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First published online July 13, 2007; 10.1104/pp.107.103143 Plant Physiology 145:62-74 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
Characterization of AgMaT2, a Plasma Membrane Mannitol Transporter from Celery, Expressed in Phloem Cells, Including Phloem Parenchyma Cells[OA]Unité Mixte de Recherche Centre National de la Recherche Scientifique 6161 Transport des Assimilats, Université de Poitiers, Bâtiment Botanique, 86022 Poitiers cedex, France
A second mannitol transporter, AgMaT2, was identified in celery (Apium graveolens L. var. dulce), a species that synthesizes and transports mannitol. This transporter was successfully expressed in two different heterologous expression systems: baker's yeast (Saccharomyces cerevisiae) cells and tobacco (Nicotiana tabacum) plants (a non-mannitol-producing species). Data indicated that AgMaT2 works as an H+/mannitol cotransporter with a weak selectivity toward other polyol molecules. When expressed in tobacco, AgMaT2 decreased the sensitivity to the mannitol-secreting pathogenic fungi Alternaria longipes, suggesting a role for polyol transporters in defense mechanisms. In celery, in situ hybridization showed that AgMaT2 was expressed in the phloem of leaflets, petioles from young and mature leaves, floral stems, and roots. In the phloem of petioles and leaflets, AgMaT2, as localized with specific antibodies, was present in the plasma membrane of three ontologically related cell types: sieve elements, companion cells, and phloem parenchyma cells. These new data are discussed in relation to the physiological role of AgMaT2 in regulating mannitol fluxes in celery petioles.
Polyols are present in a number of plant species, where they are primary products of photosynthesis and are translocated between organs in the phloem. Polyols act as carbon storage compounds and are widely distributed among genera in the plant kingdom (Moing, 2000  ). Polyols are also found in algae, fungi, and lichens. The most commonly found polyols are mannitol (reduced form of Man), sorbitol (reduced form of Fru), and dulcitol (reduced form of Glc; Stoop et al., 1996). On top of their function as translocated sugars, polyols also have antioxidative and osmoprotectant properties that could be related to resistance to several abiotic stresses (drought, salt, cold) and they may even play a role in some biotic stresses (Stoop et al., 1996). This was partly confirmed by plants engineered to produce polyols that showed enhanced resistance to stress (Tarczynski et al., 1993). However, the polyol content of these transgenic plants was low compared to naturally polyol-producing plants (Karakas et al., 1997). Therefore, the evolutionary, if any, advantage for a plant to produce polyol has still to be proven. A better description of plants synthesizing polyols is a preliminary step toward understanding the role of polyols in plants. Although the synthesis and degradation pathways have been extensively described and characterized at the molecular level (Williamson et al., 1995; Everard et al., 1997), only recently were transport mechanisms unraveled through the first cloning of a mannitol transporter in plants (Noiraud et al., 2001a). All these initial clonings have been achieved on celery (Apium graveolens L. var. dulce), which was taken as model plant for these studies.
In species where polyols are products of photosynthesis and are translocated between source and sink organs through the phloem, it was initially proposed that phloem loading of polyols was symplastic (Flora and Madore, 1993
These initial sequences were used to identify polyol transporters in species translocating sorbitol, such as cherry (Prunus avium) trees (Gao et al., 2003 To gain information on AgMaT2, its function and localization were investigated by several methods. This transporter was successfully expressed in yeast (Saccharomyces cerevisiae), where it displayed uptake characteristics similar to other H+/polyol transporters, and in tobacco (Nicotiana tabacum), a plant that does not synthesize polyols. Preliminary results indicate that AgMaT2 expression led to decreased sensitivity to the mannitol-secreting fungi Alternaria longipes. In celery, expression of the gene and localization of the protein were determined. The results suggest that AgMaT2 has a role in phloem loading of mannitol, as it is present in the plasma membrane of sieve elements (SEs), companion cells (CCs), and also of phloem parenchyma cells. The data are discussed in relation with the function of transport phloem in a temporary sink organ such as celery petiole.
Cloning and Functional Expression of AgMaT2 in Baker's Yeast
AgMaT2 cDNA was initially identified during the screening of a cDNA library made from celery phloem (initially named M7 in Noiraud et al., 2001a Phylogenic analysis of the deduced protein AgMaT2 showed highest homology with AgMaT1 and polyol transporters from Plantago and the parasitic plant Orobanche ramosa (OrMaT1; Fig. 1 ). Polyol transporters from legume species group together and so do transporters from Rosaceous species. Putative sequences from rice (OsPST1 and OsPST2), the only monocot species included in the comparison, were more distantly related.
To study the function of AgMaT2, AgMaT2 cDNA was cloned into a yeast expression vector (pDR 196) and expressed in the Saccharomyces strain RS453 (Noiraud et al., 2001a ). Figure 2
shows that the uptake rate of 0.55 mM radiolabeled mannitol is 6 times higher in yeast expressing AgMaT2. To further characterize the uptake of mannitol, the kinetic parameters were determined (Fig. 3
). Uptake as a function of mannitol concentration displays a saturation over the concentration range studied (Fig. 3A). Lineweaver/Burk representation (Fig. 3B) allows the calculation of a KM of 1.8 mM and a Vmax of 28 nmol mg protein–1 min–1. A 2 mM mannitol concentration was therefore used to study the pH dependency of mannitol uptake. The optimal pH for mannitol uptake is around 4.5 (data not shown) as for AgMaT1. This, together with the large inhibition (almost 80%) of mannitol uptake by the protonophore carbonylcyanide m-chlorophenylhydrazone (Table I
), was taken as evidence for a mannitol/H+ cotransport. Several substrates (polyols and hexoses) were tested for their effect on mannitol uptake at a substrate/mannitol concentration ratio of 10. Suc had no effect on mannitol uptake, whereas all other sugars inhibited mannitol uptake. The most inhibitory were xylitol, Glc, and Fru (around 65% inhibition). As already noted for AgMaT1, p-chloromercuriphenylsulfonic acid (PCMBS) had no significant effect on mannitol transport.
AgMaT2 Was Successfully Expressed in Tobacco and Gives Protection to Alternaria
To study the activity of AgMaT2 in a plant cell system, AgMaT2 was introduced into tobacco plants as tobacco does not produce and transport polyol (Karakas et al., 1997
No significant difference in growth parameters could be detected between control plants and those expressing AgMaT2 (B6 plants, 12 plants for each condition), except that the latter had slightly longer internodes (48 ± 8 mm versus 39 ± 6 mm) and thus the plants were higher at flowering (856 ± 120 mm versus 750 ± 98 mm). However, the number and size of leaves were not different between control and transgenic plants. No mannitol could be detected by HPLC measurements in transformed or control plants. To confirm that the protein was active in transgenic plants, leaf discs were punched from leaves of in vitro-grown plants (control and transgenics) and incubated on liquid medium with radiolabeled mannitol (see "Materials and Methods"). The radioactivity taken up after incubation times ranging from 15 to 75 min was assayed (Fig. 5 ). It is clear that uptake of mannitol was much higher in plants expressing AgMaT2: The slopes of the curves are 1.31 nmol min–1 disk–1 for control plants, 4.76 for B5 plants, and 9.31 for B6 plants. This indicates that the capacity to take up mannitol is linked to the presence of the transgene.
Autoradiographs of leaf discs after incubation with radiolabeled mannitol were compared with those obtained with radiolabeled Suc, the main form of carbon transport in tobacco. For Suc, taken as a control, the white signal associated with the radioactivity was similar in both control (Fig. 6A ) and B6 (Fig. 6B) plants, indicating that AgMaT2 expression did not alter Suc uptake. In the case of mannitol, the signal was only seen in B6 plants (Fig. 6D), not in control plants (Fig. 6C). Interestingly, in B6 plants the localization of the signal in discs after uptake of mannitol was similar to the one after uptake of Suc. Under our experimental conditions (leaf discs from in vitro-grown plants where the lower epidermis has not been removed), the cut edges and the major veins of the discs were labeled. AgMaT2 was expressed under the control of the ubiquitous cauliflower mosaic virus 35S promoter, but mannitol was accumulated in the major vein as Suc. This confirmed the high expression level of the 35S promoter in the phloem cells, as already noted (Kühn et al., 1996 ), and demonstrated that exogenously supplied mannitol followed the same transport path as Suc. The presence of the lower epidermis prevented a good labeling of the minor vein network compared to, for example, Vicia faba leaf discs after incubation on Suc (Delrot and Bonnemain, 1981). Labeling is clearly linked to the expression of a transporter: It is seen in the both types of plant in the case of Suc (Fig. 6, A and B), but for mannitol only in plants expressing AgMaT2.
Mannitol secreted by some fungal pathogens has been proposed to protect them against reactive oxygen species-mediated host defense during infection (Jennings et al., 1998 ). Jennings et al. (2002) demonstrated that the constitutive expression of a celery mannitol dehydrogenase activity in tobacco led to a decreased sensitivity toward the mannitol-secreting pathogenic fungus Alternaria alternata. As mannitol dehydrogenase was demonstrated to be cytosolic (Yamamoto et al., 1997), we hypothesized that a mannitol transport activity had to be present at the same time. To test this possibility, AgMaT2-transformed and untransformed plants were challenged with A. longipes (formerly A. alternata). At 4 d after inoculation, the development of typical symptoms, i.e. necrotic lesions surrounded by chlorotic halos, was observed on both wild-type and transformed inoculated plants (Fig. 7
). No symptom was observed on leaves of control plants treated with sterile distilled water (data not shown). While the two tested genotypes could be considered as susceptible, the percentage of necrotic tissues was significantly lower in leaves expressing AgMaT2 (B6 plants) than in leaves from untransformed plants. The mean necrotic areas were 14.6% ± 0.9% and 3.0% ± 0.2% of the total leaf surface for control and transformed plants, respectively (mean of five measurements on representative leaves from two independent tests).
Localization of AgMaT2 Gene Expression in the Phloem of Celery
Although the functional expression of AgMaT2 in yeast and tobacco are in favor of plasma membrane localization, no such information was available about the localization in celery. As a first step to characterize the expression pattern of AgMaT2, in situ hybridization experiments were run on sections made from different celery organs embedded in paraffin. Preliminary experiments were run to ensure that no cross-reaction of the probe AgMaT2 with other mannitol transporters from celery (AgMaT1: Noiraud et al., 2001a
The vascular bundles in the petiole and leaflet midribs have the same structure. Phloem can be divided in two regions: adjacent to the cambium, the conducting zone rich in CCs and SEs, then the bundle cap made of phloem parenchyma cells. Bundle cap is much wider in the petiole (Fig. 8C). The conducting zone was evidenced by hybridization with an AgPP2-1 probe (Fig. 8B): AgPP2-1 is only expressed in CCs (Dinant et al., 2003 ).
Interestingly, oil ducts are present in the inner part of the bundle cap and also in the conducting zone of the phloem (arrows in Fig. 8, C and G). This may facilitate exchange of molecules between phloem cells and cells at the border of the oil duct. The conducting zone and bundle cap are very tightly linked and then can easily be separated from surrounding tissues (xylem and storage parenchyma in the petiole) by mechanical dissection, allowing a range of studies on phloem function (Daie, 1986 AgMaT2 expression was detected in the conducting zone of petiole vascular bundles at two different development stages: young yellowish leaves (Fig. 8D) and fully mature green leaves (Fig. 8E). It has to be noted that oil ducts were not labeled. In petioles from young leaves, the phloem parenchyma cells in the bundle cap showed AgMaT2 expression (pc in Fig. 8D), which was not the case in petiole from mature leaves (Fig. 8E). Staining was not found in the xylem or storage parenchyma cells (Fig. 8, D and E). AgMaT2 was also expressed in the conducting zone in the midrib of leaflets from young and mature leaves (Fig. 8, F and G, respectively). It has to be noted that the size of leaflets did not change significantly during this development period, although the vascular bundle increased in size (compare Fig. 8, F and G). As noted previously, no label was found in oils ducts and other tissues. The parenchyma cell zone is very thin in young leaflets (Fig. 8F) and larger in mature leaflets (Fig. 8G); however, no staining was detected in that zone in both cases. Experiments were also conducted on roots from young plants. Staining was located in the phloem zone of the stele and at a lower level in the cortical parenchyma (Fig. 8H). As celery is a biennial plant, flowering was induced by subjecting 2-month-old plants to 4 weeks vernalization in a cold room (5°C, same light conditions as described in "Materials and Methods"). Plants were then transferred back to the greenhouse, where they flowered after 3 to 4 months. In the floral stalk, only the conducting zone of the phloem was labeled with AgMaT2 (Fig. 8I).
The data from gene expression studies localized AgMaT2 expression in the conducting zone of phloem. However, this did not give indication on the expression of AgMaT2 protein. Polyclonal antibodies were raised against a peptide in the C terminus of AgMaT2 (see "Materials and Methods"). Care was taken in the choice of the peptide to avoid cross-reaction with AgMaT1. This was further confirmed by challenging plasma membrane vesicles from yeast expressing AgMaT1 or AgMaT2 with anti-AgMaT2 antibodies (Fig. 9 , lanes 2 and 6). A signal was detected only in AgMaT2-expressing yeast (lane 2). Furthermore, the apparent molecular mass of AgMaT2 (48 kD) was the same in yeast (Fig. 9, lane 1) and in plasma membrane vesicles from celery leaves (Fig. 9, lane 2). In the plasma membrane fraction from celery leaves, a signal was occasionally detected at a higher molecular mass, which may indicate interactions with other proteins or multimerization (see "Discussion"). In comparison, no signal was detected for AgMaT2 in microsomal membranes or in the soluble proteins fraction from celery leaves, but a reaction was noted for a lower molecular mass (27 kD; Fig. 9, lanes 3 and 4). Altogether, these data indicated that the antibodies raised against AgMaT2 were suitable for use in localization studies.
Initial experiments were made on sections from the same tissues (unfixed, Wang et al., 1995 ), as for in situ hybridization experiments. The results are presented on Figure 10
using a fluorescent second antibody. The signal in young leaves was very faint (Fig. 10A) and limited to the conducting zone of the phloem. The same localization was noted in mature leaflets (Fig. 10B), but the signal was much stronger. Signals were also clearly visible in the conducting zone of cross sections of petioles from young and mature leaves (Fig. 10, D and E). In all cases, no signal was detected in either xylem or parenchyma cells (bundle cap), or in oil ducts. Phloem cells were also labeled in floral stalk (Fig. 10C). Except in young petioles, those results correspond to the localization of AgMaT2 expression (Fig. 8). Longitudinal sections were also prepared from petiole phloem after embedding. The results in Figure 10F show a clear localization of the signal at the periphery of cells, in accordance with a plasma membrane localization. However, it was not possible to identify the cell type where the label was found, so the localization of AgMaT2 was studied in electron microscopy (EM) experiments. Ultrathin sections were prepared and challenged with AgMaT2 antibodies. Taking into account the different data already obtained, the investigations were limited to the conducting zone of phloem, both in petioles and leaflets. Omitting the secondary antibody led to an absence of staining, confirming the efficiency of the blocking protocol (Fig. 11G
).
The SE and CCs were easily distinguished both in leaflet veins (Fig. 11A) and petiole conducting bundles (Fig. 11B). As already reported, the SE diameter is smaller than the CC in leaflets (loading phloem; Fig. 11A), whereas both cell types are of similar diameter in petioles (conducting phloem; Fig. 11B). In the conducting zone of petiole phloem, several SEs are often found connected together, forming large conducting units (Fig. 11C). The gold particles were clearly aligned to the plasma membrane of different cell types (arrows in Fig. 11, D–J). The plasma membrane localization was confirmed in Figure 11J, where the plasma membrane of the lower cell was detached from the cell wall due to cell plasmolysis: Gold particles clearly followed the plasma membrane. The immunolabeling was studied at the interface between the different cell types in the phloem of leaflets (Fig. 11, D–F) and in petioles (Fig. 11, H–J) from mature leaves. Plasma membranes were stained in SEs and CCs at their interface in leaflets (Fig. 11D) and petioles (Fig. 11H). Moreover, plasma membrane of CCs was also labeled at the interface between two CCs (Fig. 11E). In Figure 11I, a sieve plate between two SEs is clearly visible with some labeling in the plasma membrane at the sieve plate level. Unexpectedly, staining was also noted in the plasma membrane of phloem parenchyma cells both in leaflets (Fig. 11F) and in petioles (Fig. 11J). This may be related to the ontogeny of the phloem parenchyma cells (see "Discussion"). No other cell types, such as storage parenchyma or xylem, were stained.
AgMaT2 Acts as a Mannitol Transporter in Yeast and Transgenic Tobacco
AgMaT2 cDNA was initially identified together with AgMaT1 (Noiraud et al., 2001a
As many other polyol transporters, AgMaT2 has a low substrate specificity. Both inhibition studies (Table I) and transport measurements with radiolabeled substrates (data not shown) indicate that sorbitol and xylitol are also substrates for AgMaT2. Despite inhibition of mannitol uptake by Glc, no Glc transport activity could be measured for AgMaT2 (data not shown), a situation similar to AgMaT1 (Noiraud et al., 2001a
Few sugar transporters have been successfully expressed in heterologous plant system, except for a potato (Solanum tuberosum) Suc transporter (Leggewie et al., 2003
An inducible mannitol dehydrogenase activity could be measured in tobacco plants challenged with the mannitol-producing pathogen A. alternata (Jennings et al., 1998
As little is known about the localization of expression of polyol transporter genes in plants, in situ hybridization experiments were performed in different organs to localize the expression of AgMaT2. In all organs tested, from leaflet to root and to floral stem, AgMAT2 was detected in the phloem, more precisely in the conducting zone of the phloem (in the case of large vascular bundles). In young leaves, AgMaT2 expression was already detected in the phloem of leaflet and petiole, suggesting that mannitol transport occurs early in development. Young leaves were harvested at approximately 10 cm in length, a size similar to the "young leaves" described to translocate mannitol in the phloem (Davis and Loescher, 1990
The expression of AgMaT2 in petioles was compared with that of AgPP2-1, a typical CC-expressed gene (Dinant et al., 2003
Specific antibodies were raised against the C terminus region of AgMaT2 to perform a precise localization of AgMaT2 in the phloem cells. In plasma membrane vesicles from leaves, a reaction was sometimes noted at high molecular mass: This could indicate multimerization of AgMaT2 or interaction with other transporters, as was noted recently in the case of Suc transporters in potato (Reinders et al., 2002
AgMaT2 was first localized at the cell level by immunofluorescent labeling. The data in Figure 10 show that AgMaT2 was found only in the conducting zone of phloem both in leaflets and petioles. In the leaflet, signal was more intense in the samples from mature leaves than young leaves, in accordance with a higher mannitol synthesis in mature leaves in celery (Davis and Loescher, 1990
The data presented in Figure 11 clearly demonstrate that AgMaT2 is localized to the plasma membrane, both in petiole and leaflet of mature leaves. Vascular bundles of petiole and major veins of leaflet have a similar collateral structure: The main difference is that the bundle cap is smaller in mature leaflet than in mature petiole (compare Fig. 8, C and F). The three cell types of the conducting zone (SE, CC, and phloem parenchyma cell) originate from the same mother cell (Esaü, 1936
In celery, petioles are storage organs that accumulate sugars (mannitol and hexoses) in large storage parenchyma cells (Keller and Matile, 1989
Mannitol transporters in phloem parenchyma cells of the conducting zone may be present to favor uptake of mannitol (unloaded or leaked from the sap stream), further distribution to phloem parenchyma cells of the bundle cap, and then to the storage parenchyma cells when the petiole is in the sink (storage) stage. Few plasmodesmata are present between phloem parenchyma cells and either SEs or CCs, in contrast with the numerous branched plasmodesmata found at the SE/SE and SE/CC interfaces (data not shown). The bundle cap could be considered as an intermediate zone between the conducting cells and storage parenchyma cells, at least in the petiole. Expression of AgMaT2, in conducting cells (SE/CC) and in phloem parenchyma cells, may indicate different functions fulfilled by the same transporter, according to the cell type where it is expressed, as suggested recently for ZmSUT1 (Carpaneto et al., 2005
In this article, the physiological function of AgMaT2 was investigated in celery where it is normally expressed and in tobacco, a plant that does not synthesize or transport mannitol. AgMaT2 was characterized in yeast as a H+/mannitol cotransport system. In celery, the expression pattern, both at the gene and at the protein level, showed that AgMaT2 is present in the conducting cells of the phloem, whatever the organ. More precisely, AgMaT2 was localized to the plasma membrane of SEs, CCs, and phloem parenchyma cells in the transport phloem. This localization in three different cell types has not been found for other transporters yet (Suc, hexose, or polyol transporters). This expression pattern may relate to complex sugar fluxes in an organ such as the petiole, where mannitol is being transferred in the phloem sap to sink organs (roots and developing leaves), while petiole is itself a sink organ accumulating mannitol. These data also confirm the function of phloem parenchyma cells as involved in local sugar fluxes. The results showing that AgMaT2 expression in tobacco led to decreased sensitivity to A. longipes, a result already noted when expressing a mannitol dehydrogenase activity in tobacco (Jennings et al., 2002 ), may indicate a new defense pathway to resist mannitol-secreting pathogens. Therefore, polyol transporters may fulfill different function whether they are expressed in species that synthesize and transport polyol, like celery, or in species like tobacco and Arabidopsis that do not.
Plant Material
Celery plants (Apium graveolens L. var. dulce, ‘Vert d'Elne’) were grown in a greenhouse as described by Noiraud et al. (2000)
The initial cloning of AgMaT2 was reported by Noiraud et al. (2001a)
The AgMaT2 cDNA was cloned into the EcoRI and XhoI sites of pDR 196 and used to transform yeast (Saccharomyces cerevisiae) strain RS453 as described by Noiraud et al. (2001a)
The entire coding region of AgMaT2 was cloned into pBI101 and transformed into Agrobacterium tumefaciens according to Atanassova et al. (1995)
The mannitol-secreting phytopathogenic fungus Alternaria longipes (strain CBS917.96 from Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands) was grown at 24°C under near UV light on a potato dextrose agar medium for 7 d. Sterile distilled water was then added to the plates and the conidial suspensions collected. Three-week-old in vitro-grown tobacco plants were inoculated by uniformly spreading with a paintbrush a calibrated conidial suspension (2 105 conidia/mL) over the abaxial sides of five to six leaves of either wild-type or transformed plants. Inoculated plants were incubated at 22°C. Noninoculated controls were performed using the same procedure by spreading sterile distilled water instead of spore suspension. Images were taken at 4 d after inoculation to score for lesion formation using a digital camera. The total and necrotic areas were measured for each leaf using the ImageJ v1.36b software (http://rsb.info.nih.gov/ij/). Values obtained for each leaf from a same genotype were averaged. Two independent experiments were performed.
In situ hybridization experiments were carried out on leaflet, petiole, root, and flower of celery. Small fragments from each organ were embedded in paraffin and processed as described by Vignault et al. (2005)
Antibodies were raised in rabbits against a peptide (CGLKNREAEEAKNA) chosen in the C-terminal-specific part of the protein to avoid cross-reaction with other mannitol transporters (AgMaT1). The antiserum was immunopurified against the peptide to increase the specificity of the response (Eurogentec). The specificity of the purified antibodies was confirmed on western blots (see "Results").
Immunolabeling was conducted on semithin (0.5–1 µm) and thin (60–80 nm) sections from chemically fixed/LRW embedded fresh tissues (leaflet and petiole) of celery, and sections were processed as described by Fleurat-Lessard et al. (1997) Immunogold reaction was performed on thin sections, using 15-nm gold particle-labeled goat anti-rabbit IgG (Biocell, 1/50 dilution). Samples were observed with a JEOL (1010) microscope operated at 80 kV.
For some experiments (Fig. 10), fresh hand sections were washed for 30 min in PBS-milk 2.5% (w/v), and incubated for 3 h in the AgMaT2 antiserum, washed for 30 min in PBS-milk 2.5% (w/v), and incubated for 1 h in Alexa Fluor 488-labeled secondary antibody (Wang et al., 1995
Plasma membrane vesicles were purified from yeast according to the protocol described by Stolz et al. (1994) Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number AAL85876.2.
We thank Dr. Pierrette Fleurat-Lessard for inestimable help and advice with EM technique, Mrs. Magali Lallemand for help during in situ hybridization experiments, Dr. Christina Kühn (Humboldt University, Berlin) and Dr. Sylvie Dinant (Laboratoire de Biologie Cellulaire, INRA Versailles, France) for helpful discussions, and Prof. Jean Louis Bonnemain for comments on EM pictures and critical reading of the manuscript. We are also grateful to Dr. Anne Cantereau (IBPC, Université de Poitiers, France) and Dr. Emile Béré (SIMIS, Université de Poitiers, France) for help with microscopic techniques. The support of the Région Poitou Charentes to M.J.-C. and of Vilmorin Clause and Cie to L.L.-A. during their Ph.D. theses is acknowledged. Received May 31, 2007; accepted July 3, 2007; published July 13, 2007.
1 Present address: UMR PAVE A77, Université d'Angers-INRA-INH, 2 Boulevard Lavoisier, 49045 Angers cedex, France. 
2 Present address: UMR INRA-UCBN 950, Ecophysiologie Végétale, Agronomie and Nutritions NCS, Université de CAEN, Esplanade de la Paix, 14032 Caen cedex, France. 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: Rémi Lemoine (remi.lemoine{at}univ-poitiers.fr).
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.107.103143 * Corresponding author; e-mail remi.lemoine{at}univ-poitiers.fr.
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Plant Physiol 115: 1397–1403[Abstract] This article has been cited by other articles:
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. The sequence of AgMaT2 indicated that it was highly related to other polyol transporters. When expressed in a different yeast strain, the transport activity could be demonstrated (
