloroplast number was lower in
comparison with the wild type and their structure was
altered. A similar phenotype was observed in a chx23
mutant plant obtained by TILLING. Interestingly, extracellular alkaline pH exacerbated the observed phenotype
but acidic pH alleviated the symptoms of the mutant. These
results indicate the involvement of CHX23 in chloroplast
pH regulation. Stromal pH oscillates depending on the
photosynthetic conditions (Jagendorf and Uribe, 1966) and
a cation/H+ antiporter located in the chloroplast envelope
was suggested to be, in part, responsible for these pH
changes (Demming and Gimmler, 1983). In agreement with
the involvement of AtCHX23 on the control of stromal pH,
it was demonstrated that cytoplasmic pH in guard cells
of the mutant was higher than in the wild type, presumably
due to malfunctioning of the chloroplast pH-stat (Song
et al., 2004). The effects of Na+ and K+, the likely substrates of AtCHX23, were also investigated. Mutant chx23
plants were more sensitive to NaCl, and Song et al. (2004)
suggested that Na+ sequestration into the chloroplast could
be a possible mechanism contributing to salt tolerance.
However, the correlation between salt sensitivity and
chloroplast disorganization possibly points to chloroplast
function as one of the targets of Na+-toxicity. Recently,
Liska et al. (2004) found that enhanced photosynthesis
might be one of the processes contributing to the halotolerance of the green alga Dunaliella salina. Therefore,
preservation of chloroplast pH-stat and functionality would
be important for salt-stress adaptation. By contrast, addition
of KCl produced better growth of the mutant roots and the
guard cell aperture was increased, presumably because the
absence of K+ antiport activity at the chloroplast envelope
could be compensated by increases in the cytosolic K+
concentration. Taken together, these observations strongly
suggest that CHX23 is a K+(Na+)/H+ antiporter.
Concluding remarks
During evolution, plants have accrued a large array of ion
transporters, many of which are exchangers of monovalent
cations. Emerging data indicate that members of the latter
group play relevant roles in mineral nutrition, vesicle
trafficking and protein sorting, plant development, ion
accumulation into the vacuole, resistance to salinity, and
response to pathogens. Most of these functions have been
unravelled by genetic data and the trend will continue in
the near future with forward, reverse, and quantitative
genetics contributing to the unequivocal assignment of
specific functions to, hopefully, each and every member
of this large group of ion transporters. This may seem an
enormous task but, considering the physiological processes
impinged by cation exchange at the various cell membranes, their importance cannot be overstated.
Acknowledgements
This work was supported by the Spanish Ministry of Science and
Education, grant no. BIO2003-08501, with additional support from
Junta de Andalucı́a, grant no. CVI-148. Beatriz Cubero is a fellow of
‘Programa Averroes’ of Junta de Andalucı́a.
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