Intra- and extra-cellular excretion ofcarboxylatesStefan Meyer1, Alexis De Angeli1, Alisdair R. Fernie2 and Enrico Martinoia1

1 Institute of Plant Biology, Zollikerstrasse 107, University of Zurich, CH-8008, Zurich, Switzerland2 Max-Planck-Institut of Molecular Plant Physiology, Am Muhlenberg 1, 14476 Potsdam-Golm, Germany

Review

Carboxylates, such as malate and citrate, are widelyacknowledged to have a central role in plant metab-olism. They are involved in the production of energyand its storage as well as contributing to the cellularosmolyte pool and participating in the regulation ofcellular pH. As we discuss here, recent research hasdemonstrated the functional importance of carboxylateexcretion into the soil, apoplast and vacuole, particularwith respect to the regulation of stomatal and rootfunction.

General considerations of carboxylate functionsPlants produce a range of mono-, di- and tri- carboxylates,most of which, such as citrate, malate or fumarate, areimplicated within several metabolic processes, whereasothers, such as oxalate, represent end products of meta-bolic pathways (reviewed in Refs [1–5]). Notwithstandingtheir independent biosynthesis, citrate, malate, fumarateand oxalate exhibit similar functions when excreted fromthe cytosol into the soil, apoplast or vacuole. This is due tothe commonality of their carboxylic groups, which arenegatively charged at neutral pH and, to a lesser extent,at acidic pH. It thus follows that the function of thesecarboxylates might alter with respect to the pH of thesolution into which they are excreted. That said, despitethe fact that these compounds are mainly exported fromthe cytosol in the charged form, their excretion is oftenaccompanied by the release of protons, a process that leadsto acidification of the soil, apoplast and vacuole. Here, wefocus on the role of carboxylate excretion outside the cellinto either the soil or the apoplast as well as their intra-cellular partitioning into the vacuole.

Physiological functions of carboxylatesRoot exudates constitute diverse compounds derived fromboth primary and secondary metabolism, including carbo-hydrates, carboxylates, phenolics and terpenoids [6–11].During the past few years, these exudates have attractedconsiderable interest, given their impact on a range ofprocesses, including communication between organisms,providing carbon (C) sources for soil–microbial commu-nities and roles in plant nutrition and protection [12–

14]. Although citrate and malate are the major carboxy-lates implicated in these processes, some plants excretelarge quantities of oxalate [15,16]. All these carboxylatescan form complexes with micronutrients and toxic metals.At neutral pH, the stability constants between citrate (pK

Corresponding author: Meyer, S. (stmeyer@botinst.uzh.ch).

40 1360-1385/$ – see front matter � 2009 Elsevier Ltd.

6.4, 4.75, 3.15) and metals, such as Fe3+, Al3+, Zn2+ andCd2+ are higher for citrate than for malate (pK 5.1 and3.45) and oxalate (pK 4.2 and 1.24) [15]. However, at acidicpH, oxalate forms stronger complexes with metals than domalate and citrate owing to its lower pK.

Phosphate nutrition

Approximately 80% of land plants harbour mycorrhizalassociations [17]. In such symbioses, the plant receivesmineral nutrients, in particular inorganic phosphate (Pi)and nitrogen from the fungus, in exchange for the provisionof photosynthate, which is essential for the survival of thebiotrophic fungus. Plants that do not formmycorrhiza havedeveloped alternative strategies to survive on Pi-deprivedsoils. Australian soils are particularly poor in Pi, butmembers of the Proteaceae, one of the largest plantfamilies present on this continent, rarely formmycorrhizalassociations [14,18]. These plants have developed a specialroot structure (proteoid or cluster roots) that is character-ised by short, densely packed lateral roots [13,14]. Onmaturity, cluster roots excrete large amounts of citrate,which serves as an anion exchanger and catalyses therelease of rock-bound Pi. The cluster root structure notonly increases the root surface area for adsorption, but alsofacilitates the production of locally high concentrations ofcarboxylates, thus facilitating efficient extraction of Pifrom the soil.

Other species from diverse families that do not formmycorrhizal, such as Fabaceae, Betulaceae and Casuari-naceae, also produce cluster roots to improve their Pistatus. Some of them also have mycorrhiza [19]. Givenits ease of cultivation, the model plant most commonlyused to study the function of cluster roots is white lupin(Lupinus albus). Interestingly, the root tips of this speciesmainly excrete malate, whereas mature cluster roots,which are responsible for the burst of carboxylate release,mainly excrete citrate. A strong correlation between theactivity of the ATP-dependent citrate lyase (ACL) andmalate excretion has been observed, indicating that thisenzyme is responsible for the switch between malate andcitrate excretion [20]. Using foliar Pi application and asplit-root system, another study investigated whether theroot or shoot Pi content determines cluster-root formationand citrate excretion [21]. The authors concluded that thePi status of the shoot is more important than that of theroots.

Carboxylate excretion is often, but not always, accom-panied by soil acidification through plasma membrane-localized proton pumps. It was recently observed that

All rights reserved. doi:10.1016/j.tplants.2009.10.002 Available online 11 November 2009

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acidification and excretion follow a diurnal rhythm thatstarts a few hours into the light phase [22]. Given thatexcretion of citrate requires a large provision of C, it islikely that sugars must first accumulate in the root tissuebefore carboxylate production and release. Intriguingly,the expression pattern of transporters responsible for theuptake of NO3

-, NH4+ and SO4

2- also follow similar diurnalcycles [23]. Carboxylate excretion is, however, notrestricted to cluster roots, but is a common strategy usedby plants that do not form mycorrhiza to improve their Pistatus; however, in terms of local concentration levels ofexcreted carboxylates, cluster roots substantially outstripother root types [14].

Toxic and heavy metals

Excretion of carboxylates, mainly citrate, might also bebeneficial for iron nutrition and, at least for some plantsproducing cluster roots, such aswhite lupin, the productionof this root type has been demonstrated under low ironavailability. It is generally assumed that Fe3+ complexedwith citrate, as well as with polyphenols, is not taken updirectly but first reduced to Fe2+. However, the relation-ship between Fe3+/Fe2+, soil composition and soil pH iscomplex and is not discussed further here (reviewed in Refs[24–26]).

Although aluminium is both toxic and a prominentelement of the Earth’s crust, it generally does not pose aproblem for plants because, at neutral pH or in alkalinesoils, the solubility of toxic Al3+ is low. However, approxi-mately one third of arable soils are acidic and, in theseregions, aluminium is present partially as Al3+, whichinhibits root growth and development, creating serioustoxicity symptoms and yield losses [27]. One way in whichplants deal with aluminium toxicity is via the excretion ofcarboxylates that form complexes with aluminium andthus reduce the free Al3+ concentration [27]. Owing to theirlower pK, citrate and oxalate aremore effective in complex-ing heavy metals; however, most plants mainly excretecitrate and malate via independent processes in responseto Al3+ [28]. That said, some plants, such as poplar (Popu-lus trichocarpa) predominantly excrete oxalate instead ofmalate when exposed to aluminium or other toxic metals[29], and a recent study comparing different rice (Oryzasativa) cultivars for lead tolerance revealed that this traitcorrelated with the amount of oxalate excreted [30]. Theresponse of roots to Al3+ is rapid and considerable amountsof malate are excreted during this process. This obser-vation suggests that the malate and citrate pools of thevacuole can be readily mobilized when the plant is exposedto aluminium, thus releasing sufficient amounts of carbox-ylate to complex Al3+ ions.

Interaction with microbes

Malate and citrate are suitable C-sources for many micro-organisms. Bacteroids living in symbiosis with plantsreceive their energy supply through the peribacteroidalmembrane largely in the form of malate. Similarly, theirsymbiotic counterparts, free-living nitrogen-fixing bac-teria, preferential use malate and citrate as C-sources.However, some pathogens also depend on citrate withinthe soil; for example,Pectobacterium atrosepticum requires

citrate uptake for full bacterial virulence. Accordingly, theabsence of a functional citrate transporter in P. atrosepti-cum renders potato relatively tolerant to inoculation withthis pathogen [31]. In a further study, it was observed thatinoculation of Arabidopsis (Arabidopsis thaliana) leaveswith the foliar pathogen Pseudomonas syringae (Pst DC3000) induced malate excretion in roots [32]. The studyrevealed that malate induces the chemotactic motility ofthe beneficialBacillus subtilis strain FB17, whichmoves tothe roots of leaf-infected plants and protects the rootfrom infection by P. syringae. The authors further demon-strated that infection of Arabidopsis leaves upregulatedAtALMT1, a plasma membrane-located malate channel.During the past few years, the interaction between plantroots and microbes has attracted much attention, becauseit has been recognized that this interaction has an import-ant role in plant nutrition and development [33]. Carbox-ylate excretion and acidification at the root surface mightthus have a strong general impact on microbial commu-nities [34,35].

Carboxylates in the apoplastTransport of iron, zinc and other heavy metals from theroot to the shoot occurs mainly in the form of metal com-plexes. Nicotianamine complexes several heavy metals,such as iron or zinc [36–38]. In addition, citrate has alsobeen found to complex toxicmetals and is required for theirlong distance transport in plants [39–41]. Reduced citrateconcentrations in the xylem, as observed in knock-outmutants of the multidrug and toxins efflux (MATE) trans-porter FRD3, results in the accumulation of iron in the rootand leaves and in the constitutive expression of genesrequired for iron uptake [40]. Despite the fact that theferric reductase defective3 ( f rd3) knock-out mutants con-tain excess amounts of iron, they are chlorotic, suggestingthat the intra-plant distribution of iron has been disrupted.Indeed, detailed analysis revealed that iron accumulates toa large extent in the vascular cylinder in the roots of thefrd3 mutants, that iron concentration in the xylem wasreduced and that, in leaves, iron was probably precipitatedin the cell wall and thus could not be delivered into thecells. These observations suggest that reasonable amountsof citrate are required in the apoplast and xylem to sustainnormal rates of root–shoot iron delivery.

In the leaf apoplast,malate is foundat concentrations of1–2 mM; however, under elevated CO2 levels result,increases apoplastic malate concentrations are observed[42]. It has been proposed that apoplastic malate releasedin response to elevated [CO2] at least partially mediatesclosure of stomata by shifting the current-voltage curve ofanion channels towards resting membrane potentials,thereby increasing the probability of the anion channelsopening [43–45]. Opening of these channels results in therelease of anions from guard cells and consequently instomatal closure. A recent study has shown that, in theabsence of themalate import proteinAtABCB14, stomatalopening is delayed and that malate-induced stomatalclosure is more efficient [46]. Defective stomatal openingwas also found in plants deficient in the expression offumarase, the enzyme catalysing the hydration of fuma-rate to malate [47].

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Figure 1. Tissue-specific and intracellular localization of the different plant carboxylate transport systems. (a) AtALMT9 and AttDT are localized to the tonoplast of the leaf

mesophyll tissue and mediate the flux of malate from the cytosol (C) into the vacuole (V) [52,66]. Although AttTD has been identified as a malate uptake transporter,

physiological experiments suggest that it also catalyses the efflux of malate from the vacuole to the cytosol [53]. In citrus fruits, an AttDT homolog is responsible for efflux

of citrate from the vacuole to the cytosol [80]. (b) Different transport systems are suggested to be involved in the efflux and influx of malate during stomatal opening and

closure, respectively. Whereas ABCB14 has been functionally described as a plasma membrane-targeted malate importer [46], the presence of several ALMT proteins in

guard cells is reported only at the RNA level (http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi) and the putative role of these proteins in stomatal movement remains to be

elucidated. Although ALMT-type channels could mediate the malate flux into the vacuole during stomatal opening, it can not be excluded that ALMTs could also function in

the efflux of malate into the cytosol during guard cell closure. Efflux of malate through the plasma membrane could also be mediated by either ALMT proteins or the SLAC1

protein, which is likely to represent at least part of the S-type channel; however, the substrate specificity for this channel has still to be established [44,45]. AttDT could also

act as an importer during stomatal opening and as an exporter during guard cell closure, given that published data suggest a similar function in mesophyll tissue [53,80].

However, nothing is known about the regulatory mechanisms that enable this transporter to facilitate import and export. (c) Iron is mobilized by excretion of citrate into the

apoplastic space of the vasculature tissue catalysed by the FRD3 transport protein; subsequently, the iron–citrate complex is translocated via the xylem (Xe) into the shoot

tissue [40]. (d) Under acidic soil conditions, the abundance of phytotoxic Al3+ increases and becomes a major factor limiting plant growth. Diverse carboxylates are excreted

from root tissues into the rhizosphere to bind with the Al3+ cations to form non-toxic complexes, which ameliorate aluminium toxicity. The most prominent carboxylates are

malate and citrate, which are released by the ALMT1 channel and MATE protein, respectively [59,62,74,75]. Abbreviations: E, endodermis; P, pericycle; VP, vascular

parenchyma.

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Figure 2. Putative topology and currents of two selected ALMT channels. (a)

Topology was deduced by comparison of the Arabidopsis ALMTs in the

Aramemnon database (http://aramemnon.botanik.uni-koeln.de/) and the topology

presented in Ref. [61]. We have inverted the topology proposed in Ref. [61]

because a recent report reveals activation of TaALMT1 via phosphorylation of

serine residue 384 (red dot; [81]). Given that no apoplastic protein kinase has been

identified to date, it is proposed that the amino- and carboxyl-termini of the protein

are located in the cytosol. (b) Representative currents of TaALMT1 and (c)

AtALMT9 shown for plasma membrane (TaALMT1)- and vacuolar membrane

(AtALMT9)-located ALMTs. Whereas TaALMT1 mediates instantaneously activated

currents, vacuolar malate currents of AtALMT9 exhibit an instantaneous and a

time-dependent component. Reproduced with permission from Ref. [82] (b) and

Ref. [66] (c).

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Internal excretion: vacuolar storageIn mature plant cells, the central vacuole occupies 80–90%of the cell volume. Vacuoles contain a large number ofinorganic ions, soluble carbohydrates, organic acids, aminoacids, secondary compounds and modified xenobiotics [48].Although malate is the predominant carboxylate in mostplants, citrate and, in some plants such as A. thaliana,fumarate can also accumulate to high levels [49]. Carbox-ylates stored within the vacuole have an importantrole in osmoregulation and as counter-ions for vacuolarpotassium, sodium and magnesium. This role is mostpronounced in guard cells, where malate is the predomi-nant anion in most plants during stomata opening andclosure [50,51].

Plants exhibiting CAM photosynthesis temporarilystore malate as a C source and, in C4 plants, it is theintermediate transported from themesophyll to the bundlesheaths, given that both of these metabolic pathways canrelease CO2 to be fixed during the C3 photosynthetic step.In C3 plants, malate and citrate is released from thevacuole during the night and transported into the mito-chondria to support respiration at night. Surprisingly,plants impaired in vacuolar malate transport did not dis-play an obvious phenotype [52]. However, a more detailedanalysis uncovered that the respiratory coefficient (molarratio of CO2 production to O2 consumption) was substan-tially altered from 1.13 to 1.26, indicating that malateenters the respiratory chain when it cannot be efficientlytransported into the vacuole [53].

As in roots, carboxylates excreted into the vacuole alsohave an important role in complexing essential yet toxicheavy metals that are stored in this compartment. Com-plexes of carboxylates with heavy metals such as zinc, ironor copper enable the plant to store amounts of essentialheavy metals that might be toxic if present at high con-centrations in the cytosol [54]. However, the exact nature ofthe signal transmitted from the cytosol to the vacuole torelease micronutrients required by the plant remainsunknown. One possibility is that alteration of the vacuolarpH stimulates the dissociation of the heavy metal–carbox-ylate complex. Alternatively, free carboxylates could firstbe released into the cytosol and the consequent shift inequilibrium between complexed and free micronutrientscould be anticipated to facilitate the availability of themetal ions. However, the release of micronutrients seemsunlikely to be highly specific given that the Nramp3 andNramp4 transporters (which are responsible for therelease of iron from the vacuole) are also permeable tohighly toxic cadmium [55].

In contrast to malate and citrate, oxalate always formsCa-oxalate crystals in vacuoles, albeit of different shapesand Ca2+:oxalate ratios [56]. Such crystals enable the plantto store large amounts of Ca2+ within the vacuole and tomaintain simultaneously low levels of free Ca2+, to limitthe concentration gradient between the vacuole and thecytosol. To facilitate crystal formation, transport of Ca2+

and oxalate must be strictly coordinated, a process thatawaits elucidation. The exact source of oxalate is still notwell established: in leaves, oxalate might be derived fromphotorespiration [57], but ascorbate is also a potentialsubstrate for oxalate production [58]. The ascorbate path-

way is probably the major pathway used for oxalate pro-duction in C4 plants and in roots [56].

Transporters involved in carboxylate excretionThree classes of exporters for malate and citrate have beenidentified so far (Figure 1). Malate excretion at the plasmamembrane occurs by ALMT (aluminium-activated malatetransporter) channels (Figure 2). Citrate excretion is cat-alysed by members of the MATE transporters (Figure 3).Two types of channel and/or transporter are implicated in

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Figure 3. MATE transporters involved in excretion of citrate into the soil and xylem. (a) Topology was deduced by comparison of the Arabidopsis MATEs in the Aramemnon

database (http://aramemnon.botanik.uni-koeln.de/). A long cytosolic loop is present between the second and the third a-helix only in MATE proteins reported to transport

citrate (AtFRD3 and AtMATE). This loop has also been found in citrate-transporting MATE proteins of other plant species [28]. (b) HvAACT1, a MATE transporter of barley

excreting citrate into the soil, is predominantly localized to epidermal root cells (scale bar = 100 mm; Mu = cultivar Murasakimochi). (c) The citrate transporter AtFRD3 is

localized to the pericycle and is responsible for keeping iron soluble in the apoplast. (d) An optical cross section of a root with AtFRD3 localized to the pericycle. (e) frd3

mutant plants exhibit a pale phenotype. (f) Absence of FRD3 leads to the precipitation of iron in cell walls whereas, in the presence of FRD3 (g), iron can be translocated to

the shoot (blue corresponds to iron content). Reproduced with permission from Ref. [74] (b) and Refs [40,77] (g).

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the internal excretion of malate into the vacuole: theALMT channels and the tDT transporter (Figure 1).

ALMTs

As mentioned above, most plants excrete carboxylates intothe surrounding soil in response to Al3+ to form non-toxiccomplexes. TaALMT1 was the first malate channel ident-ified in bread wheat (Triticum aestivum L.) [59]. It isconstitutively expressed in root apices, but further acti-vated by apoplastic aluminium, thus conferring resistanceto wheat cultivars. Expression of TaALMT1 in heter-ologous systems such as Xenopus oocytes, rice, tobacco(Nicotiana tabacum) and barley (Hordeum vulgare)mediated an Al3+-activated efflux of malate and increasedAl3+ resistance [59,60]. TaALMT1 encodes a hydrophobicprotein containing six to seven transmembrane spanningdomains [61]. It was suggested that both the amino andcarboxyl termini are located on the extracellular side of theplasma membrane. Homologous proteins have been ident-ified in other plant species (e.g. AtALMT1, [62]; BnALMT1/BnALMT2, [63]; ZmALMT1, [64]; and ScALMT1, [65])and several studies have demonstrated that theseproteins perform similar functions as TaALMT1. However,

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functional analysis of ZmALMT1 in Xenopus oocytessuggests that this transporter is involved in general anionmineral nutrition and homeostasis processes (i.e. Cl-)rather than mediating the specific release of carboxylatesin response to Al3+ [64].

InArabidopsis theAtALMTprotein family consists of 14members and can be subdivided into three clades.AtALMT1 belongs to the same clade as TaALMT1 (44%similarity) and also localizes to the plasma membrane ofthe root, where it mediates an Al3+-activated malate extru-sion into the rhizosphere [62]. Al3+ is required not only toactivate AtALMT1, but also to induce expression of thisprotein, a mechanism also described for BnALMT1 andBnALMT2 [63]. By contrast, AtALMT9, a member of thesecond clade, is targeted to the tonoplast and is highlyexpressed in the mesophyll tissue of leaves [66]. Deletionmutants of AtALMT9 exhibited a reduced current densityin vacuoles isolated from mesophyll protoplasts, whereastobacco cells overexpressing AtALMT9 exhibited stronglyenhanced malate currents. The high expression ofAtALMT9 in the mesophyll tissue indicates a function inmalate homeostasis rather than in Al3+ tolerance; how-ever, Al3+ activation could be demonstrated in Xenopus

Figure 4. Topology and amino acids possibly determining the sodium dependency

of dicarboxylate–tricarboxylate carrier (DTC) transporters. (a) Hydrophobicity plot

analysis (http://www.expasy.ch/cgi-bin/protscale.pl) predicts that DTC transporters

form 12 transmembrane domains with the amino- and carboxyl-termini facing the

cytosol. Contrary to the animal dicarboxylate (DC) transporters, the plant AttDT has

been shown to transport DCs in a Na+-independent manner [52]. (b) Region of the

DTC proteins involved in Na+ binding (numbering of amino acids corresponds to

AttDT). The T509 of the human hNaDC1 as well as the S512 of the RbNaDC1 of

rabbit have been shown to be involved in Na+ binding [83]. In an equivalent

position, the plant DTC transporters contain an alanine as opposed to the polar

residue of their animal counterparts. It is possible that this difference in amino acid

sequence is involved in determining the dependency on Na+.

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oocytes. In addition to AtALMT9, othermembers of clade IIalso localize to the tonoplast (S. Meyer, unpublished data).It thus seems reasonable to speculate that the intracellularlocalization of the ALMT proteins is largely uniformwithina clade and depends on slight differences in sequence andtopology between members of the different clades.

Interestingly other members of the AtALMT proteinfamily might fulfil additional as yet unknown functionsgiven that data from microarray experiments indicate theaccumulation of their mRNA levels in many other planttissues (http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi). Onesuch example is the guard cell (AtALMT6, At2g17470;AtALMT12, At4g17970; and AtALMT5, At1g68600),wherein malate largely functions as a charge-balancinganion for potassium [67]. In general, anion channels func-tion as central regulators of stomatal movements, becauseopening and closure is driven by uptake or release ofosmotically active anions and organic metabolites [68].In respect to malate, anion currents have been reportedfor the stomatal plasmamembrane and the vacuolar mem-brane, but despite the important role of malate in guardcell regulation, little is known about molecular identity ofmalate transport systems in guard cells. In addition, nomalate channels have been identified for this cell type atthe genetic level. It is conceivable, therefore, that membersof the AtALMT family could be involved in the regulation ofstomatal movement either as vacuolar or as plasma mem-brane localized anion channels (Figure 1b).

MATE transporters

Multidrug and toxic compound extrusion (MATE) proteinswere first described in bacteria as transporters that canexport several antimicrobial agents and therefore confermultidrug resistance [69]. They have subsequently beenfound in all eukaryotes. In bacteria, export of drugs byMATE transporters is coupled either to the antiport of Na+

or H+; by contrast, in eukaryotes, only H+-coupled antiportactivity has been described so far. MATE transporters inanimals and bacteria are also part of the detoxificationpathway. In bacteria, 9–13 genes encoding MATE trans-porters are present in the genome, whereas there are fewermammalian MATE transporters, with two in humans andmice, and three in dogs [70]. In plants,MATE transporters,similar to ABC transporters, are overrepresented. This isprobably due to the large number of diverse secondarycompounds, which can be regarded as toxic compoundsproduced by the plant cell and which must be detoxified, byextrusion into either the apoplast or the vacuole.

In Arabidopsis, the MATE transporter family contains56 members with locations in the plasma membrane, thevacuolar membrane and the chloroplast envelope. Thetransporters are involved in functions as diverse as detox-ification and lateral root formation, salicylic-dependentsignaling and transport of secondary metabolites, suchas catechin glucosides, anthocyanins and nicotine [71–

73]. Two MATE transporters excrete citrate out of thecytosol. AtMATE/AtDTX42 (At1g51340), as its closest sor-ghum homolog (SbMATE), confers aluminium tolerance byexcretion of citrate into the soil in response to the exposureto Al3+ [28,74,75]. By contrast, its closest intraspecifichomolog AtFRD3 is implicated in the excretion of citrate

into the xylem, and is required for long-distance irontransport and uptake into the leaf cells [40,76,77]. Thefunction of most plant MATE proteins has yet to be estab-lished; however, it is surprising that a primary metabolitesuch as citrate is transported by this class of proteins.

The tonoplast dicarboxylate transporter

The vacuolar malate transporter (Arabidopsis tonoplastdicarboxylate transporter, AttDT) has been identified byinvestigating the plant homologue of the human sodium-dependent dicarboxylate transporter HsNaDC-1 [52](Figure 4). This human transporter is a member of thesolute carrier family 13A2 (SLC13A2), which comprisessodium-coupled sulphate and carboxylate transporters[78]. SLC13A2 in turn belongs to the large transporterfamily of the solute carrier family 13. In humans, SLC13A2transporters are localized to the plasma membrane ofmany different organs. The carboxylate transporters ofthis subfamily can be subdivided into three clades(NaDC1–3) that, although they differ in their sensitivityto pH and the affinity to the substrate, are all electrogenicbecause the transport of one, generally divalent anion iscoupled to that of three Na+ [78].

Arabidopsis contains only one homologue of the NaDCfamily, which exhibits 65.6% similarity and 38.4% identitywith HsDC1, whereas two members of this family havebeen annotated in rice andBrassica oleracea (Aramemnon;http://aramemnon.botanik.uni-koeln.de/index.html). Con-trary to the animal NaDcs, AttDT is localized to thevacuolar membrane and its transport activity is not

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sodium dependent. Accumulation of malate is energized bythe electrochemical gradient and, in the case of vacuolarmalate transport, it is thought that the driving force forvacuolar accumulation is the difference in the electricalmembrane potential between the cytosol and the vacuole.Despite the fact that biochemical analysis showed com-petitive inhibition of malate by citrate and vice versa,deletion mutants of AttDT were not impaired in citratetransport [79].

A homologue of AttDT, CsCit1, has been identified incitrus juice sac cells [80]. The highest expression of CsCit1coincided with the decrease in vacuolar citrate concen-tration and increase in pH. By heterologous expressionin yeast, the authors provided a convincing result thatimplicated CsCit1 in the H+-dependent efflux of citratefrom the vacuole. This result is in line with physiologicalobservations that AttDT can act also as a malate exporter[53]. It will therefore be interesting to determine howAttDT is regulated to act as both importer and exporter.

Conclusions and outlookThe central importance of carboxylates in plant metab-olism and physiological processes has long been recog-nized. However, the post-genome era has led to rapidadvances, with several transporters being identified thatmediate the excretion of carboxylates into the soil, apoplastand vacuole. The relatively easy acquisition of knock-outmutants and transgenics in Arabidopsis has acceleratedunderstanding of the precise function and importance ofseveral of these transport proteins, with studies onaluminium tolerance, iron and Pi nutrition being ofparticular note. Despite these crucial advances, severalquestions remain. These include: (i) what is the nature ofvacuolar citrate import? (ii) What are the mechanisms ofoxalate transport across the plasma and tonoplast mem-branes? (iii) How are the transporter activities physiologi-cally regulated? (iv) What is the cue that initiates rootexudation? And (v) which channels are responsible forobserved increases in apoplastic malate? Once the entirecomplement of carboxylate transporters have been ident-ified, the challenge remains to understand how they areregulated in concert and how they are hierarchically con-trolled to accommodate the many responses that plants, assessile organisms, require to adjust to environmental cir-cumstances.

AcknowledgementsThe work of the Zurich laboratory related to this review was supported bygrants of the Swiss National Foundation and the EU project VaTEP. Wethank Maik Hadorn for help with the figures.

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