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Plant Physiology 134:204-214 (2004) © 2004 American Society of Plant Biologists AmSUT1, a Sucrose Transporter in Collection and Transport Phloem of the Putative Symplastic Phloem Loader Alonsoa meridionalis1Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Abteilung Biochemie der Pflanze, Universität Göttingen, Justus-von-Liebig-Weg 11, 37077 Göttingen, Germany (C.K., G.L.); and Universität Erlangen-Nürnberg, Lehrstuhl Botanik II-Molekulare Pflanzenphysiologie, Staudtstrasse 5, 91058 Erlangen, Germany (R.S., N.S.)
A sucrose (Suc) transporter cDNA has been cloned from Alonsoa meridionalis, a member of the Scrophulariaceae. This plant species has an open minor vein configuration and translocates mainly raffinose and stachyose in addition to Suc in the phloem (C. Knop, O. Voitsekhovskaja, G. Lohaus [2001  ] Planta 213: 80–91). These are typical properties of symplastic phloem loaders. For functional characterization, AmSUT1 cDNA was expressed in bakers' yeast (Saccharomyces cerevisiae). Substrate and inhibitor specificities, energy dependence, and Km value of the protein agree well with the properties measured for other Suc transporters of apoplastic phloem loaders. A polyclonal antiserum against the 17 N-terminal amino acids of the A. meridionalis Suc transporter AmSUT1 was used to determine the cellular localization of the AmSUT1 protein. Using fluorescence labeling on sections from A. meridionalis leaves and stems, AmSUT1 was localized exclusively in phloem cells. Further histological characterization identified these cells as companion cells and sieve elements. p-Chloromercuribenzenesulfonic acid affected the sugar exudation of cut leaves in such a way that the exudation rates of Suc and hexoses decreased, whereas those of raffinose and stachyose increased. The data presented indicate that phloem loading of Suc and retrieval of Suc in A. meridionalis are at least partly mediated by the activity of AmSUT1 in addition to symplastic phloem loading.
Assimilates produced during photosynthesis in mature leaves are distributed by the phloem system to support the growth of heterotrophic organs. The flow of assimilates such as Suc starts with symplastic cell-to-cell transport through mesophyll cells and bundle sheath cells, after which they are loaded into the sieve element (SE)-companion cell (CC) complex.
Plasmodesmal frequencies between the SE-CC complex and the adjacent cells in minor veins have been studied in many plant species. Gamalei (1989
The minor vein structure is correlated with the mode of phloem loading. Based on the plasmodesmal connections between the phloem and the surrounding cells and the presence of intermediary cells, transfer cells, or ordinary CCs, two types of phloem loading have been proposed: the apoplastic (Giaquinta, 1983
The apoplastic phloem loading mode is based on: (a) the steep uphill Suc gradient between the phloem sap and the cytosol of the surrounding cells (Geiger et al., 1973
In apoplastic phloem loaders, the long-distance transport form of carbohydrates is almost exclusively Suc. In the last decade, several genes of the proton-coupled Suc uptake transporter (SUT) family have been cloned and characterized from apoplastic phloem loaders (Riesmeier et al., 1992
In symplastic phloem loaders, such as members of the Cucurbitaceae, Lamiaceae, or several species of the Scrophulariaceae, carbohydrates are translocated in form of raffinose and stachyose (Zimmermann and Ziegler, 1975
Until now, the existence of Suc transporters has been much less investigated in plant species with open minor vein configuration than in those with closed minor vein configuration. Therefore, we analyzed a member of the Scrophulariaceae, A. meridionalis. In a previous study, we have shown that the minor vein phloem of this species contains intermediary cells and some ordinary CCs (Knop et al., 2001 The aim of the present study was to elucidate the functional properties of the Suc transporter from A. meridionalis (AmSUT1) by expression of the AmSUT1 cDNA in yeast. Furthermore, we studied the localization of the AmSUT1 protein with polyclonal antisera. The protein could be localized in the SEs and CCs of the collection and transport phloem of A. meridionalis. Therefore, the functions of the Suc transporter in this putative symplastic phloem loader will be discussed.
Characterization of AmSUT1 in Yeast
Previous physiological studies indicated that A. meridionalis is a putative symplastic phloem loader (Knop et al., 2001
For functional analysis and for studies of the kinetic parameters, AmSUT1 was expressed in yeast. The AmSUT1 cDNA was cloned into the Escherichia coli/yeast shuttle vector pNEV-E in sense or antisense orientation downstream of the strong promoter of the PMA1 plasma membrane ATPase from yeast and transformed into the yeast strain DBY2617 (Kaiser and Botstein, 1986 Yeast cells harboring the sense construct (CKY-Am1s) transported 14C-Suc at high rates, whereas the Suc transport into yeast cells harboring the antisense construct (CKY-Am1as) was negligible (Fig. 1A). These results indicate that the AmSUT1 protein is a transporter for Suc. The pH dependence of Suc transport by AmSUT1 was determined in the range of pH 5 to 8 (Fig. 1B). Transport rates increased continuously with decreasing external pH. At pH 8, AmSUT1 was almost completely inactive. Kinetic analysis of 14C-Suc uptake by yeast cells expressing AmSUT1 revealed an apparent Km value for Suc of 1.8 mM at pH 5.5 (Fig. 1C).
To determine the substrate specificity of AmSUT1, transport of 14C-labeled Suc was studied in the 10-fold excess of other sugars that might be potential substrates for this transporter like raffinose and stachyose because these are the predominant translocated sugars in A. meridionalis. The only sugar competing with Suc uptake was maltose, whereas the trisaccharide raffinose and the tetrasaccharide stachyose had no effect on the transport rate (Table I).
14C-Suc uptake via AmSUT1 was almost completely inhibited by the protonophore carbonyl cyanide CCCP (Table I). These data are consistent with a proton-coupled Suc uptake mechanism. All Suc transporters from higher plants characterized so far are highly sensitive to the SH-modifying agent PCMBS (Giaquinta, 1983
Polyclonal antisera were raised in rabbits by using synthetic peptides corresponding to the 17 N-terminal amino acids of AmSUT1. Protein extracts from plasma membranes of transgenic yeast cells expressing AmSUT1 and from yeast cells transformed with AmSUT1 in antisense direction were separated by SDS-PAGE, blotted, and treated with anti-AmSUT1 antiserum. A single specific band at an apparent molecular mass of 43 kD, which corresponds to the AmSUT1 protein, was recognized only in lane A of Figure 2, and the corresponding band was absent from plasma membrane preparations of yeast cells transformed with AmSUT1 in antisense orientation (Fig. 2, lane B). The difference of 9 kD between the molecular mass calculated from the DNA sequence (52.3 kD) and from SDS gels (43 kD) agrees well with the results from other plant Suc transporters such as AtSUC2 and PmSUC2 (Gahrtz et al., 1994
Does the anti-AmSUT1 antiserum recognize proteins other than AmSUT1 in A. meridionalis? To test this possibility, proteins were solubilized from A. meridionalis leaves and petioles. Western-blot analysis of these fractions gave no bands, probably as a result of low and cell-specific expression of AmSUT1 (Fig. 2, lanes E and F). Only in plasma membrane proteins isolated from A. meridionalis leaves was AmSUT1 detected as a band at a molecular mass of 43 kD (Fig. 2, lane D). Some weaker bands could also be detected at higher molecular masses (Fig. 2, lane D). Controls in which the anti-AmSUT1 antibody was replaced by pre-immune serum showed no bands (Fig. 2, lane G).
AmSUT1 was localized at the cellular level by using anti-AmSUT1 antiserum. Sections from stems or leaves of A. meridionalis were incubated with the anti-AmSUT1 antiserum, stained with a fluorescein isothiocyanate (FITC) isomer 1-conjugated secondary antibody, and investigated under fluorescent light. Cross sections of the A. meridionalis midrib are presented in Figure 3, A to E. In these sections, the cells labeled with the anti-AmSUT1 antiserum are located within the phloem (Fig. 3, A and C). The labeled cells have a dense cytoplasm as can be seen in white light (Fig. 3B). Controls in which the anti-AmSUT1 antibody was omitted showed no FITC labeling (Fig. 3D). The yellow-green staining of the xylem vessels is due to the phenolic compounds in the cell wall of the respective cells.
Figure 3, F to I, show sections of veins of the third order (major veins) and minor veins that were also labeled with anti-AmSUT1 antiserum and stained with FITC. Fluorescence microscopy of labeled sections shows green, anti-AmSUT1 antiserum-dependent fluorescence in defined cells opposite of the xylem cells (Fig. 3, F and H), indicating localization of the AmSUT1 protein in phloem cells of major (third order, Fig. 3F) or minor veins (Fig. 3H). In Figure 3H, the internal part of the phloem of the minor veins is more heavily labeled, probably the CCs and sieve tubes, than the laterally positioned cells of the minor vein, probably the intermediary cells (Fisher, 1986 Immunofluorescent detection of AmSUT1 was also performed on stem tissue of A. meridionalis. Sections incubated with anti-AmSUT1 antiserum show again that the AmSUT1 protein was detected in cells of the transport phloem (Fig. 4A). Control sections incubated with the secondary antibody alone gave no immunofluorescence signal (Fig. 4B).
To determine in which cell types of the phloem AmSUT1 was located, the fluorescence and light microscopic pictures were compared (Fig. 4, C and E; stars mark the same cells). At higher magnification, pairs of labeled cells were visible (Fig. 4D). One cell type had dense cytoplasm that showed green fluorescence, probably the CCs, and was adjacent to the other AmSUT1 expressing cell type that had a less dense cytoplasm and non-labeled parts, probably the SEs or young SEs; here, the green fluorescence was limited to the outer parts of the cells. For a more precise characterization of the cell type, it was necessary to look at additional properties of the labeled cells. One of the characteristics of mature SEs is denucleation during development. Double staining of longitudinal sections with the anti-AmSUT1 antiserum/FITC conjugate and with DAPI, which binds to DNA and results in a blue fluorescent staining of the nucleus, revealed that one type of the AmSUT1-containing cells has a well-developed nucleus. The cells are specifically located in the phloem and are very long in size, suggesting that these cells are CCs or very young SEs (Fig. 4F). In addition, double staining of longitudinal sections was performed using anti-AmSUT1 antiserum/FITC conjugate and aniline blue, which is SE specific, because it binds to callose that is deposited in the sieve plates of the SEs. The green FITC and the blue anilin label the same cell (Fig. 4H). The fact that both labels marked the same cell revealed that the other AmSUT1-containing cell type is the SE (Fig. 4H).
To gain further information on the mode of phloem loading in A. meridionalis, the effect of PCMBS on sugar exudation of cut leaves was tested. The extent of PCMBS sensitivity of Suc translocation has been used to distinguish between apoplastic and symplastic phloem loaders in several studies (Turgeon and Gowan, 1990
In previous studies, we collected phloem sap of A. meridionalis with the laser-aphid-stylet technique (Knop et al., 2001
In Table II, the effects of PCMBS on A. merdionalis and potato are shown. The apoplastic PCMBS concentration was determined as follows (van Bel et al., 1994
With control leaves, the exudation rate of sugars was much higher in potato than in A. meridionalis. PCMBS treatment had different effects on sugar exudation of the two plant species: Although the exudation rate was strongly reduced in potato (4% of control), it was nearly the same in A. meridionalis (89% of control). Although the exudation rate of the sum of sugars was almost unchanged in A. meridionalis, the exudation rates of single sugars changed in different directions. Although the exudation rates of raffinose and stachyose significantly increased, the rates of Glc, Fru, and Suc significantly decreased. However, the exudation rate of hexoses decreased more pronounced than that of Suc. In contrast to the exudate of cut petioles (Table II; Heineke et al., 1992
Comparison of the Biochemical Characteristics of AmSUT1 with Those of Other Suc Transporters
The cDNA of a Suc transporter was isolated from the putative symplastic phloem loader A. meridionalis (Knop et al., 2001
The SH-modifying agent PCMBS is an inhibitor of Suc transporters. Also, the Suc transporter from A. meridionalis is inhibited by this agent. In the presence of 50 or 100 µM PCMBS, Suc uptake in yeast expressing AmSUT1 was decreased to 51% and 19% (Table I), respectively, which is similar to the inhibition of other Suc transporters tested by heterologous expression in yeast cells (SoSUT1, 100 µM PCMBS, 21% residual activity, Riesmeier et al., 1992
The substrate specificity observed for AmSUT1 is in agreement with the already described characteristics of Suc transporters in typical apoplastic phloem loaders, like spinach (Riesmeier et al., 1992
To study the cell-specific localization of AmSUT1, antibodies were raised against the N terminus of the protein. The antiserum recognized a single polypeptide of 43 kD in plasma membranes from yeast expressing AmSUT1 and some weaker bands in addition to the 43-kD polypeptide in plasma membranes from A. meridionalis leaves. By immunofluorescence, AmSUT1 was localized in the phloem of leaves and stems of A. meridionalis. Additional use of histochemical techniques showed that the antiserum/FITC conjugate-labeled cells were CCs and either mature and/or young SEs (Fig. 4, F–I). In several other plant species, Suc transporters were only localized either in the CCs, e.g. in Arabidopsis and P. major (PmSUC2, Stadler et al., 1995
It is possible that A. meridionalis contains more than one Suc transporter and that the anti-AmSUT1 antiserum cross-reacted with these proteins. This could be the reason for the label in two cell types, the CCs and the sieve tubes. However, cross-reaction is unlikely because the antibodies were raised against the N terminus of the protein, which is a highly variable region of Suc transporters (Sauer and Stolz, 1994
The polymer trap model (Turgeon, 1991
The minor- and medium-sized veins are the major sites of primary phloem loading. Similar to other herbaceous putative symplastic phloem loaders (e.g. squash [Cucurbita pepo], Turgeon et al., 1975
Mixed apoplastic and symplastic phloem loading also has been assumed for other plants with an open vein anatomy (Turgeon et al., 1975
Although the phloem of the midrib and the stem is primarily responsible for long-distance transport and allocation, phloem loading of Suc is also required along this path (retrieval) because the high concentration of Suc and other sugars within the sieve tubes causes a permanent passive leakage of Suc into the apoplast. Moreover, the apoplastic Suc concentration in A. meridionalis (2.1 mM, Knop et al., 2001
The results presented here show that phloem loading of Suc or Suc retrieval in A. meridionalis, a putative symplastic phloem loader (Knop et al., 2001
Plant Material
Alonsoa meridionalis O. Kuntze and potato (Solanum tuberosum cv Désirée) were grown in compost soil under greenhouse conditions as described by Knop et al. (2001
For heterologous expression of AmSUT1, the Escherichia coli/yeast shuttle vector pNEV-E (Sauer and Stolz, 1994
Plasmids with inserts in sense and antisense orientations (pCK-Am1s and pCK-Am1as) were used to transform yeast strain DBY 2617 (Kaiser and Botstein, 1986
Plasma membrane proteins from yeast cells were enriched (Winzer, 1999
Synthetic peptides corresponding to the N terminus of AmSUT1 (MEVGNEAKSTALPPAQA) were synthesized and coupled to keyhole limpet hemocyanin via the C terminus. These proteins were used to immunize three rabbits (Dr. Julio Pineda, Pineda-Antikörper-Service, Berlin). Immunization was repeated every month until d 120. The quality of the antisera was tested using protein extracts of plasma membranes from transgenic yeast cells expressing AmSUT1 in sense (CKY-Am1s) or antisense (CKY-Am1as; negative control) orientation (Fig. 2). The specificity of the antiserum was also tested by reaction of plasma membrane proteins from transgenic yeast strains expressing AmSUT1 in sense orientation with anti-AmSUT1 antiserum that had been pre-incubated overnight with the antigenic peptide for saturating the antiserum. The final concentration of the antigenic N-terminal peptide was 3.5 mg mL-1. The serum was used directly for western blots at a dilution of 1:500 (v/v) and for immunocytochemistry at a dilution of 1:500 (v/v) to 1:5,000 (v/v).
The fixation of the material was carried out according to Stadler et al. (1995
For removal of methacrylate from the semithin sections, coverslips were incubated for 2 min in 100% (v/v) acetone. Rehydration proceeded in a series of ethanol (100%, 70%, and 30% [v/v]) for each 30 s. The coverslips were washed with Tris-buffered saline buffer (50 mM Tris-HCl [pH 7.5] and 150 mM NaCl) for 30 s and incubated in blocking buffer (1% [w/v] skimmed milk powder in Tris-buffered saline) for 45 min. After overnight incubation with anti-AmSUT1 antiserum (diluted 1:500–1:5,000 [v/v] in blocking buffer), the coverslips were washed three times with blocking buffer and incubated for 1 h with anti-rabbit IgG-FITC isomer 1 conjugate (diluted 1:300 [w/v] in blocking buffer, Sigma, St. Louis). For control sections, the anti-AmSUT1 antiserum was omitted. After five final washes with blocking buffer for 5 min each, the coverslips were rinsed with water and mounted in 10 µL of ProLong-Antifade Kit (Molecular Probes, Leiden, The Netherlands). Photographs were taken with a fluorescence phase microscope (Carl Zeiss, Göttingen, Germany) with an excitation light of 450 to 490 nm. For double staining of the AmSUT1 protein with antiserum/FITC conjugate and of nuclei with DAPI (Serva, Heidelberg), sections were treated as described above. After the final rinsing with water, coverslips were incubated for 1 h at room temperature in DAPI (0.2 µg mL-1). DAPI fluorescence was detected with an excitation light of 365 nm. For double staining of the AmSUT1 protein with antiserum/FITC conjugate and of sieve plates with aniline blue (Water Blue, Fluka, Buchs, Switzerland), the coverslips were incubated for 5 min in aniline blue (0.5% [w/v] in 200 mM NaPO4 buffer [pH 7.2]). Aniline blue fluorescence was detected with an excitation light of 365 nm.
Exudates were collected by the EDTA-facilitated exudation method described by King and Zeevaart (1974 To study the effect of PCMBS, the cut petioles of leaves were placed in 0.5 mL of tap water with or without 1 mM PCMBS and dissolved in 0.1% (v/v) DMSO for 1 h before exudation. Control assays were performed with the same concentration of DMSO. The Eppendorf cups along with their contents were preweighed. After 1 h, the leaves were removed, and the cups were weighed again. The difference in weight before and after the transpiration period enabled calculation of the volume transpired and the corresponding amount of PCMBS absorbed by the treated leaves.
Sugars in phloem exudates were assayed by HPLC with pulsed amperometric detection using a CarboPAC10 (Dionex, Sunnyvale, CA) column and precolumn and an NaOH eluent as described previously (Lohaus et al., 1995
We thank Katharina Pawlowski and Jens Tilsner for critical reading of the manuscript and Carola Schröder for excellent technical assistance. Received June 27, 2003; returned for revision September 10, 2003; accepted October 5, 2003.
Article, publication date, and citation information can be found at http://www.plantphysiol.org/cgi/doi/10.1104/pp.103.029264.
1 This work was supported by the Deutsche Forschungsgemeinschaft.
2 Present address: Roche Diagnostics GmbH, Nonnenwald 2, 82372 Penzberg, Germany. * Corresponding author; e-mail glohaus{at}gwdg.de; fax 49–551–395749.
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ABSTRACT
RESULTS
1–6 galactosyln-Suc) in Cucumis melo, Coleus blumei, and Ajuga reptans (Holthaus and Schmitz, 1991
) and Suc uptake into control cells transformed with AmSUT1 in antisense orientation (yeast strain CKY-Am1as; 
). Transport assays were performed with an initial outside Suc concentration of 1 mM at pH 5.5. B, pH dependence of Suc uptake by yeast cells expressing AmSUT1 (yeast strain CKY-Am1s). C, Lineweaver-Burk plot of Suc uptake as a function of concentration in yeast cells expressing AmSUT1 (yeast strain CKY-Am1s). Reciprocal uptake rates are plotted against reciprocal Suc concentrations in the assays.
des phloemtransports auf das speicherverhalten von verschiedenen rübensorten (Beta vulgaris L.) unter besonderer berücksichtigung des saccharosetransporters BvSUT1. PhD thesis. University of Göttingen, Germany
