the plant journal the arabidopsis vacuolar malate channel ... · the arabidopsis vacuolar malate...

The Arabidopsis vacuolar malate channel is a member ofthe ALMT family

Peter Kovermann1,†,‡, Stefan Meyer1,†,*, Stefan Hortensteiner1, Cristiana Picco2, Joachim Scholz-Starke2,

Silvia Ravera3, Youngsook Lee4 and Enrico Martinoia1,4

1Institute for Plant Biology, University of Zurich, CH-8008 Zurich, Switzerland,2Institute of Biophysics, National Research Council, I-16149 Genoa, Italy,3Institute of Physiology, University of Zurich, CH-8057 Zurich, Switzerland, and4POSTECH-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 path-

ways 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 there-

fore be present in the cytosol, chloroplasts, mitochondria,

peroxisomes, glyoxysomes and vacuole. Furthermore,

malate plays a role in pH regulation, is an important

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

ª 2007 The Authors 1169Journal compilation ª 2007 Blackwell Publishing Ltd

The Plant Journal (2007) 52, 1169–1180 doi: 10.1111/j.1365-313X.2007.03367.x

malate have been investigated extensively, which reflects

the interest in this metabolite. Several chloroplast and

mitochondrial carboxylate transporters have been identi-

fied, with most of them exchanging malate with another

carboxylate (Menzlaff and Flugge, 1993; Palmieri et al.,

1993). Vacuolar malate transport has also been investigated

in detail using flux analysis, membrane potential- and pH-

dependent fluorescence probes, and the patch-clamp tech-

nique (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 pro-

ton pumps (Maeshima, 2001; Rea and Sanders, 1987).

Observations of malate currents across the tonoplast indi-

cate 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 Kalanchoe

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 noc-

turnal malate transport (Hafke et al., 2003). Recently, a vac-

uolar malate transporter from Arabidopsis has been

identified at the molecular level (Arabidopsis thaliana

tonoplast dicarboxylate transporter, AttDT, At5g47560; Em-

merlich et al., 2003). This carrier is an ortholog of the renal

Na+/dicarboxylate transporter present in the proximal tub-

ulus 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 wild-

type (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 coef-

ficient 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 alu-

minum-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 epider-

mal 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 demon-

strated 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 expres-

sion 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

1170 Peter Kovermann et al.

ª 2007 The AuthorsJournal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 52, 1169–1180

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

topologies, which are implemented in the ARAMEMNON

database (Schwacke et al., 2003), whereas the consensus

scoring strongly enhances the reliability of topology pre-

dictions (Nilsson et al., 2000). A graphical overlay of an

alignment based on the amino acid sequences of TaALMT1,

AtALMT1, AtALMT5 and AtALMT9 by putative transmem-

brane 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

In order to verify our hypothesis that members of the

AtALMT family are targeted to membranes of different

organelles, we undertook subcellular localization experi-

ments with two members of the second clade: AtALMT5 and

AtAMLT9 (Figure 1a). GFP was fused in frame to the C-ter-

minal end of AtALMT5 and AtALMT9. The transient expres-

sion 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 mi-

stargeted because of the lack of chloroplasts in epidermal

cells, we conducted the same experiments in chloroplast-

containing 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 cyto-

plasm. Taken together these observations clearly demon-

strate that in contrast to AtALMT1, AtALMT9 is a tonoplast

protein. It is tempting to speculate that the difference in the

(a)

(b)

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 trans-

membrane 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 pro-

vided by the ARAMEMNON plant membrane protein database (Schwacke

et al., 2003).

Arabidopsis vacuolar malate channel 1171


subcellular localization between these two proteins relies on

differences in transmembrane topology within the C-termi-

nal 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

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 (Fig-

ure 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 indi-

cated 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 experi-

ments (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.

(a) (b)

(c)

(d)

(e)

(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.

(a) (b)

(c) (d)

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.



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 anal-

ysis 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 electrophy-

siological investigations on CAM and C3 plants. Thus far the

available data indicate that malate currents across the

tonoplast of different plant species share common charac-

teristics, 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 move-

ments 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 volt-

ages. These currents consisted of an instantaneous element

(Iinst) superimposed at negative voltages by a slow time-

dependent element (Istd, Figure 5a). The current–voltage

(I–V) relationships for Iinst (data not shown) and Istd (Fig-

ure 5c, ) showed that both elements were inward-rectify-

ing, 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 pheno-

typically 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

(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.



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

To obtain further proof for the channel activity of AtALMT9

we transiently overexpressed the AtALMT9-GFP fusion con-

struct in leaves of Nicotiana benthamiana under the control

of the CaMV 35S promoter. Vacuoles isolated from these

leaves revealed clearly detectable fluorescence in the tono-

plast, thereby confirming the results of our transient locali-

zation 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 com-

ponent, background currents in WT tobacco vacuoles lacked

a pronounced time dependence (Figure 6a,b). The compari-

son of malate currents from WT vacuoles and AtALMT9-

overexpressing 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 vac-

uolar 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 spec-

ificity, we also measured fumarate- and chloride-mediated

total currents in vacuoles isolated from the AtALMT9-GFP-

expressing 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 stud-

ies on K. daigremontiana have shown that mesophyll

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.



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).

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; Desbrosses-

Fonrouge 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 environ-

ment or missing post-translational modifications in the

heterologous oocyte system (Stuhmer 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) (b)

(c) (d)

Figure 6. Patch-clamp analysis of AtALMT9-GFP

heterologously expressed in Nicotiana benth-

amiana.

(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) AtALMT9-

GFP under asymmetric ionic conditions (100 mM

anions out/10 mM anions in). Scale bar: (c)

15 lm.



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 tur-

gor. To fulfil these functions malate must be accumulated

within the large central vacuole. In this work we have iden-

tified 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 dimin-

ished. 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

Figure 7. Voltage-clamp experiments on Xeno-

pus 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 contain-

ing 0 mM (a) or 10 mM malate (b).

(c) Mean current–voltage relationships con-

structed from recordings like the ones shown in

(a) and (b). Currents derive from AtALMT9 cRNA-

injected oocytes (circles) and control oocytes

(squares) in the presence of 0 mM malate (open

symbols) or 10 mM malate (filled symbols). Val-

ues 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.



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 CLU-

STALW algorithm using default parameters (http://www.ebi.ac.uk/clustalw; Higgins et al., 1994). Hydrophobicity profiles were calcu-lated according to the method described by Kyte and Doolittle(1982). Structural consensus scores and the nomenclature of theAtALMT protein family was adopted from the ARAMEMNON plantmembrane protein database (http://aramemnon.botanik.uni-koeln.de; Schwacke et al., 2003). Dendrograms were constructedusing PHYLODRAW (Graphics Application Laboratory, http://pearl.cs.pusan.ac.kr).

Strains and growth conditions

Escherichia coli (DH5a; Hanahan, 1983) was used for cloning.A. thaliana (Col-0) plants were grown in controlled environmentchambers or on agar medium (8-h light//16-h dark, 22�C, 55% rela-tive humidity). N. benthamiana plants were grown in potting soil(16-h light//8-h dark, 22�C, 55% relative humidity). The transforma-tion of Arabidopsis was performed with Agrobacterium tumefac-iens (GV3101; Holsters et al., 1980).

Tissue-specific expression and subcellular localization

of AtALMT9 in Arabidopsis

A 1785 bp promoter region upstream of AtALMT9 was amplifiedfrom genomic DNA of Arabidopsis (Col-0) by high-fidelity PCR usingthe primers At3g18440g-1847f (5¢-GGGGACAAGTTTGTACAAAAA-AGCAGGCT-GTTTCTCTCTGTGCCTGAGTTTG-3¢) and At3g18440g-30r (5¢-GGGGACCACTTTGTACAAGAAAGCTGGGTGACGGATTCT-CAAAGAGAATTAAGC-3¢). This region was cloned into the GATE-WAY entry vector pDONR207 (Invitrogen, http://www.invitrogen.com) before recombination cloning into pMDC163 (Cur-tis and Grossniklaus, 2003). The vector construct was transformedinto Arabidopsis using the Agrobacterium-mediated floral-dippingmethod (Clough and Bent, 1998). T2 progeny of hygromycin-resis-tant transformants were GUS-stained at various developmentalstages. Embedding of GUS-stained leaves was performed in Tech-novit (Heraeus Kulzer GmbH, http://www.heraeus-kulzer.com). Tolocalize AtALMT9 at the subcellular level, the AtALMT9 cDNA(1797 bp) was amplified from RIKEN clone pda08640 (cDNA cloneRAFL09-66-G16; Sakurai et al., 2005; Seki et al., 1998, 2002; Yamadaet al., 2003) with the primers Mc8forw (5¢-GGTACCATGGCGGCG-AAGCAAGGTTCCTTC-3¢) and Mc8backw (5¢-GGTACCCATCCC-AAAACACCTACGAATCTT-3¢), and then were ligated at the KpnI siteinto the pGFP2 vector (Haseloff and Amos, 1995) to create a con-stitutively expressed AtALMT9-GFP fusion protein. The resultingAtALMT9-GFP construct was transiently expressed in Arabidopsisand onion (Allium cepa) epidermal cells, as well as in guard cells of

V. faba using a Helium Biolistic Particle Delivery system (Bio-RadLaboratories, 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 Ara-bidopsis 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 At3g18440-TDNA-LB (5¢-GTCACCGAATAAAGTGGAAAGC-3¢) binding 110-bpupstream of the start ATG. Lines with homozygous T-DNAinsertions were identified by genomic PCR with a set of AtAL-MT9-specific primers (At3g18440-TDNA-LB and At3g18440-TDNA-RB: 5¢-AGGTCCACCACCACTTCATAAC-3¢). The abundance of theAtALMT9 transcript in homozygous knock-out lines and in WTplants was assayed by isolation of total RNA from whole leavesfollowed by RT-PCR (Amersham kit, http://www.amersham.com)using the AtALMT9-specific primers AtALMT9forw1 (5¢-GGTAC-CATGGCGGCGAAGCAAGGTTCCTTC-3¢) and AtALMT9backw1(5¢-GGTACCCATCCCAAAACACCTACGAATCTT-3¢) and the controlprimers Actin-forw (5¢-GGAACAGTGTGACTCACACCATC-3¢) andActin-backw (5¢-AAGCTGTTCTTTCCCTCTACGC-3¢).

Quantification of malate concentrations

For quantification experiments, a-mannosidase and malate weredetermined in the protoplasts according to the method described byHurth et al. (2005).

Expression of AtALMT9-GFP in N. benthamiana

For transient overexpression of an AtALMT9-GFP construct intobacco leaves, the AtALMT9 cDNA (1797 bp) was cloned under thecontrol of the CaMV 35S promoter using the pART7/pART27 clon-ing/expression system (Gleave, 1992). The Agrobacterium-medi-ated infiltration of N. benthamiana leaves was conducted asdescribed by Yang et al. (2001), with slight modifications. Afteragroinfiltration, tobacco plants were grown in the greenhouse at22�C under 16 h of light. Isolation of vacuoles and patch-clampexperiments were performed 48 h after infiltration.

Isolation of vacuoles

Protoplasts were isolated from leaf mesophyll as previously des-cribed by Song et al. (2003), with minor modifications. After incu-bation at 30�C (45–60 min) protoplasts were liberated by gentleagitation, and a small aliquot (20 ll) of protoplast suspension wastransferred into a patch-clamp chamber filled with 200 ll of a lysissolution 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 standardpatch-clamp technique according to the method described byHamill et al. (1987), in whole-vacuole configuration using an EPC-10 amplifier (HEKA Electronics, http://www.heka.com). Data



acquisition and analysis were conducted with PULSE (HEKAElectronics) 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 aDMZ-Universal puller (Carl Zeiss, Inc., http://www.zeiss.com).The bath chamber was mounted on an inverted fluorescencemicroscope (Eclipse TE2000-U; Nikon Instruments, http://www.nikoninstruments.com). For whole-cell experiments, vacuoles witha diameter of 25–40 lm were selected. The applied voltages referto the cytoplasmic side of the vacuole, whereas the vacuolar sidewas at the ground (Bertl et al., 1992). The vacuolar surface areaswere determined from capacitance currents measured in responseto short (10-ms) voltage steps of 10-mV amplitude (Gillis, 1995).All measurements were made at room temperature (20–22�C).Osmolality of all solutions was calibrated to 440 mosmol kg)1

with mannitol. In all solutions malic acid was buffered with Bis-Tris Propane (BTP), a relatively impermeable cation, to ensure thatthe currents observed were principally attributable to movementsof the malate2) ions (Hafke et al., 2003). The standard bath solu-tion contained 100 mM malic acid, 1 mM CaCl2, 1 mM EDTA and3 mM MgCl2, pH 7.5, adjusted with BTP. The pipette solutioncontained 10 mM malic acid, 1 mM CaCl2 and 3 mM MgCl2, pH 5.5,adjusted with BTP. For patch-clamp experiments on substratespecificity, malic acid was replaced by fumaric acid and hydro-chloric acid. Whole-cell configuration was made by a short bipolarvoltage pulse (� 900 mV, 600 ls each). Current density plots(pA pF)1) of Arabidopsis vacuoles were obtained by plotting theisochronal current amplitude differences between the first and last20 ms of the voltage step, normalized by the tonoplast capaci-tance, against the applied test voltage. The comparison of WT andAtALMT9-overexpressing Nicotiana vacuoles was performed bythe determination of total steady-state current during the last20 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¢-TATCAAATCATATGTTACATCCCAAAA-CAC-3¢), and was subcloned into the pCF3 expression vector (at NotIand NdeI restriction sites) for efficient expression in oocytes, asdescribed previously (Preston et al., 1992; Shitan et al., 2003). Theplasmid was linearized using the unique site AscI, and was used as atemplate for the synthesis of capped cRNA using a Message Ma-chine T7 kit (Ambion, http://www.ambion.com). Stage V–VI defolli-culated oocytes from Xenopus were isolated and maintained asdescribed previously (Virkki et al., 2006). TEVC experiments onoocytes expressing AtALMT9 were conducted according to themethod described by Baumgartner et al. (1999). Oocytes were in-jected with 50 nl cRNA (0.2 lg ll)1) encoding AtALMT9. Controloocytes were injected with 50 nl of double-distilled water. Afterinjection, the oocytes were incubated at 18�C in modified Barth’ssolution 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 500EAmplifier (Molecular Devices Corporation, http://www.molecu-lardevices.com). The voltage clamp was controlled, and data wereacquired using a computer running PCLAMP8 software (Molecular

Devices Corporation), which also controlled the valves for solutionswitching. 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. Allexperiments were performed at room temperature under continu-ous flow of experimental solutions. Solutions in the recordingchamber were changed at a rate of 5 ml min)1. Bath solutions werechosen 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 to220 mosmol kg)1 with mannitol. To test for aluminum-dependentactivation, 0.1 mM Al2(SO4)3 was added to a bath solution contain-ing 0 mM malate and 0.1 mM LaCl3.

Data analysis was performed using the CLAMPFIT software(Molecular Devices Corporation) and GRAPHPAD software (GraphPadSoftware, http://www.graphpad.com). Current–voltage curves (I–V)were constructed by plotting the isochronal instantaneous currentsat 75 ms of the voltage steps versus the test voltages. Each data setwas obtained from at least two batches of oocytes from twodifferent donor frogs.

Acknowledgements

We thank Franco Gambale for advice and discussion on the oocyteexperiments, S. W. Peters for careful reading of the manuscript andI. C. Forster for help with experiments on oocytes. This work wassupported 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 ‘Va-TEP’ (EM) and Global Research program of the Ministry of Scienceand Technology of Korea (grant no. 4.0001795.01) (YL, EM). Wethank the Salk Institute Genomic Analysis Laboratory for providingthe sequence-indexed Arabidopsis T-DNA insertion mutants. Theauthors wish to thank NASC for providing seeds.

Supplementary Material

The following supplementary material is available for this articleonline:Figure S1. Differences in the C-terminal transmembrane topology ofclade-I and clade-II members from the aluminum-activated malatetransporter (ALMT) family.Figure S2. Subcellular localization of AtALMT5 by transient expres-sion of AtALMT5-GFP fusion proteins.Figure S3. Analysis of AtALMT9 expression by AtALMT9promoter:GUS reporter plants.This material is available as part of the online article from http://www.blackwell-synergy.comPlease note: Blackwell Publishing are not responsible for thecontent or functionality of any supplementary materials suppliedby the authors. Any queries (other than missing material) should bedirected to the corresponding author for the article.

References

Baumgartner, W., Islas, L. and Sigworth, F.J. (1999) Two-micro-electrode voltage-clamp of Xenopus oocytes: voltage errorsand compensation for local current flow. Biophys. J. 77, 1980–1991.

Becker, D., Geiger, D., Dunkel, M. et al. (2004) AtTPK4, an Arabid-opsis tandem-pore K+ channel, poised to control the pollenmembrane voltage in a pH- and Ca2+-dependent manner. Proc.Natl Acad. Sci. U.S.A. 101, 15621–15626.



Bertl, A.E., Blumwald, R., Coronado, R. et al. (1992) Electrical mea-surements on endomembranes. Science, 258, 873–874.

Brandizzi, F., Hanton, S., Da Silva, L.L., Boevink, P., Evans, D.,

Oparka, K., Denecke, J. and Hawes, C. (2003) ER quality controlcan lead to retrograde transport from the ER lumen to the cytosoland the nucleoplasm in plants. Plant J. 34, 269–281.

Carter, C., Pan, S., Zouhar, J., Avila, E.L., Girke, T. and Raikhel, N.V.

(2004) The vegetative vacuole proteome of Arabidopsis thalianareveals predicted and unexpected proteins. Plant Cell, 16, 3285–3303.

Cerana, R., Giromini, L. and Colombo, R. (1995) Malate-regulatedchannels permeable to anions in vacuoles of Arabidopsis thali-ana. Aust. J. Plant Physiol. 22, 115–121.

Chen, P.Y. (2005) Structure and function of CLC channels. Annu.Rev. Physiol. 67, 809–839.

Chenna, R., Sugawara, H., Koike, T., Lopez, R., Gibson, T.J., Higgins,

D.G. and Thompson, J.D. (2003) Multiple sequence alignmentwith the Clustal series of programs. Nucleic Acids Res. 31, 3497–3500.

Clough, S. J. and Bent, A.F. (1998) Floral dip: a simplified method forAgrobacterium-mediated transformation of Arabidopsis thaliana.Plant J. 16, 735–743.

Curtis, M. and Grossniklaus, U. (2003) A gateway cloning vector setfor high-throughput functional analysis of genes in planta. PlantPhys. 133, 462–469.

Czempinski, K., Frachisse, J.M., Maurel, C., Barbier-Brygoo, H. and

Mueller-Roeber, B. (2002) Vacuolar membrane localization ofthe Arabidopsis ‘two-pore’ K+ channel KCO1. Plant J. 29, 809–820.

Desbrosses-Fonrouge, A.G., Voigt, K., Schroder, A., Arrivault, S.,

Thomine, S. and Kramer, U. (2005) Arabidopsis thaliana MTP1 is aZn transporter in the vacuolar membrane which mediates Zndetoxification and drives leaf Zn accumulation. FEBS Lett. 539,4165–4174.

Emmerlich, V., Linka, N., Reinhold, T., Hurth, M., Traub, M.,

Martinoia, E. and Neuhaus, H.E. (2003) The plant homolog to thehuman sodium/dicarboxylic cotransporter is the vacuolar malatecarrier. Proc. Natl Acad. Sci. U.S.A. 100, 11122–11126.

Endler, A., Meyer, S., Schelbert, S., Schneider, T., Weschke, W.,

Peters, S.-W., Keller, F., Baginsky, S., Martinoia, E. and Schmidt,

U.G. (2006) Identification of a vacuolar sucrose transporter inbarley and Arabidopsis mesophyll cells by a tonoplast proteomicapproach. Plant Physiol. 141, 196–207.

Epimashko, S., Meckel, T., Fischer-Schliebs, E., Luttge, U. and Thiel,

G. (2004) Two functionally different vacuoles for static anddynamic purposes in one plant mesophyll leaf cell. Plant J. 37,294–300.

Fluckiger, R., De Caroli, M., Piro, G., Dalessandro, G., Neuhaus, J.M.

and Di Sansebastiano, G.P. (2003) Vacuolar system distribution inArabidopsis tissues visualized using GFP fusion proteins. J. Exp.Bot. 54, 1577–1584.

Gao, X.Q., Li, C.G., Wei, P.C., Zhang, X.Y., Chen, J. and Wang, X.C.

(2005) The dynamic changes of tonoplasts in guard cells areimportant for stomatal movement in Vicia faba. Plant Physiol.139, 1207–1216.

Gerhardt, R., Stitt, M.N. and Heldt, H.W. (1987) Subcellular metab-olite levels in spinach leaves: regulation of sucrose synthesisduring diurnal alterations in photosynthetic partitioning. PlantPhysiol. 83, 339–407.

Gillis, K.D. (1995) Techniques for membrane capacitance measure-ments. In Single-Channel Recording. (Neher, E. and Sakmann, B.eds). New York: Plenum Press, pp. 155–199.

Gleave, A.P. (1992) A versatile binary vector system with a T-DNAorganisational structure conducive to efficient integration of

cloned DNA into the plant genome. Plant Mol. Biol. 20, 1203–1207.

Gout, E., Bligny, R., Pascal, N. and Douce, R. (1993) 13C nuclearmagnetic resonance studies of malate and citrate synthesis andcompartmentation in higher plant cells. J. Biol. Chem. 268, 3986–3992.

Hafke, J.B., Hafke, Y., Smith, J.A.C., Luttge, U. and Thiel, G. (2003)Vacuolar malate uptake is mediated by an anion-selective inwardrectifier. Plant J. 35, 116–128.

Hamill, O.P., Marty, A., Neher, E., Sakmann, B. and Sigworth, F.J.

(1987) Improved patch-clamp techniques for high-resolutioncurrent recording from cells and cell-free membrane patches.Pfluegers Arch. 391, 85–100.

Hanahan, D. (1983) Studies on transformation of Escherichia coliwith plasmids. J. Mol. Biol. 166, 557–580.

Haseloff, J. and Amos, B. (1995) GFP in plants. Trends Genet. 11,328–329.

Higgins, D.G., Thompson, J.D. and Gibson, T.J. (1994) CLUSTAL W:improving the sensitivity of progressive multiple sequencealignment through sequence weighting, position-specific gappenalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680.

Hoekenga, O.A., Maron, L.G., Pineros, M.A., Cancado, G.M.A.,

Shaff, J, Kobayashi, Y., Ryan, P.R., Dong, B., Delhaize, E. and

Sasaki, T. (2006) AtALMT1, which encodes a malate transporter,is identified as one of several genes critical for aluminum toler-ance in Arabidopsis. Proc. Natl Acad. Sci. U.S.A. 103, 9738–9743.

Holsters, M., Silva, B., Van Vliet, F., Genetello, C., De Block, M.,

Dhaese, P., Depicker, A., Inze, D., Engler, G. and Villaroel, R.

(1980) The functional organization of the nopaline A. tumefaciensplasmid pTiC58. Plasmid, 3, 212–230.

Hurth, M.A., Suh, S.J., Kretzschmar, T., Geis, T., Bregante, M.,

Gambale, F., Martinoia, E. and Neuhaus, H.E. (2005) Impaired pHhomeostasis in Arabidopsis lacking the vacuolar dicarboxylatetransporter and analysis of carboxylic acid transport across thetonoplast. Plant Physiol. 137, 901–910.

Jaquinod, M., Villiers, F., Kieffer-Jaquinod, S., Hugouvieux, V.,

Bruley, C., Garin, J. and Bourguignon, J. (2007) A proteomicsdissection of Arabidopsis thaliana vacuoles isolated from cellculture. Mol. Cell Proteomics, 6, 394–412.

Kyte, J. and Doolittle, R.F. (1982) A simple method for displaying thehydropathic character of a protein. J. Mol. Biol. 157, 105–132.

Ligaba, A., Katsuhara, M., Ryan, P.R., Shibasaka, M. and

Matsumoto, H. (2006) The BnALMT1 and BnALMT2 genes fromrape encode aluminum-activated malate transporters thatenhance the aluminum resistance of plant cells. Plant Physiol.142, 1294–1303.

Maeshima, M. (2001) Tonoplast transporters: organization andfunction. Ann. Rev. Plant Physiol. 52, 469–497.

Martinoia, E., Flugge, U.I., Kaiser, G., Heber, U. and Heldt, H.W.

(1985) Functional reconstitution of the malate carrier of barleymesophyll vacuoles in liposomes. Biophys. Biochim. Acta, 806,311–319.

Maurel, C., Reizer, J., Schroeder, J.I. and Chrispeels, M.J. (1993) Thevacuolar membrane protein y-TIP creates water specific channelsin Xenopus oocytes. EMBO J. 12, 2241–2247.

Menzlaff, E. and Flugge, U.I. (1993) Purification and functionalreconstitution of the 2-oxoglutarate/malate translocator fromspinach chloroplasts. Biophys. Biochim. Acta, 1147, 13–18.

Neumann, G. and Martinoia, E. (2002) Cluster roots-an undergroundadaptation for survival in extreme environments. Trends PlantSci. 7, 162–167.

Nilsson, J., Persson, B. and von Heijne, G. (2000) Consensus pre-dictions of membrane protein topology. FEBS Lett. 486, 267–269.



Palmieri, F., Bisaccia, F., Capobianco, L., Dolce, V., Fiermonte, G.,

Iacobazzi, V. and Zara, V. (1993) Transmembrane topology,genes, and biogenesis of the mitochondrial phosphate and oxo-glutarate carriers. J. Bioenerg. Biomembr. 25, 493–591.

Pantoja, O. and Smith, J.A.C. (2002) Sensitivity of the plant vacuolarmalate channel to pH, Ca2+ and anion-channel blockers.J. Membr. Biol. 186, 31–42.

Pardo, L.A., Heinemann, S.H., Terlau, H., Ludewig, U., Lorra, C.,

Pongs, O. and Stuehmer, W. (1992) Extracellular K+ specificallymodulates a rat brain K+ channel. Proc. Natl Acad. Sci. U.S.A. 89,2466–2470.

Pei, Z.M., Ward, J.M., Harper, J.F. and Schroeder, J.I. (1996)Magnesium sensitizes slow vacuolar channels to physiologicalcytosolic calcium and inhibits fast vacuolar channels in Fava beanguard cell vacuoles. EMBO J. 15, 6564–6574.

Picco, C., Naso, A., Soliani, P. and Gambale, F. (2007) The zincbinding site of the Shaker channel KDC1 from Daucus carota.Biophys J., doi:10.1529/biophysj.107.114009.

Preston, G.M., Carroll, T.P., Guggino, W.B. and Agre, P. (1992)Appearance of water channels in Xenopus oocytes expressingred cell CHIP28 protein. Science, 256, 385–387.

Ratajczak, R., Richter, J. and Luttge, U. (1994) Adaptation of thetonoplast V-type H+-ATPase of Mesembryanthemum crystalli-num to salt stress, C3-CAM transition and plant age. Plant CellEnviron. 17, 1101–1112.

Rea, P. and Sanders, D. (1987) Tonoplast energization: twoH+-pumps one membrane. Physiol. Plantarum, 71, 131–141.

Ryan, P.R., Delhaize, E. and Jones, D.L. (2001) Function and mech-anism of organic anion exudation from plant roots. Annu. Rev.Plant Physiol. Plant Mol. Biol. 2, 527–560.

Sakurai, T., Satou, M., Akiyama, K., Iida, K., Seki, M., Kuromori, T.,

Ito, T., Konagaya, A., Toyoda, T. and Shinozaki, K. (2005) RARGE:a large-scale database of RIKEN Arabidopsis resources rangingfrom transcriptome to phenome. Nucleic Acids Res. 33, D647–650. (Database issue).

Sasaki, T., Yamamoto, Y., Ezaki, B., Katsuhara, M., Ahn, S.J., Ryan,

P.R., Delhaize, E. and Matsumoto, H. (2004) A wheat geneencoding an aluminum-activated malate transporter. Plant J. 37,645–653.

Scholl, R.L., May, S.T. and Ware, D.H. (2000) Seed and molecularresources for Arabidopsis. Plant Physiol. 124, 1477–1480.

Schwacke, R., Schneider, A., Van Der Graaff, E., Fischer, K., Catoni,

E., Desimone, M., Frommer, W.B., Flugge, U.I. and Kunze, R.

(2003) ARAMEMNON, a novel database for Arabidopsis integralmembrane proteins. Plant Physiol. 131, 16–26.

Seki, M., Carnici, P., Nishiyama, Y., Hayashizaki, Y. and Shinozaki,

K. (1998) High-efficiency cloning of Arabidopsis full-length cDNAby biotinylated CAP trapper. Plant J. 15, 707–720.

Seki, M., Narusaka, M., Ishida, J. et al. (2002) Monitoring theexpression profiles of 7000 Arabidopsis genes under drought,cold and high-salinity stresses using a full-length cDNA micro-array. Plant J. 31, 279–292.

Shitan, N., Bazin, I., Dan, K., Obata, K., Kigawa, K., Ueda, K., Sato, F.,

Forestier, C. and Yasaki, K. (2003) Involvement of CjMDR1, a plantmultidrug-resistance-type ATP-binding cassette protein, in alka-loid transport in Coptis japonica. Proc. Natl Acad. Sci. U.S.A. 100,751–756.

Song, W.Y., Sohn, E.J., Martinoia, E., Lee, Y.J., Young, Y.Y.,

Jasinski, M., Forestier, C., Hwang, I. and Lee, Y. (2003) Engi-neering tolerance and accumulation of lead and cadmium intransgenic plants. Nat. Biotech. 21, 914–919.

Stuhmer, W. and Parekh, A.B. (1995) Electrophysiological record-ings from Xenopus oocytes. In Single Channel Recording, 2ndedn. (Sakmann, B. and Neher, E., eds). New York: Plenum Press,pp. 341–356.

Tokimasa, T. and North, R.A. (1996) Effects of barium, lanthanumand gadolinium on endogenous chloride and potassium currentsin Xenopus oocytes. J. Physiol. 496, 677–686.

Virkki, L.V., Murer, H. and Forster, I.C. (2006) Voltage clamp fluo-rometric measurements on a Type II Na+-coupled Pi cotrans-porter: shedding light on substrate binding order. J. Gen. Phys.127, 539–555.

Wood, M.J. and Korn, S.J. (2000) Two Mechanisms of K+-dependentpotentiation in Kv2.1 potassium channels. Biophys. J. 79, 2535–2546.

Yamada, K., Lim, J., Dale, J.M. et al. (2003) Empirical analysis oftranscriptional activity in the Arabidopsis genome. Science, 302,842–846.

Yang, K.Y., Liu, Y. and Zhang, S. (2001) Activation of amitogen-activated protein kinase pathway is involved indisease resistance in tobacco. Proc. Natl Acad. Sci. U.S.A. 98,741–746.



the plant journal the arabidopsis vacuolar malate channel ... · the arabidopsis vacuolar malate...

Documents