The Plant Journal (2007) 52, 1169–1180
doi: 10.1111/j.1365-313X.2007.03367.x
The Arabidopsis vacuolar malate channel is a member of
the ALMT family
Peter Kovermann1,†,‡, Stefan Meyer1,†,*, Stefan Hörtensteiner1, Cristiana Picco2, Joachim Scholz-Starke2,
Silvia Ravera3, Youngsook Lee4 and Enrico Martinoia1,4
1
Institute for Plant Biology, University of Zürich, CH-8008 Zürich, Switzerland,
2
Institute of Biophysics, National Research Council, I-16149 Genoa, Italy,
3
Institute of Physiology, University of Zürich, CH-8057 Zürich, Switzerland, and
4
POSTECH-UZH Cooperative Laboratory, Division of Molecular Life Sciences, Pohang University of Science
and Technology, Pohang, 790-784, Korea
Received 24 April 2007; revised 15 October 2007; accepted 29 October 2007.
*
For correspondence (fax +41 4463 48286; e-mail [email protected]).
†
These authors contributed equally to this study.
‡
Present address: Institute of Neurophysiology, Hannover Medical School, D-30625 Hannover, Germany.
Summary
In plants, malate is a central metabolite and fulfills a large number of functions. Vacuolar malate may reach
very high concentrations and fluctuate rapidly, whereas cytosolic malate is kept at a constant level allowing
optimal metabolism. Recently, a vacuolar malate transporter (Arabidopsis thaliana tonoplast dicarboxylate
transporter, AttDT) was identified that did not correspond to the well-characterized vacuolar malate channel.
We therefore hypothesized that a member of the aluminum-activated malate transporter (ALMT) gene family
could code for a vacuolar malate channel. Using GFP fusion constructs, we could show that AtALMT9
(A. thaliana ALMT9) is targeted to the vacuole. Promoter-GUS fusion constructs demonstrated that this gene is
expressed in all organs, but is cell-type specific as GUS activity in leaves was detected nearly exclusively in
mesophyll cells. Patch-clamp analysis of an Atalmt9 T-DNA insertion mutant exhibited strongly reduced
vacuolar malate channel activity. In order to functionally characterize AtALMT9 as a malate channel, we
heterologously expressed this gene in tobacco and in oocytes. Overexpression of AtALMT9-GFP in Nicotiana
benthamiana leaves strongly enhanced the malate current densities across the mesophyll tonoplasts.
Functional expression of AtALMT9 in Xenopus oocytes induced anion currents, which were clearly
distinguishable from endogenous oocyte currents. Our results demonstrate that AtALMT9 is a vacuolar
malate channel. Deletion mutants for AtALMT9 exhibit only slightly reduced malate content in mesophyll
protoplasts and no visible phenotype, indicating that AttDT and the residual malate channel activity are
sufficient to sustain the transport activity necessary to regulate the cytosolic malate homeostasis.
Keywords: AtALMT9, malate transport, tonoplast, anion channel, Arabidopsis thaliana L.
Introduction
Malate is implicated in a large number of metabolic pathways in all living organisms. In plants it plays a central role in
a multitude of functions. As a metabolite of the Krebs cycle it
is involved in the production of ATP. In the glyoxylate cycle
malate is closely linked to the b-oxidation of fatty acids and
the production of NADH. Malate also serves as a temporary
carbon store and provides reduction equivalents in C4 and
Crassulacean acid metabolism (CAM) plants. It must therefore be present in the cytosol, chloroplasts, mitochondria,
peroxisomes, glyoxysomes and vacuole. Furthermore,
malate plays a role in pH regulation, is an important
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd
osmoticum and acts as a major anion compensating the
positive charges of potassium and sodium. A metabolite
that is implicated in such a complex network has to be tightly
controlled. Using non-aqueous fractionation and in vivo
NMR it has been shown that cytosolic malate concentrations
are kept very constant, whereas vacuolar malate contents
fluctuate diurnally or in response to environmental changes
(Gerhardt et al., 1987; Gout et al., 1993). In roots, malate is
excreted into the apoplast and may complex and detoxify
aluminum or release rock-bound phosphate (Neumann and
Martinoia, 2002; Ryan et al., 2001). Transport processes for
1169
1170 Peter Kovermann et al.
malate have been investigated extensively, which reflects
the interest in this metabolite. Several chloroplast and
mitochondrial carboxylate transporters have been identified, with most of them exchanging malate with another
carboxylate (Menzlaff and Flügge, 1993; Palmieri et al.,
1993). Vacuolar malate transport has also been investigated
in detail using flux analysis, membrane potential- and pHdependent fluorescence probes, and the patch-clamp technique (Cerana et al., 1995; Hafke et al., 2003; Martinoia et al.,
1985; Pantoja and Smith, 2002; Pei et al., 1996; Ratajczak
et al., 1994). According to these studies, malate uptake is
driven by the electrochemical potential difference between
the cytosol and the vacuole generated by the vacuolar proton pumps (Maeshima, 2001; Rea and Sanders, 1987).
Observations of malate currents across the tonoplast indicate that they are strongly inward-rectifying, thus favoring
the movement of malate from the cytosol into the vacuole
(Cerana et al., 1995; Epimashko et al., 2004; Hafke et al.,
2003; Hurth et al., 2005; Pantoja and Smith, 2002). It was
shown that macroscopic currents observed on Kalanchoë
daigremontiana vacuoles can be attributed to the activity of
a small 3-pS malate-selective channel, and that channel
density and open probability suffice for the required nocturnal malate transport (Hafke et al., 2003). Recently, a vacuolar malate transporter from Arabidopsis has been
identified at the molecular level (Arabidopsis thaliana
tonoplast dicarboxylate transporter, AttDT, At5g47560; Emmerlich et al., 2003). This carrier is an ortholog of the renal
Na+/dicarboxylate transporter present in the proximal tubulus of mammalian kidney. Homozygous T-DNA insertional
knock-out mutants lacking a functional AttDT did not show
an obvious phenotype, but contained less malate in leaves.
Leaf malate contents were reduced to 25–50% of the wildtype (WT) contents in Attdt deletion mutants, whereas the
residual vacuolar malate transport activity in the mutants
was reduced to about 30% of that observed for vacuoles
isolated from WT plants. Furthermore, the respiratory coefficient was increased in the deletion mutants, indicating a
higher consumption of carboxylates in the absence of the
malate transporter. Surprisingly, further investigations
using the patch-clamp method revealed that Attdt mutants
still exhibited the well-described vacuolar malate channel,
as well as citrate transport activity (Hurth et al., 2005). Hence,
vacuolar malate transport is catalyzed by a transporter and
at least one channel. However, the molecular nature of the
vacuolar malate channel remained to be elucidated. Former
vacuolar proteomic studies identified a large number of
putative vacuolar membrane proteins (Carter et al., 2004;
Endler et al., 2006; Jaquinod et al., 2007); however, no
putative candidate emerged from these data. Instead, malate
channels localized in the plasma membrane have recently
been described in wheat (TaALMT1, Triticum aestivum aluminum-activated malate transporter 1; Sasaki et al., 2004),
Arabidopsis (AtALMT1; Hoekenga et al., 2006) and rape
(BnALMT1 and BnALMT2, Brassica napus AMLT1 and
AMLT2; Ligaba et al., 2006). These malate channels confer
aluminum tolerance by extruding malate from root epidermal cells into the surrounding soil. In Arabidopsis these
AtALMTs form a small protein family of 14 members
(Hoekenga et al., 2006). Recent data have clearly demonstrated that gene products of the same protein family are
often targeted to different membranes (Becker et al., 2004;
Chen, 2005; Czempinski et al., 2002; Endler et al., 2006). This
led us to hypothesize that one or several members of the
AtALMT protein family could be targeted to the tonoplast,
where they function as malate channels.
We describe in this work that an AtALMT9-GFP fusion
protein localizes to the tonoplast of Arabidopsis and onion
epidermal cells, as well as to the tonoplast of Vicia faba
guard cells. Patch-clamp experiments using mesophyll
vacuoles isolated from Atalmt9 deletion mutants revealed
that the current density was attenuated by approximately
70%, whereas the malate concentrations in the protoplasts
were slightly decreased. Furthermore, control patch-clamp
measurements on vacuoles derived from AtALTM9-GFP
overexpressing tobacco cells showed strongly enhanced
malate channel activities. In addition, the functional expression of AtALMT9 in Xenopus oocytes further confirmed the
identity of AtALMT9 as a bona fide malate channel. With the
exception of a strongly reduced malate channel activity and
a slightly reduced vacuolar malate concentration, we could
not observe any obvious phenotype for Atalmt9, suggesting
possible functional redundancy of the vacuolar malate
transporter AttDT and vacuolar ALMTs in Arabidopsis.
Results and discussion
Arabidopsis ALMTs are hydrophobic proteins
with slight differential topologies
A dendrogram based on an amino acid sequence alignment
(Chenna et al., 2003) showed high similarities at the amino
acid level within the AtALMT protein family and TaALMT1
(65.8 1.3% average pair distance, Figure S1). Furthermore,
the gene structure is largely conserved within all members
with respect to the number of introns (ARAMEMNON plant
membrane protein database, http://aramemnon.botanik.
uni-koeln.de; Schwacke et al., 2003). The dendrogram based
on amino acid sequence similarity indicated that the
AtALMT family is grouped into three distinct clades
(Figure 1a). TaALMT1 (AB081803) belongs to clade 1, which
includes AtALMT1, 2, 7, 8 and 10 (At1g08430, At1g08440,
At2g27240, At3g11680 and At4g00910), whereas clade 2
includes AtALMT3, 4, 5, 6 and 9 (At1g18420, At1g25480,
At1g68600, At2g17470 and At3g18440). The protein family
members AtALMT11, 12, 13 and 14 (At4g17585, At4g17970,
At5g46600, At5g46610) belong to clade 3. A comparison of
the hydrophobicity profiles of the well-described AtALMT1
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Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 52, 1169–1180
Arabidopsis vacuolar malate channel 1171
(a)
(b)
topologies, which are implemented in the ARAMEMNON
database (Schwacke et al., 2003), whereas the consensus
scoring strongly enhances the reliability of topology predictions (Nilsson et al., 2000). A graphical overlay of an
alignment based on the amino acid sequences of TaALMT1,
AtALMT1, AtALMT5 and AtALMT9 by putative transmembrane regions showed that the protein members of clades 1
and 2 differ in the C-terminal transmembrane topology, with
respect to number and position (see Figure S1); whereby a
possible role of this difference in function and localization
can only be assumed.
Hoekenga et al. (2006) previously characterized the
clade-1 member AtALMT1 as an aluminum-activated malate
channel localized in the plasma membrane of Arabidopsis
roots. Because of the high sequence homology within the
ALMT gene family, it seemed possible that all protein
members fulfil analogous functions as malate channels
within the plant cell. Moreover, taking the fact that gene
products of the same protein family may be targeted to
different membranes, in this study we investigated AtALMT5
and AtALMT9 as members of clade 2, which is slightly more
similar to the previously characterized tonoplast transporter
AttDT, compared with the other two clades (Figure 1a).
AtALMT9-GFP localizes in the tonoplast
Figure 1. Dendogram and hydrophobicity analysis of the aluminum-activated
malate transporter (ALMT) protein family.
(a) The dendrogram of 15 members of the ALMT protein family and the
Arabidopsis thaliana tonoplast dicarboxylate transporter (AttDT), based on an
amino acid sequence alignment with CLUSTALW (Chenna et al., 2003), shows
that AtALMTs cluster in three main groups, whereas the plasma membrane
localized proteins TaALMT1 and AtALMT1 both share the same clade. Branch
lengths are proportional to the level of interfered evolutionary change, and
are given in relative units.
(b) Hydrophobicity analysis of AtALMT5, AtALMT9, AtALMT1 and TaALMT1
shows slight differences in transmembrane topology. Hydrophobicity values
were calculated according to the Kyte and Doolittle method (window size,
11 amino acids; Kyte and Doolittle, 1982) and were plotted against the amino
acid position. Positive values indicate hydrophobic regions. Putative transmembrane segments are indicated by horizontal bars that are colored
according to the consensus scores, which represent a scale for the accordance
between different algorithms for transmembrane structure prediction provided by the ARAMEMNON plant membrane protein database (Schwacke
et al., 2003).
and TaALMT1 with members of clade 2, e.g. AtALMT5 and
AtALMT9, indicated that all these proteins are hydrophobic
with two transmembrane domains: one of which is localized
to the N-terminus, consisting of between five and seven
transmembrane a-helices, and the other is localized to the
C-terminus, spanning the tonoplast only once (Figure 1b).
This prediction was achieved by consulting consensus
scores, which represent percentage accordances between
different algorithms for the elucidation of transmembrane
In order to verify our hypothesis that members of the
AtALMT family are targeted to membranes of different
organelles, we undertook subcellular localization experiments with two members of the second clade: AtALMT5 and
AtAMLT9 (Figure 1a). GFP was fused in frame to the C-terminal end of AtALMT5 and AtALMT9. The transient expression of these constructs in Arabidopsis and onion epidermal
cells by particle bombardment demonstrated that AtALMT9
was targeted to the tonoplast in both cases (Figure 2a–e),
whereas fluorescence for AtALMT5-GFP was observed
exclusively in the endoplasmic reticulum (ER; Figure S2). To
preclude the possibility that the fusion proteins were mistargeted because of the lack of chloroplasts in epidermal
cells, we conducted the same experiments in chloroplastcontaining guard cells of V. faba. The GFP fluorescence
pattern of these cells confirmed our observations that
AtALMT9 was targeted to the tonoplast (Figure 2f,g), and
that AtALMT5 was targeted to the ER (Figure S2). In contrast
to epidermal cells, guard cells contain a far more complex
vacuolar membrane system, consisting of a large number of
invaginations, playing an important role in rapid changes of
vacuolar volume during stomatal movement (Gao et al.,
2005). Because of this structure, it was easier to visualize the
tonoplastic localization of AtALMT9-GFP by a fluorescence
signal from the chloroplasts, which are located in the cytoplasm. Taken together these observations clearly demonstrate that in contrast to AtALMT1, AtALMT9 is a tonoplast
protein. It is tempting to speculate that the difference in the
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Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 52, 1169–1180
1172 Peter Kovermann et al.
(a)
(c)
(b)
(a)
(b)
(c)
(d)
(e)
(d)
(f)
(g)
Figure 2. Subcellular localization of AtALMT9 by transient expression of the
AtALMT9-GFP fusion construct.
In Arabidopsis epidermal cells (a,b) as well as in onion epidermal cells (c–e)
fluorescence of the AtALMT9-GFP fusion protein is targeted to the tonoplast,
which separates the vacuolar lumen from the cytoplasm containing the
nucleus (marked by an arrow). The transmission picture (c) is the same onion
epidermal cell as in the fluorescent image (d). GFP clearly surrounds the
nucleus of the Arabidopsis and onion cells (b and e). Cell walls and nuclei of
Arabidopsis epidermal cells were stained in red with propidium iodide,
whereas the nucleus in the onion epidermal cell is visible by an overlay of (c)
and (d). In chloroplast-containing guard cells of Vicia faba the fluorescence of
the AtALMT9-GFP fusion protein (f, g) is also visible in the tonoplast
(chloroplasts are shown by red fluorescence in the transmission picture, f).
The GFP fluorescence surrounds the chloroplast (marked by an arrow) located
in the cytoplasm between the vacuole and the plasma membrane of the cell.
Scale bars: (a) 25 lm; (c) 20 lm; (f) 10 lm.
subcellular localization between these two proteins relies on
differences in transmembrane topology within the C-terminal region, as described above. The fact that AtALMT5, also a
member of the second clade (Figure 1a), is not localized in
the vacuolar membrane may result from slight differences in
the predicted structure between AtALMT5 and AtALMT9
(Figure 1b, S1).
Analysis of AtALMT9 expression by AtALMT9
promoter:GUS plants
Detailed tissue expression pattern analysis is a prerequisite
to understanding the function of a given gene product. We
Figure 3. Analysis of AtALMT9 expression by AtALMT9 promoter:GUS
reporter plants.
(a) GUS-histochemical staining of young Arabidopsis seedlings (4 days after
germination) showing GUS activity in the hypocotyls. GUS-histochemical
staining of young (b, 8 days after germination) and older (c, 20 days after
germination) Arabidopsis plants, showing GUS expression in the leaves and
the stem, and also along the root.
(d) Cross-section of a rosette leaf showing GUS expression mainly in the
mesophyll cells, and weak expression in the upper and lower epidermal cell
layers. Scale bars: (a) 1 mm; (b, c) 0.1 cm; (d) 100 lm.
thus investigated the tissue specificity of AtALMT9 using
plants transformed with a b-glucuronidase gene (GUS)
under the control of a 1785 bp promoter region upstream of
AtALMT9. Analysis of these transgenic plants revealed GUS
activity in the hypocotyl of young seedlings (Figure 3a), and
strong activity in the leaves of young and older plants (Figure 3b,c). GUS activity was also detected in the roots of
younger plants (Figure 3b). This activity was higher in the
later developmental stages (not shown). In the flower tissue,
GUS staining was found in both the sepals and the stamina
(Figure S3). To investigate the expression profile in leaves in
more detail, leaves were embedded and cross-sectioned
(Figure 3d). Microscopical analysis of these sections indicated that GUS activity was concentrated in the mesophyll
tissue of leaves, whereas only weak activity was visible in
the upper and lower epidermal cell layers. Our observations
concur with gene expression data from microarray experiments (average of at least 231 chip experiments) available in
the Genevestigator A. thaliana microarray database (http://
https://www.genevestigator.ethz.ch), which indicated that
AtALMT9 mRNA accumulated similarly in all tissues of the
plant, with the highest levels in flowers, roots and leaves.
Summarizing our localization data, we clearly demonstrate
that AtALMT9 is a vacuolar protein expressed in nearly all
organs of the plant, with a cell-type specificity in leaves, as
GUS staining was observed nearly exclusively in mesophyll
cell layers. Therefore, we decided to focus our interest on the
vacuole of the leaf mesophyll cells for further functional
studies.
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Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 52, 1169–1180
Arabidopsis vacuolar malate channel 1173
(a)
(b)
Figure 4. Analysis of an AtALMT9 T-DNA insertion mutant plant.
(a) The genomic structure of AtALMT9, and the position and orientation of the
T-DNA insertion in the AtALMT9 gene of the mutant line SALK_590362. The
gene has six exons (black arrows) and the insertion is located at position
+19 bp downstream of the start codon. The orientation of the left border (LB)
is indicated. (b) Semiquantitative RT-PCR analysis of the abundance of the
AtALMT9 transcript demonstrated the lack of AtALMT9 transcript in the
homozygous knock-out line.
Malate currents are decreased in Atalmt9 knock-out plants
To reveal the physiological role of the tonoplast-localized
AtALMT9 protein, we obtained a SALK T-DNA insertion
mutant line for AtALMT9 from the Nottingham Arabidopsis
stock center (NASC, http://arabidopsis.info; N590362; Scholl
et al., 2000), carrying a T-DNA insertion in the first exon of
the AtALMT9 gene (SALK_090362, Figure 4a). Homozygous
knock-out plants were identified by PCR using appropriate
primers (not shown). The absence of the AtALMT9 transcript
in these mutant plants was demonstrated by RT-PCR analysis with primers amplifying the entire coding region
(1797 bp, Figure 4b). This homozygous line was used to
perform electrophysiological analyses, and to investigate
whether differences in malate channel activity could be
observed compared with vacuoles isolated from WT plants.
The presence of a vacuolar malate import channel in the
mesophyll of WT plants was already shown by electrophysiological investigations on CAM and C3 plants. Thus far the
available data indicate that malate currents across the
tonoplast of different plant species share common characteristics, such as inward rectification, selectivity for anions
over cations and activation at high membrane potentials
(Epimashko et al., 2004; Pantoja and Smith, 2002).
In our study we quantified the malate channel activity at
the mesophyll tonoplast with the patch-clamp technique in
the whole-vacuole mode. We clamped Arabidopsis WT
vacuoles to test voltages of 3-s duration between +100 and
)140 mV. The test voltages follow the sign convention
proposed by Bertl et al. (1992). To ensure that currents
observed at pH 7.5 (bath) were principally caused by movements of the malate2) ions, we buffered malic acid with an
impermeable cation according to the method described by
Hafke et al. (2003) and Hurth et al. (2005). Under asymmetric
ionic conditions (100 mM malate2) out/10 mM malate2) in),
large inward currents were observed at negative test voltages. These currents consisted of an instantaneous element
(Iinst) superimposed at negative voltages by a slow timedependent element (Istd, Figure 5a). The current–voltage
(I–V) relationships for Iinst (data not shown) and Istd (Figure 5c, ) showed that both elements were inward-rectifying, which is in agreement with the previously described
properties of tonoplastic malate currents (Epimashko et al.,
2004; Hafke et al., 2003; Pantoja and Smith, 2002). At a test
voltage of )140 mV the vacuoles exhibited a mean current
density (Istd) of 13.6 1.9 pA pF)1 (n = 5, Figure 5c, ). This
fits well with previously observed values obtained under the
same ionic conditions with Arabidopsis WT mesophyll
vacuoles (Hurth et al., 2005). In order to further characterize
these currents, we performed a tail current analysis under
asymmetric ionic conditions (100 mM malate2) out/10 mM
malate2) in, data not shown). A reversal potential of +12 mV
could be calculated. This reversal potential is closer to the
theoretical potential for malate2) (+23 mV) than to the
Nernst potentials for other ions in the solution
(EBPTH+ = )63 mV, ECl- = 0 mV, ECa2+ = 0 mV and EMg2+ =
0 mV; Hafke et al., 2003), which strongly suggests that Istd
originates from inward-rectifying malate channels. Similar
observations have already been described for Arabidopsis
and other plants (Cerana et al., 1995; Hafke et al., 2003; Hurth
et al., 2005; Martinoia et al., 1985; Pantoja and Smith, 2002;
Pei et al., 1996).
Compared with WT vacuoles, vacuoles isolated from
Atalmt9 deletion mutants exhibited strongly diminished
inward currents compared with WT vacuoles (Figure 5b,c).
At a test voltage of )140 mV we observed a strong current
reduction compared with WT plants (T3 progeny,
Istd = 3.13 0.96 pA pF)1, n = 7; T4 progeny, Istd = 4.1 0.89 pA pF)1, n = 5; Figure 5c). Despite this difference, the
mutant plants grown under normal conditions were phenotypically indistinguishable. Determination of malate content
in isolated mesophyll protoplasts showed that deletion
mutants contained 19.5 7.4% and 20.0 6.0% (SE) less
malate, compared with the WT obtained from the seed batch
of the SALK mutant line and the normal Col-0 WT plants,
respectively.
In Attdt deletion mutants, leaf malate content was
reduced by 50–75%, and vacuoles isolated from Attdt
mutants still contained 30% of the cellular malate in their
vacuoles, indicating that under normal conditions AttDT is
the major malate importer (Hurth et al., 2005). The high
capacity of AtALMT9 at high potential differences indicates
that this channel is important under specific conditions
when the vacuole is hyperpolarized. However, it also has
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Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 52, 1169–1180
1174 Peter Kovermann et al.
to be kept in mind that Attdt deletion mutants exhibited
only a slight phenotype, which could be observed under
certain stress conditions. In addition, our results support
the presence of an additional vacuolar malate channel,
which together with AttDT can ensure the import of malate
in Atalmt9 deletion mutants. In summary, the observations
presented above suggest that AtALMT9 is either a vacuolar
malate channel or is at least involved in a regulatory
function on the channel.
Overexpression of AtALMT9-GFP in tobacco leaves
enhances malate currents across the tonoplast
Figure 5. Patch-clamp analysis of vacuoles obtained from Arabidopsis
Atalmt9 knock-out plants compared with wild-type (WT) vacuoles.
(a) Whole-vacuole malate current density traces of an Arabidopsis WT
vacuole, obtained by dividing the raw currents by the tonoplast capacitance
under asymmetric ionic conditions (100 mM malate2) out/10 mM malate2) in).
(b) Whole-vacuole current density traces of a vacuole isolated from Atalmt9
knock-out plants under the same ionic conditions as in (a) showed strongly
diminished malate current densities.
(c) Corresponding current density plots derived from whole-vacuole malate
currents across the tonoplast from WT plants ( , n = 5), and from Atalmt9
knock-out plants from the T3 (m, n = 7) and the T4 (d, n = 6) progenies.
To obtain further proof for the channel activity of AtALMT9
we transiently overexpressed the AtALMT9-GFP fusion construct in leaves of Nicotiana benthamiana under the control
of the CaMV 35S promoter. Vacuoles isolated from these
leaves revealed clearly detectable fluorescence in the tonoplast, thereby confirming the results of our transient localization studies (Figure 6c). Patch-clamp measurements on
these fluorescent vacuoles and tobacco WT vacuoles
demonstrated that the overexpression of AtALMT9-GFP
enhanced malate current density across the tobacco
mesophyll tonoplast. Whereas the AtALMT9-GFP-mediated
malate currents exhibited the well-described characteristics,
consisting of an instantaneous and a time-dependent component, background currents in WT tobacco vacuoles lacked
a pronounced time dependence (Figure 6a,b). The comparison of malate currents from WT vacuoles and AtALMT9overexpressing vacuoles was achieved by the determination
of total current amplitudes. Overall, a 7.4-fold increase of
total malate current density was observed in vacuoles
derived from overexpressing plants at )120 mV (Figure 6d;
WT, 14.2 1.5 pA pF)1, n = 5; AtALMT9-GFP, 104.9 23.9 pA pF)1, n = 8). The relatively large variability results
from different levels of transgene expression. Strongly
fluorescent vacuoles exhibited a high malate current density,
whereas the malate current densities in non-fluorescent
vacuoles from the same sample behaved as WT vacuoles
(Figure 6d). Furthermore, to exclude the possibility that the
high expression of a vacuolar membrane protein fused to
GFP could unspecifically induce malate currents, we also
performed control experiments by overexpressing the vacuolar sucrose transporter construct SUT4-GFP (Endler et al.,
2006). No increase in malate currents could be detected (data
not shown).
To obtain more information about the substrate specificity, we also measured fumarate- and chloride-mediated
total currents in vacuoles isolated from the AtALMT9-GFPexpressing tobacco plants (Figure 6d). The increase in
fumarate current densities at 120 mV was 3.7-fold (WT,
22.0 1.6 pA pF)1, n = 5; AtALMT9-GFP, 81.8 12.3 pA
pF)1, n = 4) compared with WT vacuoles. Former studies on K. daigremontiana have shown that mesophyll
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Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 52, 1169–1180
Arabidopsis vacuolar malate channel 1175
Figure 6. Patch-clamp analysis of AtALMT9-GFP
heterologously expressed in Nicotiana benthamiana.
(a) Whole-vacuole malate current density traces
of a tobacco wild-type (WT) vacuole obtained
under the same conditions as in Figure 5a).
(b) Whole-vacuole malate current density traces
of a strongly fluorescent vacuole isolated from
AtALMT9-GFP-overexpressing tobacco (same
conditions as in Figure 5a) exhibited largely
increased malate current densities.
(c) Transmission and fluorescence picture of a
vacuole released from a tobacco mesophyll
protoplast overexpressing AtALMT9-GFP.
(d) Bar graphs representing current densities at
)120 mV obtained by the patch-clamping of WT
tobacco vacuoles and vacuoles derived from
tobacco plants overexpressing (OE) AtALMT9GFP under asymmetric ionic conditions (100 mM
anions out/10 mM anions in). Scale bar: (c)
15 lm.
(a)
(c)
vacuoles exhibit higher fumarate current densities than
malate current densities (Hafke et al., 2003). This is
apparently also true for N. benthamiana WT vacuoles
(Figure 6d). In vacuoles from AtALMT9-GFP-expressing
tobacco plants the increases in fumarate current densities
as well as the absolute values are lower compared with
those for malate, which indicates that AtALMT9 has a
higher selectivity for malate than for fumarate. However,
because of the high variability of the fluorescence of the
vacuoles, reflecting the expression level of ALMT9, an
exact permeability ratio for malate and fumarate can not
be deduced. Lower current densities compared with the
dicarboxylates could be detected for chloride in tobacco
WT vacuoles. The AtALMT9-GFP-mediated increase in
chloride currents was by a factor of 2.2 at 120 mV (WT,
10.6 2.2 pA pF)1, n = 6; AtALMT9-GFP, 23.5 1.5 pA
pF)1, n = 4). This result indicates that AtALMT9 also
exhibits a weak chloride conductance. The AtALMT9-GFP
mediated currents can also be calculated by substracting
the WT current densities, and further underlines the
results presented above, even assuming that two
charges are transferred in the case of the dicarboxylates
and only one charge is transferred for chloride
(malate, 90.7 pA pF)1; fumarate, 59.8 pA pF)1; chloride,
12.9 pA pF)1).
(b)
(d)
AtALMT9-expressing oocytes show malate currents
The expression of AtALMT9 in Xenopus oocytes served
to confirm that the protein indeed has channel-forming
capability. Plasma membrane localized ALMT proteins from
wheat, Arabidopsis and rape mediated malate fluxes when
the respective genes were heterologously expressed in
Xenopus oocytes (Hoekenga et al., 2006; Ligaba et al., 2006;
Sasaki et al., 2004). Furthermore, it has been shown that
plant tonoplast proteins can be targeted to the plasma
membrane of oocytes (Maurel et al., 1993; DesbrossesFonrouge et al., 2005).
When oocytes injected with AtALMT9 cRNA were
challenged with a series of voltage steps from +20 to
)160 mV, instantaneously activating currents were
recorded (Figure 7a). In contrast to the observations in
the tonoplast system (Figures 5 and 6), no currents were
activated in a time-dependent manner in response to 3-s
voltage pulses (data not shown). The membrane environment or missing post-translational modifications in the
heterologous oocyte system (Stühmer and Parekh, 1995)
may change the characteristics of the AtALMT9 protein.
Current amplitudes increased when the external malate
concentration was changed from 0 to 10 mM (Figure 7a,b).
This behavior is surprising for the range of negative test
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 52, 1169–1180
1176 Peter Kovermann et al.
Figure 7. Voltage-clamp experiments on Xenopus laevis oocytes expressing AtALMT9.
(a, b) Representative current traces recorded in
response to voltage pulses from +20 to )160 mV
in )20-mV steps. The holding and tail potential
was )60 mV. AtALMT9 cRNA-injected oocytes
were challenged with external solutions containing 0 mM (a) or 10 mM malate (b).
(c) Mean current–voltage relationships constructed from recordings like the ones shown in
(a) and (b). Currents derive from AtALMT9 cRNAinjected oocytes (circles) and control oocytes
(squares) in the presence of 0 mM malate (open
symbols) or 10 mM malate (filled symbols). Values are the means of at least four experiments;
error bars represent the SE. Reversal potentials
for currents from cRNA-injected oocytes in 0 mM
(1) and 10 mM malate (2) are indicated by arrows.
potentials (as it implies that more malate moves out the
cell in the presence of an oppositely directed gradient),
but could indicate a regulatory role of malate in channel
functioning. For example, such a type of regulation has
been found for some voltage-gated potassium channels
(Pardo et al., 1992; Wood and Korn, 2000). Current–
voltage relationships for all conditions tested are shown
in Figure 7c. Compared with AtALMT9-expressing
oocytes, current amplitudes in control oocytes were much
smaller and did not significantly increase upon the
addition of 10 mM malate. Upon stepping from 0 to
10 mM external malate, currents in cRNA-injected oocytes
increased about 1.9-fold at )160 mV, and shifted towards
more negative reversal potentials (DErev = )11 2.4 mV,
n = 4). This behaviour is consistent with the activation of
anion-selective inward currents in AtALMT9-expressing
oocytes.
We used lanthanum (La3+) to exclude the possibility that
the observed differences were caused by endogenous
oocyte currents appearing at negative potentials (Tokimasa
and North, 1996; Picco et al., 2007). The addition of 1 mM
LaCl3 to the bath solution containing 10 mM malate did not
alter instantaneous current amplitudes, but effectively
blocked the typical time-dependent endogenous currents
appearing at high negative potentials (data not shown). A
common feature shared by ALMT proteins localized at the
plasma membrane of plant roots is the slow activation by
external aluminum (Al3+; Sasaki et al., 2004; Ligaba et al.,
2006; Hoekenga et al., 2006). When AtALMT9-expressing
oocytes were challenged with 0.1 mM Al2(SO4)3 in the bath
solution without malate, negative currents at )120 mV
slowly increased, before reaching a maximum value
(1.5-fold 0.1; n = 4) within 10 min. By contrast, there was
no change in current amplitude in control oocytes
(0.98 0.05, n = 4; data not shown).
Conclusion
A large number of studies have shown that malate plays an
essential role in the metabolism of the plant cell, and is
implicated in ion homeostasis and maintenance of cell turgor. To fulfil these functions malate must be accumulated
within the large central vacuole. In this work we have identified AtALMT9, a homolog of TaALMT1 and AtALMT1, as a
tonoplastic malate channel. In Arabidopsis knock-out
mutants, the vacuolar malate currents were strongly
reduced, and the malate concentration was slightly diminished. We confirmed that AtALMT9 was a bona fide malate
channel by functional expression of the channel in
N. benthamiana and in Xenopus oocytes. The fact that no
obvious phenotype could be observed is very probably
caused by malate transport activity of AttDT and the residual
channel activity observed in knock-out plants. Furthermore,
the observation that AtALMT9 is mainly expressed in the
mesophyll argues that an epidermis-specific malate channel
is also present. Therefore, we are presently investigating
other members of the AtALMT family in Arabidopsis to
determine if any others localize to the tonoplast. Functional
characterization of these additional malate channels, and the
generation of double and triple knock-out plants, also in
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 52, 1169–1180
Arabidopsis vacuolar malate channel 1177
combination with the Attdt deletion mutant, will allow us to
elucidate in detail the role of the vacuole in cytosolic malate
homeostasis. Furthermore, our study will also allow us to
identify and characterize the vacuolar malate channels of
CAM plants, and to investigate whether this channel is a
prerequisite for CAM metabolism, as is often assumed.
Experimental procedures
Hydrophobicity analysis and comparison
of primary structures
Amino acids sequence alignments were performed with the CLUalgorithm using default parameters (http://www.ebi.ac.uk/
clustalw; Higgins et al., 1994). Hydrophobicity profiles were calculated according to the method described by Kyte and Doolittle
(1982). Structural consensus scores and the nomenclature of the
AtALMT protein family was adopted from the ARAMEMNON plant
membrane protein database (http://aramemnon.botanik.unikoeln.de; Schwacke et al., 2003). Dendrograms were constructed
using PHYLODRAW (Graphics Application Laboratory, http://pearl.cs.
pusan.ac.kr).
STALW
V. faba using a Helium Biolistic Particle Delivery system (Bio-Rad
Laboratories, http://www.bio-rad.com).
Selection of Atalmt9 knock-out lines
Seeds of Atalmt9 knock-out lines (stock number SALK_090362;
Salk Institute Genomic Analysis Laboratory, http://signal.salk.edu/
cgi-bin/tdnaexpress) were obtained from the Nottingham Arabidopsis stock center (N590362; NASC, http://arabidopsis.info).
Genomic DNA was extracted from 4-week-old soil-grown plants,
and the T-DNA insertion (+19 bp downstream of the ATG)
was verified by PCR with the T-DNA-specific primer LBb1
(5¢-GCGTGGACCGCTTGCTGCAACT-3¢) and the primer At3g18440TDNA-LB (5¢-GTCACCGAATAAAGTGGAAAGC-3¢) binding 110-bp
upstream of the start ATG. Lines with homozygous T-DNA
insertions were identified by genomic PCR with a set of AtALMT9-specific primers (At3g18440-TDNA-LB and At3g18440-TDNARB: 5¢-AGGTCCACCACCACTTCATAAC-3¢). The abundance of the
AtALMT9 transcript in homozygous knock-out lines and in WT
plants was assayed by isolation of total RNA from whole leaves
followed by RT-PCR (Amersham kit, http://www.amersham.com)
using the AtALMT9-specific primers AtALMT9forw1 (5¢-GGTACCATGGCGGCGAAGCAAGGTTCCTTC-3¢) and AtALMT9backw1
(5¢-GGTACCCATCCCAAAACACCTACGAATCTT-3¢) and the control
primers Actin-forw (5¢-GGAACAGTGTGACTCACACCATC-3¢) and
Actin-backw (5¢-AAGCTGTTCTTTCCCTCTACGC-3¢).
Strains and growth conditions
Escherichia coli (DH5a; Hanahan, 1983) was used for cloning.
A. thaliana (Col-0) plants were grown in controlled environment
chambers or on agar medium (8-h light//16-h dark, 22C, 55% relative humidity). N. benthamiana plants were grown in potting soil
(16-h light//8-h dark, 22C, 55% relative humidity). The transformation of Arabidopsis was performed with Agrobacterium tumefaciens (GV3101; Holsters et al., 1980).
Quantification of malate concentrations
For quantification experiments, a-mannosidase and malate were
determined in the protoplasts according to the method described by
Hurth et al. (2005).
Expression of AtALMT9-GFP in N. benthamiana
Tissue-specific expression and subcellular localization
of AtALMT9 in Arabidopsis
A 1785 bp promoter region upstream of AtALMT9 was amplified
from genomic DNA of Arabidopsis (Col-0) by high-fidelity PCR using
the primers At3g18440g-1847f (5¢-GGGGACAAGTTTGTACAAAAAAGCAGGCT-GTTTCTCTCTGTGCCTGAGTTTG-3¢) and At3g18440g30r (5¢-GGGGACCACTTTGTACAAGAAAGCTGGGTGACGGATTCTCAAAGAGAATTAAGC-3¢). This region was cloned into the GATEWAY entry vector pDONR207 (Invitrogen, http://www.
invitrogen.com) before recombination cloning into pMDC163 (Curtis and Grossniklaus, 2003). The vector construct was transformed
into Arabidopsis using the Agrobacterium-mediated floral-dipping
method (Clough and Bent, 1998). T2 progeny of hygromycin-resistant transformants were GUS-stained at various developmental
stages. Embedding of GUS-stained leaves was performed in Technovit (Heraeus Kulzer GmbH, http://www.heraeus-kulzer.com). To
localize AtALMT9 at the subcellular level, the AtALMT9 cDNA
(1797 bp) was amplified from RIKEN clone pda08640 (cDNA clone
RAFL09-66-G16; Sakurai et al., 2005; Seki et al., 1998, 2002; Yamada
et al., 2003) with the primers Mc8forw (5¢-GGTACCATGGCGGCGAAGCAAGGTTCCTTC-3¢) and Mc8backw (5¢-GGTACCCATCCCAAAACACCTACGAATCTT-3¢), and then were ligated at the KpnI site
into the pGFP2 vector (Haseloff and Amos, 1995) to create a constitutively expressed AtALMT9-GFP fusion protein. The resulting
AtALMT9-GFP construct was transiently expressed in Arabidopsis
and onion (Allium cepa) epidermal cells, as well as in guard cells of
For transient overexpression of an AtALMT9-GFP construct in
tobacco leaves, the AtALMT9 cDNA (1797 bp) was cloned under the
control of the CaMV 35S promoter using the pART7/pART27 cloning/expression system (Gleave, 1992). The Agrobacterium-mediated infiltration of N. benthamiana leaves was conducted as
described by Yang et al. (2001), with slight modifications. After
agroinfiltration, tobacco plants were grown in the greenhouse at
22C under 16 h of light. Isolation of vacuoles and patch-clamp
experiments were performed 48 h after infiltration.
Isolation of vacuoles
Protoplasts were isolated from leaf mesophyll as previously described by Song et al. (2003), with minor modifications. After incubation at 30C (45–60 min) protoplasts were liberated by gentle
agitation, and a small aliquot (20 ll) of protoplast suspension was
transferred into a patch-clamp chamber filled with 200 ll of a lysis
solution consisting of patch-clamp bath solution plus 8 mM EDTA.
After 4 min the lysis buffer was replaced by the bath solution.
Patch-clamp measurements
Whole-vacuole malate currents were recorded using the standard
patch-clamp technique according to the method described by
Hamill et al. (1987), in whole-vacuole configuration using an EPC10 amplifier (HEKA Electronics, http://www.heka.com). Data
ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 52, 1169–1180
1178 Peter Kovermann et al.
acquisition and analysis were conducted with PULSE (HEKA
Electronics) and ORIGIN (OriginLab, http://www.originlab.com),
in combination with the PULSEFIT software (HEKA Electronics).
Patch pipettes were prepared from borosilicate glass capillaries
(Harvard Apparatus, http://www.harvardapparatus.com) with a
DMZ-Universal puller (Carl Zeiss, Inc., http://www.zeiss.com).
The bath chamber was mounted on an inverted fluorescence
microscope (Eclipse TE2000-U; Nikon Instruments, http://www.
nikoninstruments.com). For whole-cell experiments, vacuoles with
a diameter of 25–40 lm were selected. The applied voltages refer
to the cytoplasmic side of the vacuole, whereas the vacuolar side
was at the ground (Bertl et al., 1992). The vacuolar surface areas
were determined from capacitance currents measured in response
to short (10-ms) voltage steps of 10-mV amplitude (Gillis, 1995).
All measurements were made at room temperature (20–22C).
Osmolality of all solutions was calibrated to 440 mosmol kg)1
with mannitol. In all solutions malic acid was buffered with BisTris Propane (BTP), a relatively impermeable cation, to ensure that
the currents observed were principally attributable to movements
of the malate2) ions (Hafke et al., 2003). The standard bath solution contained 100 mM malic acid, 1 mM CaCl2, 1 mM EDTA and
3 mM MgCl2, pH 7.5, adjusted with BTP. The pipette solution
contained 10 mM malic acid, 1 mM CaCl2 and 3 mM MgCl2, pH 5.5,
adjusted with BTP. For patch-clamp experiments on substrate
specificity, malic acid was replaced by fumaric acid and hydrochloric acid. Whole-cell configuration was made by a short bipolar
voltage pulse ( 900 mV, 600 ls each). Current density plots
(pA pF)1) of Arabidopsis vacuoles were obtained by plotting the
isochronal current amplitude differences between the first and last
20 ms of the voltage step, normalized by the tonoplast capacitance, against the applied test voltage. The comparison of WT and
AtALMT9-overexpressing Nicotiana vacuoles was performed by
the determination of total steady-state current during the last
20 ms of the voltage step.
Two-electrode voltage clamp (TEVC) on Xenopus oocytes
The AtALMT9 cDNA was amplified from the RIKEN clone pda08640
(cDNA clone RAFL09-66-G16; Sakurai et al., 2005; Seki et al., 1998,
2002; Yamada et al., 2003) with the primers Mc8forwCF3
(5¢-GGGAATTCGCGGCCGCATGGCGGCGAAGCAAGGTTCCTTC-3¢)
and Mc8CF3-backw-1 (5¢-TATCAAATCATATGTTACATCCCAAAACAC-3¢), and was subcloned into the pCF3 expression vector (at NotI
and NdeI restriction sites) for efficient expression in oocytes, as
described previously (Preston et al., 1992; Shitan et al., 2003). The
plasmid was linearized using the unique site AscI, and was used as a
template for the synthesis of capped cRNA using a Message Machine T7 kit (Ambion, http://www.ambion.com). Stage V–VI defolliculated oocytes from Xenopus were isolated and maintained as
described previously (Virkki et al., 2006). TEVC experiments on
oocytes expressing AtALMT9 were conducted according to the
method described by Baumgartner et al. (1999). Oocytes were injected with 50 nl cRNA (0.2 lg ll)1) encoding AtALMT9. Control
oocytes were injected with 50 nl of double-distilled water. After
injection, the oocytes were incubated at 18C in modified Barth’s
solution containing 88 mM NaCl, 1 mM KCl, 0.41 mM CaCl2, 0.82 mM
MgSO4, 2.5 mM NaHCO3, 2 mM Ca(NO3)2 and 7.5 mM HEPES,
pH 7.5, adjusted with 2-amino-2-(hydroxymethyl)-1,3-propanediol
(TRIS), supplemented with penicillin (5 mg ml)1) and spectomycin
(5 mg ml)1). Electrophysiological experiments were performed 4–
5 days after injection. TEVC was made using the Geneclamp 500E
Amplifier (Molecular Devices Corporation, http://www.moleculardevices.com). The voltage clamp was controlled, and data were
acquired using a computer running PCLAMP8 software (Molecular
Devices Corporation), which also controlled the valves for solution
switching. Oocytes were initially superfused with ND-100 solution
(100 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2 and 10 mM
HEPES, pH 7.4) before switching to experimental solutions. All
experiments were performed at room temperature under continuous flow of experimental solutions. Solutions in the recording
chamber were changed at a rate of 5 ml min)1. Bath solutions were
chosen in order to reveal malate-dependent currents: 0 or 10 mM
malic acid, 0.3 mM CaCl2, buffered with BTP to pH 7.5, adjusted to
220 mosmol kg)1 with mannitol. To test for aluminum-dependent
activation, 0.1 mM Al2(SO4)3 was added to a bath solution containing 0 mM malate and 0.1 mM LaCl3.
Data analysis was performed using the CLAMPFIT software
(Molecular Devices Corporation) and GRAPHPAD software (GraphPad
Software, http://www.graphpad.com). Current–voltage curves (I–V)
were constructed by plotting the isochronal instantaneous currents
at 75 ms of the voltage steps versus the test voltages. Each data set
was obtained from at least two batches of oocytes from two
different donor frogs.
Acknowledgements
We thank Franco Gambale for advice and discussion on the oocyte
experiments, S. W. Peters for careful reading of the manuscript and
I. C. Forster for help with experiments on oocytes. This work was
supported by the Alexander von Humboldt Stiftung (PK,
1116390gadodin77), the Deutsche Forschungsgemeinschaft (SM,
ME 1955/2-1; JS-S, SCHO 1238/1-1), the Swiss National Foundation,
the Roche Research Foundation (PK, 2006/101), the EU-Project ‘VaTEP’ (EM) and Global Research program of the Ministry of Science
and Technology of Korea (grant no. 4.0001795.01) (YL, EM). We
thank the Salk Institute Genomic Analysis Laboratory for providing
the sequence-indexed Arabidopsis T-DNA insertion mutants. The
authors wish to thank NASC for providing seeds.
Supplementary Material
The following supplementary material is available for this article
online:
Figure S1. Differences in the C-terminal transmembrane topology of
clade-I and clade-II members from the aluminum-activated malate
transporter (ALMT) family.
Figure S2. Subcellular localization of AtALMT5 by transient expression of AtALMT5-GFP fusion proteins.
Figure S3. Analysis of AtALMT9 expression by AtALMT9
promoter:GUS reporter plants.
This material is available as part of the online article from http://
www.blackwell-synergy.com
Please note: Blackwell Publishing are not responsible for the
content or functionality of any supplementary materials supplied
by the authors. Any queries (other than missing material) should be
directed to the corresponding author for the article.
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ª 2007 The Authors
Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 52, 1169–1180