This Article Abstract Full Text (PDF) All Versions of this Article: 141/1/196    most recent pp.106.079533v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (30) Citing Articles via Google Scholar Google Scholar Articles by Endler, A. Articles by Schmidt, U. G. Search for Related Content PubMed PubMed Citation Articles by Endler, A. Articles by Schmidt, U. G. Agricola Articles by Endler, A. Articles by Schmidt, U. G.

SYSTEMS BIOLOGY, MOLECULAR BIOLOGY, AND GENE REGULATION

Identification of a Vacuolar Sucrose Transporter in Barley and Arabidopsis Mesophyll Cells by a Tonoplast Proteomic Approach¹

University of Zurich, Institute of Plant Biology, CH–8008 Zurich, Switzerland (A.E., S.M., S.S., T.S., S.W.P., F.K., E.M., U.G.S.); Institute of Plant Genetics and Crop Plant Research, D–06466 Gatersleben, Germany (W.W.); and Swiss Federal Institute of Technology, Institute of Plant Science and Functional Genomics Center Zurich, CH–8092 Zurich, Switzerland (S.B.)

	ABSTRACT

TOP ABSTRACT RESULTS AND DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 
The vacuole is the main cellular storage pool, where sucrose (Suc) accumulates to high concentrations. While a limited number of vacuolar membrane proteins, such as V-type H⁺-ATPases and H⁺-pyrophosphatases, are well characterized, the majority of vacuolar transporters are still unidentified, among them the transporter(s) responsible for vacuolar Suc uptake and release. In search of novel tonoplast transporters, we used a proteomic approach, analyzing the tonoplast fraction of highly purified mesophyll vacuoles of the crop plant barley (Hordeum vulgare). We identified 101 proteins, including 88 vacuolar and putative vacuolar proteins. The Suc transporter (SUT) HvSUT2 was discovered among the 40 vacuolar proteins, which were previously not reported in Arabidopsis (Arabidopsis thaliana) vacuolar proteomic studies. To confirm the tonoplast localization of this Suc transporter, we constructed and expressed green fluorescent protein (GFP) fusion proteins with HvSUT2 and its closest Arabidopsis homolog, AtSUT4. Transient expression of HvSUT2-GFP and AtSUT4-GFP in Arabidopsis leaves and onion (Allium cepa) epidermal cells resulted in green fluorescence at the tonoplast, indicating that these Suc transporters are indeed located at the vacuolar membrane. Using a microcapillary, we selected mesophyll protoplasts from a leaf protoplast preparation and demonstrated unequivocally that, in contrast to the companion cell-specific AtSUC2, HvSUT2 and AtSUT4 are expressed in mesophyll protoplasts, suggesting that HvSUT2 and AtSUT4 are involved in transport and vacuolar storage of photosynthetically derived Suc.

Increasing evidence shows that impaired vacuolar deposition or retrieval affects plant metabolism. Catala et al. (2003) showed that the vacuolar calcium-proton exchanger CAX1 is induced during cold treatment and is involved in the regulation of genes responsible for cold tolerance. In the case of the vacuolar AtNRAMP3, Thomine et al. (2003) demonstrated that knockout plants exhibited an increased cadmium tolerance. It was postulated that AtNRAMP3 is an iron exporter and that under iron deficiency the plant is no longer able to mobilize vacuolar iron. The vacuolar malate carrier has recently been identified (Emmerlich et al., 2003). Cellular and vacuolar malate and fumarate contents were strongly reduced in deletion mutants of this transporter, despite the fact that a functional malate channel was still present in the tonoplast (Hurth et al., 2005). Knockout plants also exhibited an increased respiratory coefficient, indicating a shift of the substrates respired from carbohydrates in wild-type plants to mainly organic acids in the mutant. Recently, the Ca²⁺-dependent Ca²⁺-release channel TCP1 has been identified and functionally characterized. Deletion mutants lack functional slow channel activity, and are defective in abscisic acid-induced repression of germination and in the response of stomata to extracellular calcium (Peiter et al., 2005). These examples show that a detailed knowledge of vacuolar transporter function and regulation is required to better understand mechanisms regulating cytosolic homeostasis. However, despite some progress in identifying novel vacuolar transporters, our present knowledge of the intrinsic transporters in the tonoplast remains vague. A case in point is that to date one of the most important vacuolar transporters, the Suc transporter (SUT), has not been identified. In leaves, a large proportion of the Suc produced by photosynthesis is transported into the vacuole during the light period for storage. At night, Suc is released and loaded into the phloem for transport to the sink tissues (Kaiser and Heber, 1984; Martinoia et al., 1987). Inhibition of vacuolar Suc transport will increase cytoplasmic Suc concentrations and inhibit photosynthesis. Furthermore, many plants, such as barley (Hordeum vulgare) and wheat (Triticum aestivum), synthesize fructans in leaves (Pollock and Cairns, 1991). Fructan synthesis occurs within the vacuole and requires Suc (Wagner et al., 1983). Fructan accumulation allows such plants to store carbohydrates within the large central vacuole, additionally limiting the increase of osmolarity. Furthermore, fructans may play an important role as cold, drought, and salt stress protectants (Ritsema and Smeekens, 2003).

One possibility to identify new vacuolar transporters is by a proteomic approach. Thus far, vacuolar proteomic approaches have been described using Arabidopsis (Arabidopsis thaliana) vacuoles (Carter et al., 2004; Sazuka et al., 2004; Shimaoka et al., 2004; Szponarski et al., 2004). Currently, no data are available for monocotyledonous cereal plants.

In this work, we present data on a vacuolar membrane proteomic approach using highly purified vacuolar membranes isolated from mesophyll cells of the crop plant barley. We identified 101 unique proteins; 45.5% of the detected barley vacuolar proteins have not been reported in the Arabidopsis vacuolar proteome. Among them is the Suc transporter HvSUT2, previously shown to catalyze Suc uptake when heterologously expressed in yeast (Saccharomyces cerevisiae; Weschke et al., 2000). HvSUT2, as well as its Arabidopsis homolog AtSUT4, were localized at the tonoplast as the first vacuolar Suc transporter using green fluorescent protein (GFP) fusion constructs.

	RESULTS AND DISCUSSION

TOP ABSTRACT RESULTS AND DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 
To understand the function of the vacuole and the interactions between the vacuole and the metabolic pathways occurring in the cytoplasm, it is important to identify vacuolar transporters that are involved in the export of solutes from the cytosol and the reimport in case of metabolically relevant compounds, such as sugars, organic acids, nitrate, or phosphate. To date, a range of transporters (Maeshima, 2001; Martinoia et al., 2002) have been identified, but, as can be deduced from vacuolar localization and transport studies, a large number remain uncharacterized. To identify novel tonoplast proteins, we isolated highly purified vacuolar membranes from barley mesophyll cells. Barley is an important crop plant closely related to wheat and rice (Oryza sativa). It contains only one type of mesophyll cells, whereas Arabidopsis has two types of mesophyll cells, spongy and palisade cells.

Purification of Barley Vacuoles

We could overcome the limiting factor of tonoplast proteomic projects, the requirement of highly purified vacuolar membranes in sufficient amounts, by improving a previously described method (Rentsch and Martinoia, 1991) for the isolation of vacuoles from barley leaves. Intact vacuoles were stained with neutral red and the purity was visually checked by bright-field microscopy. No obvious chloroplast or protoplast contaminations were observed (Fig. 1A ). As described by Kaiser et al. (1986), the purity of the vacuole preparations was analyzed by the measurement of marker enzymes found in cytoplasm, mitochondria, endoplasmic reticulum (ER), and chloroplast (Table I ). Contamination with these organelles was less than 1%, except for the ER (8%).

View larger version (47K): [in this window] [in a new window]   Figure 1. Bright-field microscopy of purified vacuoles from barley mesophyll protoplasts (A). Western-blot analysis (B) with compartment-specific antibodies: TIP (dimer 45 kD), PIP (dimer 50 kD), ATP- (55 kD), BiP (73 kD), and AOX (36 kD). Aliquots (5 µg) of vacuolar membrane proteins (V) and total membrane proteins of protoplasts (P) were loaded on each lane, respectively. vac, Vacuole; PM, plasma membrane; chl, chloroplast; ER, endoplasmic reticulum; mito, mitochondria.

Analysis of Tonoplast Proteins by Liquid Chromatography-Tandem Mass Spectrometry

An untreated vacuolar membrane fraction as well as vacuolar membrane fractions washed with either 0.5 M NaOH or 0.3 M KI were analyzed by liquid chromatography-tandem mass spectrometry (MS/MS). Alkaline (NaOH) and saline (KI) treatment reduced the proportion of bound peripheral proteins from 53% in the untreated fraction to 38% in the KI-treated fraction and 34% in the NaOH-treated fraction. Cumulatively, we identified 101 proteins, 49 proteins with at least one transmembrane domain and 52 proteins with no transmembrane domain (Tables II–IV ).

We identified 11 of 12 subunits of the V-type H⁺-ATPase (Sze et al., 2002). In comparison with published Arabidopsis vacuolar proteomic projects, only Carter et al. (2004) and Shimaoka et al. (2004) obtained such a complete set of subunits for the V-type H⁺-ATPase. Despite this, our proteomic analysis resulted in a limited number of known vacuolar proteins. This discrepancy may be explained by the absence of some known vacuolar transporters in barley mesophyll cells, or barley proteins are not similar enough to permit an unequivocal attribution of peptides during the database searches.

Soluble Proteins and Nonvacuolar Proteins
In our investigation of the tonoplast, we found 52 proteins with no transmembrane domain. Most of these putative membrane-associated proteins are involved in stress response or in membrane fusion and remodeling (Table II).

We identified a few known nonvacuolar proteins, including four membrane proteins (Table III) and nine soluble proteins (Table IV). Soluble proteins can associate with membranes, which might explain their detection in the tonoplast preparation. The view that these proteins were peripherally associated with the membrane is supported by the fact that the proportion of soluble known nonvacuolar proteins was the highest in the nontreated vacuolar membrane fraction (8.8%). However, since the vacuole is a lytic organelle, a proportion of these apparent contaminants may be a result of the ongoing degradation processes. Interestingly, only one plasma membrane protein, aquaporin 1, was detected, demonstrating the high purity of our preparations. Most of the known nonvacuolar proteins (61.5%) were previously reported in one of the Arabidopsis vacuolar proteomic studies (Tables III and IV).

Novel Vacuolar Membrane Proteins
Besides well-known vacuolar membrane proteins, a large number of annotated membrane proteins with an unknown subcellular localization were identified, including several sugar transporters (gi|7024413, gi|26986186, gi|49388943, gi|51854311, and gi|31433313) and ABC proteins (gi|55773917, gi|34912536, and gi|34915038), an amino acid transporter (gi|47497044), a peptide transporter (gi|15076661), an organic anion transporter (gi|34898286), a putative calcium translocating P-type ATPase (gi|14275750), and a putative chloride channel (gi|34015349) of the CLC family (Nakamura et al., 2006). Moreover, there were also nine proteins with at least one transmembrane helix for which a function has yet not been elucidated (Table II).

The closest Arabidopsis homolog of the identified peptide transporter (gi|15076661) is AtPTR2-B (At2g02040), which transports dipeptides and tripeptides and is constitutively expressed in all plant organs (Song et al., 1996). AtPTR2-B was also found by Carter et al. (2004) and Shimaoka et al. (2004) during their Arabidopsis vacuolar proteome analysis. AtPTR1, another peptide transporter of the gene family, has been localized in the plasma membrane (Dietrich et al., 2004). These results indicate that in a multigene family, different members may not be localized at the same membrane. This has also been demonstrated in studies on the subfamily of the MRP-type ABC transporters (Geisler et al., 2004; Klein et al., 2004).

One of the identified sugar transporters (gi|51854311) is a close homolog of a hexose transporter (U43629) in sugar beet (Beta vulgaris), which has previously been postulated to catalyze facilitated diffusion of Glc across the vacuolar membrane of sugar beet (Chiou and Bush, 1996). Facilitated diffusion of Glc has also been reported to occur in barley vacuoles (Martinoia et al., 1987). Therefore, it is highly likely that this hexose transporter is indeed localized at the vacuolar membrane.

Interestingly, an additional sugar transporter identified in our proteomic approach was HvSUT2. HvSUT2 was previously expressed in yeast and shown to catalyze Suc uptake in intact yeast cells (Weschke et al., 2000). The facilitated diffusion of Suc across the vacuolar membrane of barley has already been demonstrated in earlier studies (Martinoia et al., 1987). Although Suc accumulation is one of the key functions of mesophyll vacuoles, the vacuolar transporters responsible for Suc uptake and release are as yet unidentified.

A Comparison of the Arabidopsis and Barley Tonoplast Proteome
The vacuole possesses specialized functions in different cell types and in different plant species. For example, in Arabidopsis, vacuoles accumulate mainly monosaccharides, whereas in barley, fructans may be synthesized and stored in vacuoles. To identify proteins that are particular for the vacuolar function in barley, we compared the vacuolar proteins found in barley to proteins identified in the four Arabidopsis vacuolar proteomic approaches (Carter et al., 2004; Sazuka et al., 2004; Shimaoka et al., 2004; Szponarski et al., 2004). We searched the 88 identified vacuolar proteins for the closest homologs in the Arabidopsis genome. If a similarity of 30% is used as a cutoff, six proteins, including the putative gag-pol precursor and five hypothetical proteins, have no homolog in Arabidopsis (Table II).

Although 34 to 402 vacuolar proteins were detected in the four Arabidopsis proteomic studies, only 48 (54.5%) identified barley vacuolar proteins have also been identified in at least one of the Arabidopsis studies (Table II; Fig. 2 ). The highest overlap was found with Carter et al. (2004; 48 proteins) and Shimaoka et al. (2004; 29 proteins). However, 40 proteins (45.5%) of 88 detected barley vacuolar proteins were not reported in the Arabidopsis vacuolar proteomic studies, suggesting a higher relative abundance of these proteins in barley leaf mesophyll vacuoles compared to Arabidopsis leaf mesophyll and cell culture vacuoles. Eighteen (43%) of these 40 proteins are membrane proteins, including three ABC transporters (gi|55773917, gi|34912536, and gi|34898286), one amino acid transporter (gi|47497044), one putative CLC-type chloride channel (gi|34015349), six membrane proteins with an unknown function (gi|50252990, gi|50726593, gi|51536186, gi|50948653, gi|50929895, and gi|50399936), and the Suc transporter HvSUT2 (gi|7024413). The detection of HvSUT2 in the vacuolar membrane fraction indicates a high expression of this Suc transporter in barley mesophyll cells. This can be explained by the fact that barley mesophyll cells accumulate large amounts of Suc during the day (Kaiser and Heber, 1984) and that fructan synthesis in barley mesophyll vacuoles requires Suc.

View larger version (23K): [in this window] [in a new window]   Figure 2. Comparison between the barley and Arabidopsis vacuolar membrane proteome. Barley tonoplast proteins were BLAST-searched for homologous proteins that were reported in the Arabidopsis tonoplast proteome. Proteins with a similarity of at least 30% were accepted as significant homologs. The number in brackets indicates the total number of identified proteins in each study and the bold numbers represent the number of overlapping proteins. Forty of the detected proteins were only identified in barley, suggesting a significant function at the vacuolar membrane for monocotyledon cereals.

The small number of nonvacuolar proteins that we detected in comparison to the published proteomic approaches using Arabidopsis vacuoles reveals that the purity of our barley tonoplast fractions is clearly higher. However, since the protein content of the vacuolar membrane constitutes less than 1% of the total cellular protein, small contaminations in the range of 2% to 4% have a strong impact on the detected proteins. It should also be taken into account that the plasma membrane contains about double the amount of protein compared to the vacuolar membrane. Furthermore, in mesophyll cells, the surface area of chloroplastic membranes by far exceeds that of the vacuole. Consequently, a contamination of 2% with chloroplasts would result in about 20% plastid proteins in the vacuolar fraction. GFP localization of the newly identified membrane proteins is therefore a prerequisite to confirm their putative localization obtained with a proteomic approach. This effort has not been undertaken in the vacuolar proteomic reports published so far.

Among the potentially novel vacuolar transporters, we were particularly interested in HvSUT2, since the vacuolar Suc transporter, which plays a central role in plant metabolism, still awaits identification. As aforementioned, HvSUT2 exhibited Suc transport activity over the plasma membrane when expressed in yeast (Weschke et al., 2000). However, heterologous expression in yeast does not necessarily provide information about the intracellular localization of the protein in planta.

The high purity of our vacuole preparation encouraged us to test whether HvSUT2 and its Arabidopsis counterpart, AtSUT4, are vacuolar Suc transporters, despite the fact that the closest homologs of HvSUT2 found in tomato (Lycopersicon esculentum; LeSUT4) and potato (Solanum tuberosum; StSUT4) have been immunolocalized in the plasma membrane of sieve elements (Weise et al., 2000).

To confirm the tonoplast localization of HvSUT2 and AtSUT4, GFP fusion proteins were transiently expressed in Arabidopsis leaves and onion (Allium cepa) epidermal cells. For both transporters, fluorescence was detected at the vacuolar membrane of Arabidopsis and onion epidermal cells (Fig. 3, A–J). As a control, we cloned AtSUC2, a well-known plasma membrane Suc transporter of the companion cells (Stadler and Sauer, 1996), as a GFP fusion protein. Based on fluorescence intensity, expression of AtSUC2-GFP was similar to AtSUT4-GFP and HvSUT2-GFP. However, unlike AtSUT4 and HvSUT2, AtSUC2 localizes to the plasma membrane (Fig. 3, K and L).

View larger version (52K): [in this window] [in a new window]   Figure 3. Subcellular localization of HvSUT2, AtSUT4, and AtSUC2 by transient expression of GFP fusion proteins in onion epidermal cells and Arabidopsis epidermal cells. Cell walls and nuclei of Arabidopsis were stained in red with propidium iodide. Nuclei are indicated by arrowheads. A to E, Tonoplast localization of HvSUT2 in onion epidermal cells (A–C) and Arabidopsis epidermal cells (D and E). Fluorescence of the HvSUT2-GFP fusion protein in onion epidermal cells (A) and transmission picture (C) of the same cell. The tonoplast is located in the internal side of the cytoplasm including the nucleus as shown in the overlay of the detail pictures (B and E). F to J, Localization of the AtSUT4-GFP fusion protein at the tonoplast, transiently expressed in onion epidermal cells (F–H) and Arabidopsis epidermal cells (I and J). As shown in detail pictures (G and J), the green fluorescent tonoplast surrounds the nucleus. K and L, Plasma membrane localization of AtSUC2-GFP in Arabidopsis epidermal cells.

Shimaoka et al. (2004) detected in their Arabidopsis tonoplast proteomic analysis the Suc transporter AtSUC1, which is strongly expressed in Arabidopsis flowers (Stadler et al., 1999). AtSUC1 was also identified in the plasma membrane proteomic approach of Alexandersson et al. (2004). To investigate if AtSUC1 is localized like AtSUT4 at the vacuolar membrane, we expressed AtSUC1-GFP in Arabidopsis leaves and onion epidermal cells. As shown in Figure 4 , AtSUC1 is targeted to the plasma membrane as AtSUC2. This observation stresses on the necessity to confirm the localization of novel candidates obtained in proteomic approaches by GFP-fusion proteins.

View larger version (116K): [in this window] [in a new window]   Figure 4. Subcellular localization of AtSUC1 by transient expression of GFP fusion proteins in onion epidermal and Arabidopsis leaf cells. AtSUC1-GFP localizes at the plasma membrane of onion epidermal cells (A–C) and Arabidopsis epidermal cells (D and E). Nuclei are indicated by arrows in overlay pictures (C and D). Cell walls and nuclei of Arabidopsis were stained in red with propidium iodide.

AtSUT4 promoter--glucuronidase fusion plants exhibited a strong expression in companion cells (Schulze et al., 2003). To investigate whether AtSUT4 is expressed exclusively in companion cells or if transcripts are also present in mesophyll cells, where photosynthetic Suc is stored inside the vacuoles, we performed a reverse transcription (RT)-PCR analysis with isolated leaf mesophyll protoplasts.

Thirty to 40 mesophyll protoplasts of source leaves were discriminated from smaller companion cell protoplasts, chloroplast-free sieve element protoplasts, and large epidermal protoplasts by bright-field microscopy and collected in a microcapillary. RT-PCR was conducted with primers specific for the amplification of HvSUT2 and AtSUT4. AtSUC2 was used as a marker gene for companion cell contaminations. As illustrated in Figure 5 , only AtSUT4 transcripts were detected in the mesophyll protoplast preparation. While in a typical leaf protoplast mixture, both AtSUC2 and AtSUT4 transcripts were present. HvSUT2 transcripts were detected in mesophyll cells (Fig. 5). These results prove that HvSUT2 and AtSUT4 are vacuolar Suc transporters of leaf mesophyll cells.

View larger version (58K): [in this window] [in a new window]   Figure 5. Expression analysis of AtSUT4 and HvSUT2 in mesophyll protoplasts. RT-PCR products of AtSUT4 (180 bp) and HvSUT2 (270 bp) are visible in mesophyll protoplast preparations (MP). AtSUC2 RT-PCR products (190 bp), a marker gene for companion cell contaminations, are only detected in the typical leaf protoplast mixture (LP). Actin RT-PCR products (100 bp) are indicated by an arrow.

	CONCLUSION

TOP ABSTRACT RESULTS AND DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS AND DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 

Plant Material

Barley (Hordeum vulgare) var. Baraka was grown in soil in a controlled environment chamber (16 h light/8 h dark, 300 µE m^–2 s^–1, 22°C, 60% relative humidity), and Arabidopsis (Arabidopsis thaliana) ecotype Columbia was cultivated in soil in a controlled environment chamber (8 h light/16 h dark, 150 µE m^–2 s^–1, 18°C, 60% relative humidity).

Tonoplast Isolation

Barley mesophyll vacuoles were isolated from 8-d-old plants according to Rentsch and Martinoia (1991), omitting bovine serum albumin in the isolation medium. Tonoplast vesicles were isolated by sonication of vacuoles and subsequent ultracentrifugation for 1 h at 100,000g. The supernatant was removed and the tonoplast fraction was resuspended in 20 mM HEPES-KOH, pH 7.2, at a concentration of approximately 200 µg protein/mL. The vacuolar membranes were either analyzed directly or treated by washing with 0.3 M KI or 0.5 M NaOH. Proteins were quantified according to Bradford (1976) using the Bio-Rad protein assay (Reinach).

Tonoplast Proteome Analysis and Interpretation of MS Data

Tonoplast proteins were separated using SDS-PAGE (Laemmli, 1970) with 10% acrylamide gels. The SDS gels were cut into 35 sections, and gel slices were immediately subjected to in-gel tryptic digestion (Shevchenko et al., 1996). Tryptic peptides were further fractionated by reverse-phase chromatography coupled online to an LCQ Deca XP ion trap mass spectrometer (Thermo Finnigan). The peptides were analyzed by MS full scan and MS/MS scans of the three most intense parent ions.

MS/MS data sets were interpreted according to the standards of Carr et al. (2004). The SEQUEST software (Thermo Finnigan) was used to search the National Center for Biotechnology Information (NCBI) protein database of the Liliopsida (GI4447) including 139,384 proteins. dta files were created by the SEQUEST software for every MS/MS scan with a total ion count of at least 5 x 10⁴, minimal peak count of 35, and a precursor ion mass in the range of 300 to 2,000 mass-to-charge ratio. Data were searched against the database restricted to tryptic peptides without modifications (except for carboxyamidomethylated Cys 57.0513 and oxidized Met 15.9994), allowing a parent mass error tolerance of 2 D and daughter ion error tolerance of 0.8 D. All SEQUEST data were analyzed by Peptide and Protein-Prophet (Nesvizhskii et al., 2003). Protein-Prophet is based on a statistical model that allows one to assess the reliability of protein identifications on the basis of MS/MS data. The Protein-Prophet score gives a probability estimate for the protein identification. We accepted scores above 0.9; at this cutoff, the rate of false positive protein identifications is less than 10% (Table II). Mostly, we identified the same protein from different biochemical fractions, further compounding the reliability of the identification (Table II).

For the identification of Arabidopsis homologs, we used the BLAST search of the Munich Information Center for Protein Sequences (MIPS) database (http://mips.gsf.de/proj/thal/db/search/search_frame.html). In Tables II, III, and IV, the closest Arabidopsis homologs are listed as well as the second and third homologs if the similarity differed not more than 10% from the closest homolog.

Western Blotting

Western blotting was carried out using antibodies for -TIP (Conceicao et al., 1997), ATP-, luminal BiP (member of the heat shock family), AOX (Elthon et al., 1989), and PIP (Frangne et al., 2001). Secondary antibodies (anti-rabbit or anti-chicken, coupled to alkaline phosphatase [AP] or horse radish peroxidase [HRP]; Promega) were diluted 1:3,000 (AP) or 1:25,000 (HRP) in TBST (0.2 M Tris-HCl, pH 7.5, 0.5 M NaCl, 0.05% Tween 20). AP detection mixture was prepared by adding 6.6 µL of nitroblue tetrazolium (Promega) and 3.3 µL of 5-bromo-4-chloro-3-indolyl phosphate (Promega) to 1 mL of AP buffer (0.1 m Tris-HCl, pH 9.5, 0.5 m MgCl₂). The activity of HRP was detected using a Chemiluminescence Blotting Substrate kit (SuperSignal Chemiluminescent Working Solution; Pierce) according to manufacturer's instructions.

Localization Study

To localize HvSUT2 (gi|7024412), AtSUC1 (At1g71880), and AtSUC2 (At1g22710), the respective cDNAs were cloned in frame to the N terminus of GFP into the vector pGFP2 (Haseloff and Amos, 1995). The AtSUT4-GFP fusion construct was cloned into the expression vector pART7 (Gleave, 1992). GFP fusion constructs were transiently expressed in onion (Allium cepa) and Arabidopsis epidermal cells using a Helium Biolistic Particle Delivery system (Bio-Rad). Cell walls and nuclei of Arabidopsis epidermal cells were stained with 1 mM propidium iodide. Fluorescent cells were imaged by confocal microscopy (Leica).

Collection of Mesophyll Protoplasts

The epidermis of Arabidopsis rosette leaves was rubbed off with glass paper P 80. Leaves were transferred into digestion buffer, pH 5.6 (0.5 M sorbitol; 1 mM CaCl₂; 10 mM MES) containing 0.75% cellulase YC (w/v) and 0.03% pectolyase Y23 (w/v) (both from Kyowa Chemical Products) and incubated for 1.5 h at 30°C. After digestion, protoplasts were recovered by centrifugation (2,000g for 5 min) and purified by a Percoll gradient. The protoplasts were mixed 3:1 (v/v) with 100% Percoll, pH 6 (0.5 M sorbitol; 1 mM CaCl₂; 20 mM MES), overlaid with 25% Percoll, pH 6, and betaine buffer, pH 6 (0.4 M betaine; 30 mM KCl; 20 mM HEPES) followed by centrifugation at 1,500g for 5 min. Protoplasts from the interface between 25% Percoll, pH 6, and betaine buffer were recovered. The leaf protoplast suspension was visualized by bright-field microscopy to distinguish between mesophyll protoplasts and other protoplasts. Thirty to 40 mesophyll protoplasts were drawn up into a microcapillary with a tip opening of approximately 100 µm. Total RNA was extracted from the protoplasts using the PicoPure RNA isolation kit (Arcturus).

RT-PCR

First-strand cDNA was prepared with the DNA-free total RNA using the First-Strand cDNA Synthesis kit (Amersham Biosciences). The following primers were used for RT-PCR: AtSUT4(for) 5' gtc atc cca cag gta att gtg tct gtt ggc 3', AtSUT4(rev) 5' gcg gcc gct cat ggg aga ggg atg gg 3', AtSUC2(for) 5' cat tgt cgt ccc tca gat ggt aat atc tg 3', AtSUC2(rev) 5' ctc gag atg aaa tcc cat agt agc ttt gaa g 3', Actin(for) 5' gga aca gtg tga ctc aca cca tc 3', Actin(rev) 5' aag ctg ttc ttt ccc tct acg c 3', HvSUT2(for) 5' cac aat ctt agg agc acc tct gtc gat cac g 3', and HvSUT2(rev) 5' cat ggg tac ctc gtt ggg tgg ttt tct tct tc 3'.

	ACKNOWLEDGMENTS

 
We thank Prof. Masayoshi Maeshima (University of Nagoya) for his kind supply of antibodies to BiP and Prof. Francis Marty (University of Bourgogne) for providing us with antibodies to -TIP and AOX.

Received February 22, 2006; returned for revision February 22, 2006; accepted March 16, 2006.

	FOOTNOTES

 
¹ This work was supported by the Plant Science Center Zurich-Basel (graduate research fellowship), by the project Novel Ion Channels in Plants (grant no. EU HPRN–CT–00245), and by the Deutsche Forschungsgemeinschaft (project no. ME 1955/2).

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Ulrike G. Schmidt (ulrike.schmidt{at}botinst.unizh.ch).

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.106.079533.

^* Corresponding author; e-mail ulrike.schmidt{at}botinst.unizh.ch; fax 41–44–6348204.

	LITERATURE CITED

TOP ABSTRACT RESULTS AND DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 
Alexandersson E, Saalbach G, Larsson C, Kjellbom P (2004) Arabidopsis plasma membrane proteomics identifies components of transport, signal transduction and membrane trafficking. Plant Cell Physiol 45: 1543–1556[Abstract/Free Full Text]

Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254[CrossRef][ISI][Medline]

Carr S, Aebersold R, Baldwin M, Burlingame A, Clauser K, Nesvizhskii A; Working Group on Publication Guidelines for Peptide and Protein Identification Data (2004) The need for guidelines in publication of peptide and protein identification data: Working Group on Publication Guidelines for Peptide and Protein Identification Data. Mol Cell Proteomics 3: 531–533[Free Full Text]

Carter C, Songqin P, Zouhar J, Avila EL, Girke T, Raikhel NV (2004) The vegetative vacuole proteome of Arabidopsis thaliana reveals predicted and unexpected proteins. Plant Cell 16: 3285–3303[Abstract/Free Full Text]

Catala R, Santos E, Alonso JM, Ecker JR, Martinez-Zapater JM, Salinas J (2003) Mutations in the Ca²⁺/H⁺ transporter CAX1 increase CBF/DREB1 expression and the cold-acclimation response in Arabidopsis. Plant Cell 15: 2940–2951[Abstract/Free Full Text]

Chiou TJ, Bush DR (1996) Molecular cloning, immunochemical localization to the vacuole, and expression in transgenic yeast and tobacco of a putative sugar transporter from sugar beet. Plant Physiol 110: 511–520[Abstract]

Conceicao ADS, Marty-Mazars D, Bassham DC, Sanderfoot AA, Marty F, Raikhel NV (1997) The syntaxin homolog AtPEP12p resides on a late post-Golgi compartment in plants. Plant Cell 9: 571–582[Abstract]

Dietrich D, Hammes U, Thor K, Suter-Grotemeyer M, Fluckiger R, Slusarenko AJ, Ward JM, Rentsch D (2004) AtPTR1, a plasma membrane peptide transporter expressed during seed germination and in vascular tissue of Arabidopsis. Plant J 40: 488–499[CrossRef][ISI][Medline]

Elthon TE, Nickels RL, McIntosh L (1989) Monoclonal antibodies to the alternative oxidase of higher plant mitochondria. Plant Physiol 89: 1311–1317[Abstract/Free Full Text]

Emmerlich V, Linka N, Reinhold T, Hurth MA, Traub M, Martinoia E, Neuhaus HE (2003) The plant homolog to the human sodium/dicarboxylic cotransporter is the vacuolar malate carrier. Proc Natl Acad Sci USA 100: 11122–11126[Abstract/Free Full Text]

Frangne N, Maeshima M, Schaffner AR, Mandel T, Martinoia E, Bonnemain JL (2001) Expression and distribution of a vacuolar aquaporin in young and mature leaf tissues of Brassica napus in relation to water fluxes. Planta 212: 270–278[CrossRef][ISI][Medline]

Geisler M, Girin M, Brandt S, Vincenzetti V, Plaza S, Kobae Y, Maeshima M, Billion K, Kolukisaoglu UH, Schulz B, et al (2004) Arabidopsis immunophilin-like TWD1 functionally interacts with vacuolar ABC transporters. Mol Biol Cell 15: 3393–3405[Abstract/Free Full Text]

Gleave AP (1992) A versatile binary vector system with a T-DNA organizational structure conducive to efficient integration of cloned DNA into the plant genome. Plant Mol Biol 20: 1203–1207[CrossRef][ISI][Medline]

Haseloff J, Amos B (1995) GFP in plants. Trends Genet 11: 328–329[CrossRef][ISI][Medline]

Hurth MA, Suh SJ, Kretzschmar T, Geis T, Bregante M, Gambale F, Martinoia E, Neuhaus HE (2005) Impaired pH homeostasis in Arabidopsis lacking the vacuolar dicarboxylate transporter and analysis of carboxylic acid transport across the tonoplast. Plant Physiol 137: 901–910[Abstract/Free Full Text]

Kaiser G, Heber U (1984) Sucrose transport into vacuoles isolated from barley mesophyll protoplasts. Planta 161: 562–568[CrossRef]

Kaiser G, Martinoia E, Schmitt JM, Hincha DK, Heber U (1986) Polypeptide pattern and enzymic character of vacuoles isolated from barley mesophyll protoplasts. Planta 169: 345–355[CrossRef]

Klein M, Geisler M, Suh SJ, Kolukisaoglu HU, Azevedo L, Plaza S, Curtis MD, Richter A, Weder B, Schulz B, et al (2004) Disruption of AtMRP4, a guard cell plasma membrane ABCC-type ABC transporter, leads to deregulation of stomatal opening and increased drought susceptibility. Plant J 39: 219–236[CrossRef][ISI][Medline]

Kreuz K, Tommasini R, Martinoia E (1996) Old enzymes for a new job. Plant Physiol 111: 349–353[ISI][Medline]

Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685[CrossRef][Medline]

Liu G, Sánchez-Fernández R, Li ZS, Rea PA (2001) Enhanced multispecificity of Arabidopsis vacuolar multidrug resistance-associated protein-type ATP-binding cassette transporter, AtMRP2. J Biol Chem 23: 8648–8656

Maeshima M (2001) Tonoplast transporters: organization and function. Annu Rev Plant Physiol Plant Mol Biol 52: 469–497[CrossRef][ISI][Medline]

Martinoia E, Grill E, Tommasini R, Kreuz K, Amrhein N (1993) ATP-dependent S-conjugate ‘export’ pump in the vacuolar membrane of plant. Nature 364: 247–249[CrossRef]

Martinoia E, Heck U, Wiemken A (1981) Vacuoles as storage compartments for nitrate in barley leaves. Nature 289: 292–294[CrossRef]

Martinoia E, Kaiser G, Schramm MJ, Heber U (1987) Sugar-transport across the plasmalemma and the tonoplast of barley mesophyll protoplasts: evidence for different transport-systems. Plant Physiol 131: 467–487

Martinoia E, Klein M, Geisler M, Forestier C, Kolukisaoglu U, Mueller-Roeber B, Schulz B (2002) Multifunctionality of plant ABC transporters: more than just detoxifiers. Planta 214: 345–355[CrossRef][ISI][Medline]

Matile P (1984) Das toxische Kompartiment Pflanzenzelle. Naturwissenschaften 71: 18–24[CrossRef]

Nakamura A, Fukuda A, Sakai S, Tanaka Y (2006) Molecular cloning, functional expression and subcellular localization of two putative voltage-gated chloride channels in rice (Oryza sativa L). Plant Cell Physiol 47: 32–42[Abstract/Free Full Text]

Nesvizhskii AI, Keller A, Kolker E, Aebersold R (2003) A statistical model for identifying proteins by tandem mass spectrometry. Anal Chem 75: 4646–4658[Medline]

Peiter E, Maathuis F, Mills L, Knight H, Pelloux J, Hetherington A, Sanders D (2005) The vacuolar Ca²⁺-activated channel TPC1 regulates germination and stomatal movement. Nature 434: 404–408[CrossRef][Medline]

Pollock CJ, Cairns AJ (1991) Fructan metabolism in grasses and cereals. Annu Rev Plant Physiol Plant Mol Biol 42: 77–101[CrossRef][ISI]

Rentsch D, Martinoia E (1991) The plant homolog to the human sodium/dicarboxylic cotransporter is the vacuolar malate carrier. Planta 184: 532–537[ISI]

Ritsema T, Smeekens S (2003) Fructan: beneficial for plants and humans. Curr Opin Plant Biol 6: 223–230[CrossRef][ISI][Medline]

Sazuka T, Keta S, Shiratake K, Yamaki S, Shibata D (2004) A proteomic approach to identification of transmembrane proteins and membrane-anchored proteins of Arabidopsis thaliana by peptide sequencing. DNA Res 11: 101–113[Abstract]

Schulze WX, Reinders A, Ward J, Lalonde S, Frommer WB (2003) Interactions between co-expressed Arabidopsis sucrose transporters in the split-ubiquitin system. BMC Biochem 4: 3[CrossRef][Medline]

Shevchenko A, Wilm M, Vorm O, Mann M (1996) Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 68: 850–858[Medline]

Shimaoka T, Ohnishi M, Sazuka T, Mitsuhashi N, Hara-Nishimura I, Shimazaki K, Maeshima M, Yokota A, Tomizawa K, Mimura T (2004) Isolation of intact vacuoles and proteomic analysis of tonoplast from suspension-cultured cells of Arabidopsis thaliana. Plant Cell Physiol 45: 672–683[Abstract/Free Full Text]

Song W, Steiner HY, Zhang L, Naider F, Stacey G, Becker JM (1996) Cloning of a second Arabidopsis peptide transport gene. Plant Physiol 110: 171–178[Abstract]

Stadler R, Truernit E, Gahrtz M, Sauer N (1999) The AtSUC1 sucrose carrier may represent the osmotic driving force for anther dehiscence and pollen tube growth in Arabidopsis. Plant J 19: 269–278[CrossRef][ISI][Medline]

Stadler R, Sauer N (1996) The Arabidopsis thaliana AtSUC2 gene is specifically expressed in companion cells. Bot Acta 109: 299–306[ISI]

Sze H, Schumacher K, Muller ML, Padmanaban S, Taiz L (2002) A simple nomenclature for a complex proton pump: VHA genes encode the vacuolar H(+)-ATPase. Trends Plant Sci 7: 157–161[CrossRef][ISI][Medline]

Szponarski W, Sommerer N, Boyer JC, Rossignol M, Gibart R (2004) Large-scale characterization of integral proteins from Arabidopsis vacuolar membrane by two-dimensional liquid chromatography. Proteomics 4: 397–406[CrossRef][ISI][Medline]

Theodoulou FL, Clark IM, He XL, Pallett KE, Cole DJ, Hallahan DL (2003) Co-induction of glutathione-S-transferases and multidrug resistance associated protein by xenobiotics in wheat. Pest Manag Sci 59: 202–214[CrossRef][Medline]

Thomine S, Lelievre F, Debarbieux E, Schroeder JI, Barbier-Brygoo H (2003) AtNRAMP3, a multispecific vacuolar metal transporter involved in plant responses to iron deficiency. Plant J 34: 685–695[CrossRef][ISI][Medline]

Wagner W, Keller F, Wiemken A (1983) Fructan metabolism in cereals: induction in leaves and compartmentation in protoplasts and vacuoles. Z Pflanzenphysiol 112: 359–372

Weise A, Barker L, Kuehn C, Lalonde S, Buschmann H, Frommer WB, Ward JM (2000) A new subfamily of sucrose transporters, SUT4, with low-affinity/high capacity localized in sieve elements of plants. Plant Cell 12: 1345–1355[Abstract/Free Full Text]

Weschke W, Panitz R, Sauer N, Wang Q, Neubohn B, Weber H, Wobus U (2000) Sucrose transport into barley seeds: molecular characterization of two transporters and implications for seed development and starch accumulation. Plant J 21: 455–467[CrossRef][ISI][Medline]

Xia T, Apse MP, Aharon GS, Blumwald E (2002) Identification and characterization of a NaCl-inducible vacuolar Na⁺/H⁺ antiporter in Beta vulgaris. Physiol Plant 116: 206–212[CrossRef][Medline]

Yamaguchi H, Nishizawa NK, Nakanishi H, Mori S (2002) IDI7, a new iron-regulated ABC transporter from barley roots, localizes to the tonoplast. J Exp Bot 53: 727–735[Abstract/Free Full Text]

This article has been cited by other articles:

				S. Hoth, R. Stadler, N. Sauer, and U. Z. Hammes Differential vascularization of nematode-induced feeding sites PNAS, August 26, 2008; 105(34): 12617 - 12622. [Abstract] [Full Text] [PDF]

				D. Pozueta-Romero, P. Gonzalez, E. Etxeberria, and J. Pozueta-Romero The Hyperbolic and Linear Phases of the Sucrose Accumulation Curve in Turnip Storage Cells Denote Carrier-mediated and Fluid Phase Endocytic Transport, Respectively J. Amer. Soc. Hort. Sci., July 1, 2008; 133(4): 612 - 618. [Abstract] [Full Text] [PDF]

				E. Antony, T. Taybi, M. Courbot, S. T. Mugford, J. A. C. Smith, and A. M. Borland Cloning, localization and expression analysis of vacuolar sugar transporters in the CAM plant Ananas comosus (pineapple) J. Exp. Bot., May 1, 2008; 59(7): 1895 - 1908. [Abstract] [Full Text] [PDF]

				A. B. Sivitz, A. Reinders, and J. M. Ward Arabidopsis Sucrose Transporter AtSUC1 Is Important for Pollen Germination and Sucrose-Induced Anthocyanin Accumulation Plant Physiology, May 1, 2008; 147(1): 92 - 100. [Abstract] [Full Text] [PDF]

				S. Schneider, D. Beyhl, R. Hedrich, and N. Sauer Functional and Physiological Characterization of Arabidopsis INOSITOL TRANSPORTER1, a Novel Tonoplast-Localized Transporter for myo-Inositol PLANT CELL, April 1, 2008; 20(4): 1073 - 1087. [Abstract] [Full Text] [PDF]

				I. A. Chincinska, J. Liesche, U. Krugel, J. Michalska, P. Geigenberger, B. Grimm, and C. Kuhn Sucrose Transporter StSUT4 from Potato Affects Flowering, Tuberization, and Shade Avoidance Response Plant Physiology, February 1, 2008; 146(2): 515 - 528. [Abstract] [Full Text] [PDF]

				G.-P. Xue, C. L. McIntyre, C. L.D. Jenkins, D. Glassop, A. F. van Herwaarden, and R. Shorter Molecular Dissection of Variation in Carbohydrate Metabolism Related to Water-Soluble Carbohydrate Accumulation in Stems of Wheat Plant Physiology, February 1, 2008; 146(2): 441 - 454. [Abstract] [Full Text] [PDF]

				U. G. Schmidt, A. Endler, S. Schelbert, A. Brunner, M. Schnell, H. E. Neuhaus, D. Marty-Mazars, F. Marty, S. Baginsky, and E. Martinoia Novel Tonoplast Transporters Identified Using a Proteomic Approach with Vacuoles Isolated from Cauliflower Buds Plant Physiology, September 1, 2007; 145(1): 216 - 229. [Abstract] [Full Text] [PDF]

				F. Chopin, M. Orsel, M.-F. Dorbe, F. Chardon, H.-N. Truong, A. J. Miller, A. Krapp, and F. Daniel-Vedele The Arabidopsis ATNRT2.7 Nitrate Transporter Controls Nitrate Content in Seeds PLANT CELL, May 1, 2007; 19(5): 1590 - 1602. [Abstract] [Full Text] [PDF]