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First published online February 22, 2005; 10.1104/pp.104.058453 Plant Physiology 137:901-910 (2005) © 2005 American Society of Plant Biologists Impaired pH Homeostasis in Arabidopsis Lacking the Vacuolar Dicarboxylate Transporter and Analysis of Carboxylic Acid Transport across the Tonoplast1Technische Universität Kaiserslautern, Pflanzenphysiologie, D–67653 Kaiserslautern, Germany (M.A.H., T.K., T.G., H.E.N.); Universität Zürich, Institut für Pflanzenbiologie, CH–8008 Zurich, Switzerland (S.J.S., E.M.); and Instituto de Biofisica, Consiglio Nazionale delle Ricerche, I–16149 Genova, Italy (M.B., F.G.)
Arabidopsis (Arabidopsis thaliana) mutants lacking the tonoplastic malate transporter AttDT (A. thaliana tonoplast dicarboxylate transporter) and wild-type plants showed no phenotypic differences when grown under standard conditions. To identify putative metabolic changes in AttDT knock-out plants, we provoked a metabolic scenario connected to an increased consumption of dicarboxylates. Acidification of leaf discs stimulated dicarboxylate consumption and led to extremely low levels of dicarboxylates in mutants. To investigate whether reduced dicarboxylate concentrations in mutant leaf cells and, hence, reduced capacity to produce OH– to overcome acidification might affect metabolism, we measured photosynthetic oxygen evolution under conditions where the cytosol is acidified. AttDT::tDNA protoplasts showed a much stronger inhibition of oxygen evolution at low pH values when compared to wild-type protoplasts. Apparently citrate, which is present in higher amounts in knock-out plants, is not able to replace dicarboxylates to overcome acidification. To raise more information on the cellular level, we performed localization studies of carboxylates. Although the total pool of carboxylates in mutant vacuoles was nearly unaltered, these organelles contained a lower proportion of malate and fumarate and a higher proportion of citrate when compared to wild-type vacuoles. These alterations concur with the observation that radioactively labeled malate and citrate are transported into Arabidopsis vacuoles by different carriers. In addition, wild-type vacuoles and corresponding organelles from AttDT::tDNA mutants exhibited similar malate channel activities. In conclusion, these results show that Arabidopsis vacuoles contain at least two transporters and a channel for dicarboxylates and citrate and that the activity of AttDT is critical for regulation of pH homeostasis.
In plant cells, excess malate is stored within the large, central vacuole. NMR studies demonstrated that newly synthesized malate first accumulates in the cytoplasm and, after reaching a certain threshold concentration, is subsequently transported into the vacuole, leading to a rapid exchange between the cytosol and the organelle (Gout et al., 1993  ). This observation, however, does not correspond to the classic storage theory, where a solute is stored within the vacuole until it is utilized by cellular processes, but implies that vacuolar malate accumulation is a complex process comprising synthesis and degradation, energization status of the vacuole, and modulation of transport.
Vacuolar malate transport has already been investigated in detail using flux analysis, membrane potential- and pH-dependent fluorescence probes, and electrophysiological analysis (Martinoia et al., 1985
Electrophysiological measurements showed that a malate channel is present in the vacuolar membrane of both C3 and Crassulacean acid metabolism species (Cerana et al., 1995
Recently, we identified the vacuolar malate transporter from Arabidopsis (Arabidopsis thaliana) on the molecular level (AttDT [A. thaliana tonoplast dicaboxylate transporter]; Emmerlich et al., 2003
Malate is involved in the tricarboxylate and glyoxylate cycle, acts as temporary storage of CO2 in C4 and Crassulacean acid metabolism plants, and is closely linked to phosphoenolpyruvate (PEP) and pyruvate-utilizing pathways through PEP carboxylase and malic enzyme. Furthermore, malate can be utilized as an osmoticum and as a counter ion for potassium or sodium (Martinoia and Rentsch, 1994
In a previous report, we identified the vacuolar malate transporter and the corresponding knock-out mutant plant. Surprisingly, we did not observe a clear phenotypic alteration of the knock-out plant grown under standard conditions. Given the high importance of malate in plant metabolism, we questioned whether mutants deleted in the vacuolar malate transporter AttDT are still able to exhibit regulatory processes depending upon a nonperturbed malate homeostasis. In this context, we focused on regulation of cytosolic pH, as this process is known to interact closely with carboxylate metabolism (Smith and Raven, 1979 From the results presented, we conclude that the tonoplastic malate transporter AttDT is critical for regulation of pH homeostasis under certain conditions, and Arabidopsis vacuoles contain at least two types of carrier proteins and a channel for transport of dicarboxylates and citrate.
Effect of Acidification on the Level of Various Carboxylates in Leaf Discs
To gain more information on the implication of carboxylates for pH homeostasis in Arabidopsis, we quantified the levels of the three major Arabidopsis carboxylic acids (malate, fumarate, and citrate; Chia et al., 2000
At the end of the light period, malate levels were 9.4 µmol/g fresh weight (FW) for wild-type and 4.1 µmol/g FW for mutant plants. After 24 h of incubation at pH 7, wild-type leaf discs contained 5.7 µmol/g FW, whereas corresponding knock-out samples contained only 3.0 µmol/g FW (Fig. 1A). A successive decrease of the external pH value correlated in both plant lines with a decrease of leaf malate levels, finally reaching 2.2 µmol/g FW at pH 3.7 in wild-type and less than 0.5 µmol/g FW in knock-out leaf discs (Fig. 1A). Similarly, fumarate levels in wild-type tissue were 17.5 µmol/g FW at pH 7 and decreased by lowering the external pH to about 9 µmol/g FW at pH 3.7 (Fig. 1B). As seen for malate, fumarate was also lower in knock-out leaf discs incubated at pH 7 (2.7 µmol/g FW), and was reduced to 1.3 µmol/g FW at pH 3.7 (Fig. 1B). The corresponding fumarate levels at the end of the light phase were 20.3 (SE ± 3.2) µmol/g FW in wild-type and 6.7 (SE ± 1.1) µmol/g FW in knock-out leaves. Interestingly, citrate is the sole carboxylic acid measured that is higher in knock-out than in wild-type leaf discs. Wild-type leaf discs incubated at pH 7 contained citrate levels of 9.5 µmol/g FW, whereas in corresponding knock-out tissue, citrate was present at a concentration of 13.5 µmol/g FW (Fig. 1C). The corresponding citrate levels at the end of the light phase were 8.7 (SE ± 0.8) µmol/g FW in wild-type and 13.0 (SE ± 0.3) µmol/g FW in knock-out leaves. It appears remarkable that lowering the pH in the incubation medium did not provoke similar relative citrate changes in both plant lines (Fig. 1C).
Photosynthetic oxygen evolution is a complex process dependent upon a close interaction of a wide number of metabolic processes located in the chloroplasts and cytosol and optimal maintenance of enzyme activity (Walker, 1992
To analyze whether knock-out mesophyll cells differ in their ability to cope with increased cytosolic acidification when compared with wild-type cells, we prepared protoplasts from both genotypes and incubated them in MES-buffered medium harboring, in addition, 0.1 mM benzoic acid. Benzoic acid is a weak acid (pK 4.2) able to pass the plasma membrane in the protonated form. After entering the cell, the proton is released and acidifies the cytosol (Lambert and Statford, 1998 Protoplasts from wild-type plants and knock-out mutants exhibit at pH 7.2 nearly the same rate of oxygen evolution, namely, 67 µmol/mg chl h–1 and 63 µmol/mg chl h–1, respectively (Fig. 2). Both types of protoplasts showed a reduced rate of oxygen evolution in response to acidification of the incubation medium (Fig. 2). However, acidification inhibited photosynthesis of knock-out protoplasts significantly stronger than in protoplasts from wild-type plants (Fig. 2). For example, at pH 4.3, knock-out protoplasts produced only about 26% of oxygen released by wild-type cells, and at pH 4.1, knock-out protoplasts were unable to perform photosynthesis, whereas wild-type protoplasts still exhibited substantial net oxygen production (Fig. 2). These results clearly underscore that knock-out protoplasts exhibited an increased sensitivity to cytosolic acidification.
Subcellular Localization of Carboxylic Acids in Arabidopsis Mesophyll Cells As reported above, total levels of carboxylic acids in wild-type and knock-out leaf tissues differ significantly. In addition, the data presented reveal that acidification correlates with an increased consumption of dicarboxylic acids, but the data do not provide information on their subcellular distribution. Assuming that the vacuole accounts for about 80% of the cell volume, it must be suggested that either the AttDT::tDNA mutant exhibits an extremely high cytosolic malate concentration or that, despite the fact that the mutant is lacking the vacuolar malate transporter AttDT, malate is still partially localized within the vacuole.
To answer this question, we isolated Arabidopsis mesophyll protoplasts from wild-type or AttDT::tDNA plants and compared malate, fumarate, and citrate contents in both intact protoplasts and corresponding vacuoles. For better comparison of these metabolite data with the photosynthetic performance of corresponding plant lines (Fig. 2), we isolated protoplasts in the morning and subsequently enriched the vacuoles by a mild hypo-osmotic treatment and flotation of intact vacuoles on a Percoll gradient (for details, see Emmerlich et al., 2003 As observed for entire leaf discs (Fig. 1), mesophyll protoplasts isolated from wild-type plants and AttDT::tDNA mutants contain more fumarate than malate (Fig. 3A, left). As in entire leaves, citrate content in isolated protoplasts is higher than malate content, and the total malate and fumarate levels in knock-out protoplasts are significantly lower than in corresponding wild-type protoplasts (Fig. 3A, left). Slight differences in the relative ratios between the individual carboxylic acids in leaf discs and protoplasts probably derive from the fact that the former have been incubated for 24 h in the dark, whereas the latter have been purified in the morning from intact leaves. Alternatively, this ratio could also be slightly different between mesophyll and epidermis cells.
Interestingly, vacuoles prepared from homozygous AttDT knock-out plants contained much lower, but still considerable, amounts of malate and fumarate (Fig. 3A, right). Calculation of the percentage of vacuolar carboxylate revealed that wild-type plant vacuoles contained 56.8% ± 8.8% malate and 35.3% ± 4.6% fumarate (Fig. 3B), whereas AttDT::tDNA vacuoles contained 25.1% ± 6.0% of the cellular malate and 26.8% ± 3.2% of the cellular fumarate (Fig. 3B). It should be mentioned that malate and fumarate content measured in vacuoles from AttDT::tDNA mutants cannot be due to contamination with intact protoplasts or other organelles, which was less than 5% (data not shown). In contrast to dicarboxylates, the vacuolar proportion of citrate was slightly increased in vacuoles isolated from AttDT::tDNA mutants, namely, 73.2% ± 8.6% and 80.3% ± 14.8% for wild-type and mutant plants, respectively (Fig. 3A, right, and B). However, considering the higher citrate content in AttDT::tDNA protoplasts (Fig. 3A, left), the vacuolar amount of citrate in mutants is about 1.6-fold that detected in wild-type plants (Fig. 3A, right).
Calculation of the cytosolic carboxylic acid concentrations, based on
In Figure 3, we show altered subcellular levels of the three main carboxylic acids in knock-out mutants. To reveal whether these alterations might correlate with changing respiratory activities in corresponding tissues, we prepared leaf discs 2 h after the end of the light period and analyzed the rates of CO2 release and oxygen consumption by Warburg manometry. As shown, knock-out leaf discs released CO2 at a rate that was 33.6% (±5.8%) faster than observed on wild-type leaf discs (Fig. 4A). In combination with the measured rates of oxygen consumption (data not shown), we not only observed increased respiratory activity in knock-out tissues but also calculated an increased respiratory quotient. Respiratory quotients above 1 indicate partial use of carboxylic acids as substrates for mitochondrial ATP synthesis. Wild-type leaf discs exhibited a respiratory quotient of 1.13 (±0.03), whereas knock-out leaf discs exhibited a respiratory quotient of 1.26 (±0.03; Fig. 4B).
Effect of Acidification on the Expression of the AttDT Gene To gain insight into the putative interaction between pH control and the expression of the vacuolar dicarboxylate transporter gene AttDT, we prepared leaf discs from Arabidopsis at the end of the light period and incubated these in MES-buffered medium at various pH values for 24 h in the dark. Subsequently, total RNA was isolated and northern-blot analysis was conducted using radioactively labeled AttDT cDNA. Incubation of leaf discs at pH 7 did not alter the level of AttDT mRNA when compared with the content of this mRNA species at the end of the light period (Fig. 5). By contrast, acidification of the medium below pH 6 strongly induced accumulation of AttDT mRNA in leaf discs, reaching a maximum at pH 4 (Fig. 5). These results clearly reveal a positive correlation between acidification of the incubation medium and accumulation of AttDT mRNA.
Transport of Carboxylates into Isolated Arabidopsis Vacuoles In conclusion, the data above indicate that vacuoles of AttDT::tDNA plants still possess transport systems for both dicarboxylates and citrate (Fig. 3, A–C) and that mutants are still able to mobilize most of the endogenous dicarboxylic acids upon demand induced by acidification (Fig. 1, A and B). For dicarboxylates, it is still a matter of debate whether the transport activity observed in flux experiments corresponds to the malate channel activity observed by electrophysiological techniques (see introduction).
The high citrate levels in knock-out vacuoles were surprising, since kinetic data raised by use of radioactively labeled carboxylates indicated that both citrate and malate are transported through the same carrier system into the vacuole (Oleski et al., 1987
Uptake experiments using 250 µM radioactively labeled malate or citrate showed, similar to our previous publication (Emmerlich et al., 2003
Robust whole-vacuole macroscopic currents could be detected by the patch-clamp technique on vacuoles isolated from Arabidopsis mesophyll cells in the presence of a buffer containing L-(–)-malic acid and 1,3-bis[tris(hydroxymethyl) methylamino] propane (BTP; Fig. 7A). These currents displayed a large time-dependent component that activated at negative transmembrane potentials (please note that the convention proposed by Bertl et al. [1992] is adopted) and a smaller time-independent component present at both positive and negative voltages. While the time-dependent component was very stable (up to 1 h or more), the time-independent current was maximal immediately after the break-in, displayed almost linear current-voltage relationships, and decreased progressively during the registration. In some cases, this component disappeared in a few minutes.
The labile time-independent component did not display any evident ionic selectivity and could not be studied in detail. Therefore, we concentrated our analysis on the time- and voltage-dependent current. The strongly rectifying properties of this component are summarized in the current density plot (pA/pF) obtained by dividing the mean value of the current in the last 20 ms by the tonoplast capacitance (Fig. 7B, white circles).
To gain more information on the nature of this current, we performed a tail analysis in asymmetric ionic solution (malate2–out/malate2–in = 100/10), which displayed a reversal potential of about +10 mV (Fig. 8, A and B), i.e. close to the theoretical potential of +23 mV expected for malate2– at these ionic activities (Hafke et al., 2003
In sum, these observations correspond to the vacuolar malate channel characteristics described for Arabidopsis cell cultures and other plants and strongly indicate that the currents observed are mediated by malate moving from the cytoplasm into the vacuole. To investigate now whether the malate channel involved corresponds to the malate transporter AttDT, we additionally patched vacuoles isolated from homozygous AttDT::tDNA deletion mutants. Interestingly, for unknown reasons, mutant vacuoles appeared to be less stable in this particular experimental setup when compared to wild-type vacuoles. Nevertheless, mutant vacuoles exhibited very similar currents and current density characteristics of the time-dependent component, as observed on wild-type vacuoles (Fig. 7B, black circles). Furthermore, replacement of malate by fumarate results in increased currents as observed for wild-type vacuoles, and citrate, given at a concentration of 10 mM, completely inhibited malate and fumarate currents, as observed in wild-type plants (data not shown). These results show that AttDT::tDNA plants still possess the vacuolar malate channel and indicate that AttDT does not correspond to the vacuolar malate channel.
Recently, we identified AttDT as a vacuolar malate transporter and showed that Arabidopsis plants lacking in AttDT exhibited only a small residual malate transport activity and significantly reduced total malate levels in leaf tissues (Emmerlich et al., 2003 ). Since malate homeostasis is thought to be a crucial element for maintaining a functional primary metabolism in plants, we were surprised that homozygous AttDT::tDNA plants did not exhibit strong phenotypic alterations when compared to wild types.
It is known from experiments on other species that cellular carboxylate metabolism, especially the malate metabolism, is important for regulation of the cytosolic pH (Smith and Raven, 1979
Interestingly, conversion of malate (and fumarate) to pyruvate not only provides the required OH– ions (Smith and Raven, 1979
In contrast to malate and fumarate, degradation of citrate does not occur in low-pH-challenged leaf discs (Fig. 1C). Although the exact reasons for this discrepancy are difficult to analyze, we speculate that tight control of the metabolic flux within the citric acid cycle, and the involvement of several enzymes for conversion of citrate to malate (Lehninger et al., 1994
As indicated above, the regulatory action of dicarboxylic acids for pH homeostasis ultimately depend upon the presence of these compounds in the cytosol and upon the concentrations of malate and fumarate in the vacuole, which can be used if required in the cytosol. Therefore, the observation that knock-out mesophyll protoplasts are more sensitive to acidification (Fig. 2) concurs with the observation that both the vacuolar as well as the cytosolic malate and fumarate concentrations were lowered in AttDT::tDNA mutants (Fig. 3A, right, and C). Obviously, a disturbed tonoplastic malate exchange as present in AttDT knock-out plants (Fig. 6; Emmerlich et al., 2003
This assumption is further reinforced by the demonstration that radioactively labeled malate fed into mutant leaf discs entered the Krebs cycle much faster than in wild-type tissues (Emmerlich et al., 2003
We showed that AttDT is able to catalyze malate import into isolated vacuoles (Fig. 6). In addition, the observation that malate feeding into leaf discs promotes expression of this AttDT (Emmerlich et al., 2003 Compartmentation analysis revealed that vacuolar concentrations as well as vacuolar proportions of both dicarboxylates were lower in AttDT::tDNA mutants when compared to wild-type plants (Fig. 3A, right, and C). This indicates that both dicarboxylates cross the tonoplast using the same transporter. In addition, this observation may be explained by our finding that a malate transporter (Fig. 6) and a malate channel (Figs. 7 and 8) are present in the tonoplast and exhibit slightly different affinities for both dicarboxylates. However, it must be assumed that the channel does not exhibit sufficient activity to accumulate dicarboxylates at concentrations required for proper metabolic functioning and therefore cannot fully compensate for the absence of AttDT.
The evidence that two dicarboxylate translocating systems exist in the tonoplast may also explain the differences observed concerning the affinity and the relative permeability of the vacuolar transport system when dicarboxylate transport was investigated on different systems (Martinoia and Ratajczak, 1997
In contrast to malate and fumarate, citrate content was increased in mutant leaves (Fig. 3A). This result was unexpected since, from flux analysis using radiolabeled malate and citrate, it has been suggested that both carboxylates cross the tonoplast using the same transporter (Oleski et al., 1987
In total, our observations indicate that AttDT does not correspond to the vacuolar malate channel and explain why substantial amounts of malate and fumarate can be found in AttDT::tDNA vacuoles (Fig. 3B). In addition, we clearly revealed that AttDT is critical for pH homeostasis under the conditions tested. The two dicarboxylate transport systems (transporter and channel) in concert with the so far unknown citrate transporter allow the plant cell to regulate the storage and possibly the release of citrate and malate independently. Furthermore, regulation of energization across the tonoplast could also lead to a preferential import or export of malate or fumarate. This gives the plant an important flexibility to adapt the metabolism to particular situations.
Plant Material and Growth Conditions
Wild-type plants and AttDT::tDNA plants were grown under short-day conditions in a growth chamber as described previously (Emmerlich et al., 2003
Protoplasts from wild-type and knock-out leaves were isolated as in Stitt et al. (1982)
CO2 release and oxygen consumption were measured in a Warburg device (Braun-GmbH, Melsungen, Germany). Leaf discs were prepared 2 h after the end of the light period from intact plants and respiratory activity (CO2 release and oxygen consumption) was quantified for the next 2 h.
RNA isolation and northern-blot analysis were performed using standard procedures (Sambrook et al., 1989
Fifty to 100 mg of leaf material were rapidly frozen in liquid nitrogen. One milliliter of water (about 80°C) was added and the probes were heated for 8 min at 95°C in an Eppendorf thermo-incubator. After centrifugation (5 min, 12,000g), the supernatant was used to determine the carboxylates. Malate, fumarate, and citrate were quantified spectrophotometrically in a combined assay according to Passonneau and Lowry (1993)
For flux analysis and localization experiments, protoplasts and vacuoles were isolated as described (Frangne et al., 2002 For patch-clamp experiments, protoplasts were loaded on a cover slide. After the cells were attached to the bottom, vacuoles were released by incubating the protoplasts in 160 mM BTP, 100 mM malic acid, 3 mM MgCl2 (adjusted to pH 7.4 with BTP), and 8 mM EDTA. The osmolarity was adjusted to 440 mosmol kg–1 with sorbitol.
Solutions for patch clamping were prepared according to Hafke et al. (2003)
Unless otherwise indicated, the pipette solution (inside vacuole) contained 16 mM BTP, 10 mM malate, 3 mM MgCl2, 1 mM CaCl2 (pH 7.5 with BTP) and the bath (cytosolic side) solution contained 160 mM BTP, 100 mM malate, 3 mM MgCl2, 1 mM CaCl2, 1 mM EGTA/2 mM TRIS (pH 7.4 with BTP). Osmolarity of the patch solution was adjusted to 440 mosmol kg–1 with sorbitol. Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession number AJ223445. Received December 16, 2004; returned for revision January 14, 2005; accepted January 17, 2005.
1 This work was supported by the Deutsche Forschungsgemeinschaft, Graduate Research School 845 (to the laboratory of H.E.N.); by the European Union project Novel Ion Channels in Plants (NICIP; EU HPRN–CT–00245; BBW 01.0598 to F.G. and E.M.); and by the Italy-Switzerland Consiglio Nazionale delle Ricerche/Swiss National Foundation bilateral cooperation program. E.M. was financially supported by the Swiss National Foundation. 
2 These authors contributed equally to the paper. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.058453. * Corresponding author; e-mail neuhaus{at}rhrk.uni-kl.de; fax 0631–205–2600.
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-mannosidase. Activities of this enzyme are known to exhibit only minimal fluctuations even under different growth conditions (E. Martinoia, unpublished data) and allow comparison, on one hand, between different plants and, on the other hand, between the vacuole and the intact protoplast (Boller and Kende, 1979
