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Sugar beet (Beta vulgaris L.) is one of the most important sugar-producing plants worldwide and provides about one third of the sugar consumed by humans. Here we report on molecular characterisation of the BvSUT1 gene and on the functional characterisation of the encoded transporter. In contrast to the recently identified tonoplast-localised sucrose transporter BvTST2.1 from sugar beet taproots, which evolved within the monosaccharide transporter (MST) superfamily, BvSUT1 represents a classical sucrose transporter and is a typical member of the disaccharide transporter (DST) superfamily. Transgenic Arabidopsis plants expressing the β-GLUCURONIDASE (GUS) reporter gene under control of the BvSUT1-promoter showed GUS histochemical staining of their phloem; an anti-BvSUT1-antiserum identified the BvSUT1 transporter specifically in phloem companion cells. After expression of BvSUT1 cDNA in bakers' yeasts (Saccharomyces cerevisiae) uptake characteristics of the BvSUT1 protein were studied. Moreover, the sugar beet transporter was characterised as a proton-coupled sucrose symporter in Xenopus laevis oocytes. Our findings indicate that BvSUT1 is the sucrose transporter that is responsible for loading of sucrose into the phloem of sugar beet source leaves delivering sucrose to the storage tissue in sugar beet taproot sinks., (© 2017 German Botanical Society and The Royal Botanical Society of the Netherlands.)

Trees are generally assumed to be symplastic phloem loaders. A typical feature for most wooden species is an open minor vein structure with symplastic connections between mesophyll cells and phloem cells, which allow sucrose to move cell-to-cell through the plasmodesmata into the phloem. Fraxinus excelsior (Oleaceae) also translocates raffinose family oligosaccharides in addition to sucrose. Sucrose concentration was recently shown to be higher in the phloem sap than in the mesophyll cells. This suggests the involvement of apoplastic steps and the activity of sucrose transporters in addition to symplastic phloem-loading processes. In this study, the sucrose transporter FeSUT1 from F. excelsior was analysed. Heterologous expression in baker's yeast showed that FeSUT1 mediates the uptake of sucrose. Immunohistochemical analyses revealed that FeSUT1 was exclusively located in phloem cells of minor veins and in the transport phloem of F. excelsior. Further characterization identified these cells as sieve elements and possibly ordinary companion cells but not as intermediary cells. The localization and expression pattern point towards functions of FeSUT1 in phloem loading of sucrose as well as in sucrose retrieval. FeSUT1 is most likely responsible for the observed sucrose gradient between mesophyll and phloem. The elevated expression level of FeSUT1 indicated an increased apoplastic carbon export activity from the leaves during spring and late autumn. It is hypothesized that the importance of apoplastic loading is high under low-sucrose conditions and that the availability of two different phloem-loading mechanisms confers advantages for temperate woody species like F. excelsior., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Arabidopsis thaliana has seven genes for functionally active sucrose transporters. Together with sucrose transporters from other dicot and monocot plants, these proteins form four separate phylogenetic groups. Group-IV includes the Arabidopsis protein SUC4 (synonym SUT4) and related proteins from monocots and dicots. These Group-IV sucrose transporters were reported to be either tonoplast- or plasma membrane-localised, and in heterologous expression systems were shown to act as sucrose/H(+) symporters. Here, we present comparative analyses of the subcellular localisation of the Arabidopsis SUC4 protein and of several other Group-IV sucrose transporters, studies on tissue specificity of the Arabidopsis SUC4 promoter, phenotypic characterisations of Atsuc4.1 mutants and AtSUC4 overexpressing (AtSUC4-OX) plants, and functional comparisons of Atsuc4.1 and AtSUC4-OX vacuoles. Our data show that SUC4-type sucrose transporters from different plant families (Brassicaceae, Cucurbitaceae and Solanaceae) localise exclusively to the tonoplast, demonstrating that vacuolar sucrose transport is a common theme of all SUC4-type proteins. AtSUC4 expression is confined to the stele of Arabidopsis roots, developing anthers and meristematic tissues in all aerial parts. Analyses of the carbohydrate content of WT and mutant seedlings revealed reduced sucrose content in AtSUC4-OX seedlings. This is in line with patch-clamp analyses of AtSUC4-OX vacuoles that characterise AtSUC4 as a sucrose/H(+) symporter directly in the tonoplast membrane., (© 2011 German Botanical Society and The Royal Botanical Society of the Netherlands.)

Active loading of sucrose into phloem companion cells (CCs) is an essential process in apoplastic loaders, such as Arabidopsis or tobacco (Nicotiana sp.), and is even used by symplastic loaders such as melon (Cucumis melo) under certain stress conditions. Reduction of the amount or complete removal of the transporters catalysing this transport step results in severe developmental defects. Here we present analyses of two Arabidopsis lines, suc2-4 and suc2-5, that carry a null allele of the SUC2 gene which encodes the Arabidopsis phloem loader. These lines were complemented with constructs expressing either the Arabidopsis SUC1 or the Ustilago maydis srt1 cDNA from the SUC2 promoter. Both SUC1 and Srt1 are energy-dependent sucrose/H(+) symporters and differ in specific kinetic properties from the SUC2 protein. Transgene expression was confirmed by RT-PCRs, the subcellular localization of Srt1 in planta with an Srt1-RFP fusion, and the correct CC-specific localization of the recombinant proteins by immunolocalization with anti-Srt1 and anti-SUC1 antisera. The transport capacity of Srt1 was studied in Srt1-GFP expressing Arabidopsis protoplasts. Although both proteins were found exclusively in CCs, only SUC1 complemented the developmental defects of suc2-4 and suc2-5 mutants. As SUC1 and Srt1 are well characterized, this result provides an insight into the properties that are essential for sucrose transporters to load the phloem successfully.

The vacuolar membrane is involved in solute uptake into and release from the vacuole, which is the largest plant organelle. In addition to inorganic ions and metabolites, large quantities of protons and sugars are shuttled across this membrane. Current models suggest that the proton gradient across the membrane drives the accumulation and/or release of sugars. Recent studies have associated AtSUC4 with the vacuolar membrane. Some members of the SUC family are plasma membrane proton/sucrose symporters. In addition, the sugar transporters TMT1 and TMT2, which are localized to the vacuolar membrane, have been suggested to function in proton-driven glucose antiport. Here we used the patch-clamp technique to monitor carrier-mediated sucrose transport by AtSUC4 and AtTMTs in intact Arabidopsis thaliana mesophyll vacuoles. In the whole-vacuole configuration with wild-type material, cytosolic sucrose-induced proton currents were associated with a proton/sucrose antiport mechanism. To identify the related transporter on one hand, and to enable the recording of symporter-mediated currents on the other hand, we electrophysiologically characterized vacuolar proteins recognized by Arabidopsis mutants of partially impaired sugar compartmentation. To our surprise, the intrinsic sucrose/proton antiporter activity was greatly reduced when vacuoles were isolated from plants lacking the monosaccharide transporter AtTMT1/TMT2. Transient expression of AtSUC4 in this mutant background resulted in proton/sucrose symport activity. From these studies, we conclude that, in the natural environment within the Arabidopsis cell, AtSUC4 most likely catalyses proton-coupled sucrose export from the vacuole. However, TMT1/2 probably represents a proton-coupled antiporter capable of high-capacity loading of glucose and sucrose into the vacuole., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)

Based on the high density of plasmodesmata interconnecting the intermediary cells and their neighboring phloem parenchyma or bundle-sheath cells, and based on the insensitivity to the sucrose transport inhibitor p-chloromercuribenzenesulfonic acid (PCMBS), cucurbits have been concluded to be symplastic loaders. In the present study, we identified and characterized the full-length sequence of sucrose transporter gene (CmSUT1) from melon (Cucumis melo L. cv. Hale's best jumbo). In vitro experiments confirmed that the identified gene product has sucrose transporter activity in baker's yeast. Healthy and cucumber mosaic virus (CMV)-infected melon plants were employed to examine sucrose transporter activity in planta. Pretreatment with PCMBS inhibited loading of newly fixed ¹⁴CO₂ into minor veins of CMV-infected plants. Moreover, CMV infection caused significant increase in CmSUT1 transcripts expression, mainly in vascular bundles of minor veins, which was associated with elevated sucrose content in phloem sap collected from source-leaf petioles. We propose that cucurbit plants contain the machinery for apoplastic phloem loading and that CMV infection causes a quantitative shift in the mode by which photoassimilates are loaded into the sieve tube., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)

Background: Plant sucrose transporter activities were shown to respond to changes in the extracellular pH and redox status, and oxidizing compounds like glutathione (GSSG) or H(2)O(2) were reported to effect the subcellular targeting of these proteins. We hypothesized that changes in both parameters might be used to modulate the activities of competing sucrose transporters at a plant/pathogen interface. We, therefore, compared the effects of redox-active compounds and of extracellular pH on the sucrose transporters UmSRT1 and ZmSUT1 known to compete for extracellular sucrose in the Ustilago maydis (corn smut)/Zea mays (maize) pathosystem., Methodology/principal Findings: We present functional analyses of the U. maydis sucrose transporter UmSRT1 and of the plant sucrose transporters ZmSUT1 and StSUT1 in Saccharomyces cerevisiae or in Xenopus laevis oocytes in the presence of different extracellular pH-values and redox systems, and study the possible effects of these treatments on the subcellular targeting. We observed an inverse regulation of host and pathogen sucrose transporters by changes in the apoplastic pH. Under none of the conditions analyzed, we could confirm the reported effects of redox-active compounds., Conclusions/significance: Our data suggest that changes in the extracellular pH but not of the extracellular redox status might be used to oppositely adjust the transport activities of plant and fungal sucrose transporters at the host/pathogen interface.

Plant pathogenic fungi cause massive yield losses and affect both quality and safety of food and feed produced from infected plants. The main objective of plant pathogenic fungi is to get access to the organic carbon sources of their carbon-autotrophic hosts. However, the chemical nature of the carbon source(s) and the mode of uptake are largely unknown. Here, we present a novel, plasma membrane-localized sucrose transporter (Srt1) from the corn smut fungus Ustilago maydis and its characterization as a fungal virulence factor. Srt1 has an unusually high substrate affinity, is absolutely sucrose specific, and allows the direct utilization of sucrose at the plant/fungal interface without extracellular hydrolysis and, thus, without the production of extracellular monosaccharides known to elicit plant immune responses. srt1 is expressed exclusively during infection, and its deletion strongly reduces fungal virulence. This emphasizes the central role of this protein both for efficient carbon supply and for avoidance of apoplastic signals potentially recognized by the host., Competing Interests: The authors have declared that no competing interests exist.

Leaf sucrose (Suc) transporters are essential for phloem loading and long-distance partitioning of assimilates in plants that load their phloem from the apoplast. Suc loading into the phloem is indispensable for the generation of the osmotic potential difference that drives phloem bulk flow and is central for the long-distance movement of phloem sap compounds, including hormones and signaling molecules. In previous analyses, solanaceous SUT1 Suc transporters from tobacco (Nicotiana tabacum), potato (Solanum tuberosum), and tomato (Solanum lycopersicum) were immunolocalized in plasma membranes of enucleate sieve elements. Here, we present data that identify solanaceous SUT1 proteins with high specificity in phloem companion cells. Moreover, comparisons of SUT1 localization in the abaxial and adaxial phloem revealed higher levels of SUT1 protein in the abaxial phloem of all three solanaceous species, suggesting different physiological roles for these two types of phloem. Finally, SUT1 proteins were identified in files of xylem parenchyma cells, mainly in the bicollateral veins. Together, our data provide new insight into the role of SUT1 proteins in solanaceous species.

Several plant families generate polyols, the reduced form of monosaccharides, as one of their primary photosynthetic products. Together with sucrose (Suc) or raffinose, these polyols are used for long-distance allocation of photosynthetically fixed carbon in the phloem. Many species from these families accumulate these polyols under salt or drought stress, and the underlying regulation of polyol biosynthetic or oxidizing enzymes has been studied in detail. Here, we present results on the differential regulation of genes that encode transport proteins involved in phloem loading with sorbitol and Suc under salt stress. In the Suc- and sorbitol-translocating species Plantago major, the mRNA levels of the vascular sorbitol transporters PmPLT1 and PmPLT2 are rapidly up-regulated in response to salt treatment. In contrast, mRNA levels for the phloem Suc transporter PmSUC2 stay constant during the initial phase of salt treatment and are down-regulated after 24 h of salt stress. This adaptation in phloem loading is paralleled by a down-regulation of mRNA levels for a predicted sorbitol dehydrogenase (PmSDH1) in the entire leaf and of mRNA levels for a predicted Suc phosphate synthase (PmSPS1) in the vasculature. Analyses of Suc and sorbitol concentrations in leaves, in enriched vascular tissue, and in phloem exudates of detached leaves revealed an accumulation of sorbitol and, to a lesser extent, of Suc within the leaves of salt-stressed plants, a reduced rate of phloem sap exudation after NaCl treatment, and an increased sorbitol-to-Suc ratio within the phloem sap. Thus, the up-regulation of PmPLT1 and PmPLT2 expression upon salt stress results in a preferred loading of sorbitol into the phloem of P. major.

Sucrose is the primary product of photosynthetic CO(2) fixation that is used for the distribution of assimilated carbon within higher plants. Its partitioning from the site of synthesis to different sites of storage, conversion into other storage compounds or metabolic degradation involves various steps of cell-to-cell movement and transport. Many of these steps occur within symplastic domains, i.e. sucrose moves passively cell-to-cell through plasmodesmata. Some essential steps, however, occur between symplastically isolated cells or tissues. In these cases, sucrose is transiently released into the apoplast and its cell-to-cell transport depends on the activity of plasma membrane-localized, energy dependent, H(+)-symporting carrier proteins. This paper reviews the current knowledge of sucrose transporter physiology and molecular biology.

This paper presents a detailed analysis of the PmSUC1 gene from plantago major, of its promoter activity in Arabidopsis, and of the tissue specific localization of the encoded protein in Plantago. PmSUC1 promoter activity was detected in the innermost layer of the inner integument (the endothel) of Arabidopsis plants expressing the gene of the green fluorescent protein (GFP) under the control of the PmSUC1 promoter. This promoter activity was confirmed with a PmSUC1-specific antiserum that identified the PmSUC1 protein in the endothel of Plantago and of Arabidopsis plants expressing the PmSUC1 gene under the control of its own promoter. PmSUC1 promoter activity and PmSUC1 protein were also detected in pollen grains during maturation inside the anthers and in pollen tubes during and after germination. These results demonstrate that PmSUC1 is involved in sucrose partitioning to the young embryo and to the developing pollen and growing pollen tube. In the innermost cell layer of the inner integument, a tissue that delivers nutrients to the endosperm and the embryo, PmSUC1 may catalyze the release of sucrose into the apoplast.

In order to understand sucrose transport in developing seeds of cereals at the molecular level, we cloned from a caryopses library two cDNAs encoding sucrose transporters, designated HvSUT1 and HvSUT2. Sucrose uptake activity was confirmed by heterologous expression in yeast. Both transporter genes are expressed in maternal as well as filial tissues. In a series of in situ hybridizations we analysed the cell type-specific expression in developing seeds. HvSUT1 is preferentially expressed in caryopses in the cells of the nucellar projection and the endospermal transfer layer, which represent the sites of sucrose exchange between the maternal and the filial generation and are characterized by transfer cell formation. HvSUT2 is expressed in all sink and source tissues analysed and may have a general housekeeping role. The rapid induction of HvSUT1 gene expression in caryopses at approximately 5-6 days after fertilization coincides with increasing levels of sucrose as well as sucrose synthase mRNA and activity, and occurs immediately before the onset of rapid starch accumulation within the endosperm. Starch biosynthesis requires sucrose to be imported into the endosperm, as direct precursor for starch synthesis and to promote storage-associated processes. We discuss the possible role of HvSUT1 as a control element for the endospermal sucrose concentration.

The Arabidopsis AtSUC1 protein has previously been characterized as a plasma membrane H+-sucrose symporter. This paper describes the sites of AtSUC1 gene expression and AtSUC1 protein localization and assigns specific functions to this sucrose transporter in anther development and pollen tube growth. RNase protection assays revealed AtSUC1 expression exclusively in floral tissue, which was confirmed by analyses of AtSUC1 promoter-beta-glucuronidase (GUS) plants. In situ hybridizations identified AtSUC1 expression in anther connective tissue, in funiculi and in fully developed pollen grains. Indirect immuno-fluorescence analyses with anti-AtSUC1 antiserum confirmed AtSUC1 protein localization in the connective tissue and funiculi. In mature pollen grains, however, despite high AtSUC1 mRNA levels no AtSUC1 protein was found. Only after pollination of stylar papillae was AtSUC1 protein detected inside the pollen and later inside the growing pollen tubes, suggesting a translation of pre-existing AtSUC1 mRNA after pollination. Pollen germination analyses underlined the important role of sucrose for pollen tube growth. The data presented suggest a role of AtSUC1 in the controlled dehiscence of Arabidopsis anthers. It is postulated that an important function of AtSUC1 is the cell-specific modulation of water potentials.

Monoclonal antibodies were raised and selected against recombinant Plantago major PmSUC2 sucrose carrier protein. Epitopes of two monoclonal antibodies (PS2-1A2 and PS2-4D4) were mapped using N-terminally truncated PmSUC2 proteins and a lambda library displaying random PmSUC2 peptides. PS2-1A2 recognizes an octapeptide close to the N-terminus of PmSUC2, PS2-4D4 binds to a decapeptide at the very C-terminus. Analyses of antibody binding to yeast protoplasts with functionally active, tagged PmSUC2 protein revealed that both epitopes are located in cytoplasmic domains of PmSUC2. These results support a model for plant sucrose transporters containing 12 transmembrane helices with the N-terminus and the C-terminus on the cytoplasmic side of the plasma membrane.

To elucidate the kinetic properties of the Arabidopsis H+/sucrose cotransporter, SUC1, with respect to transmembrane voltage and ligand concentrations, the transport system was heterologously expressed in Xenopus laevis oocytes. Steady-state plasma membrane currents associated with transport of sucrose were measured with two-electrode voltage clamp over the voltage range -180 to +40 mV as a function of extracellular pH and sugar concentrations. At any given voltage, currents exhibited hyperbolic kinetics with respect to extracellular H+ and sugar concentrations, and this enabled determination of values for the maximum currents in the presence of each ligand (iHmax, iSmax for H+ and sucrose) and of the ligand concentrations eliciting half-maximal currents (KHm, KSm). The iHmax and iSmax exhibited marked and statistically significant increases as a function of increasingly negative membrane potential. However, the KHm and KSm decreased with increasingly negative membrane potential. Furthermore, at any given voltage, iSmax increased and KSm decreased as a function of the external H+ concentration. Eight six-state carrier models-which comprised the four possible permutations of intracellular and extracellular ligand binding order, each with charge translocation on the sugar-loaded or -unloaded forms of the carrier-were analyzed algebraically with respect to their competence to account for the ensemble of kinetic observations. Of these, two models (first-on, first-off and last-on, first-off with respect to sucrose binding as it passes from outside to inside the cell and with charge translocation on the loaded form of the carrier) exhibit sufficient kinetic flexibility to describe the observations. Combining these two, a single model emerges in which the binding on the external side can be random, but it can only be ordered on the inside, with the sugar dissociating before the proton.

In this paper the cloning of a full-length cDNA clone encoding the PmSUC2 sucrose-H+ symporter from Plantago major is described. This plant allows the simple preparation of vascular bundles from the basal regions of fully developed source leaves and thus a separation of vascular and non-vascular tissue. A cDNA library was constructed from poly(A)+ RNA isolated from vascular bundles and used for the subsequent cloning of cDNAs. The respective mRNA is specifically expressed in the vascular bundles as shown on Northern blots of total RNA from vascular and non-vascular tissues. The PmSUC2 protein has 12 putative transmembrane helices and is highly homologous to other plant sucrose transporters. Substrate specificity and energy dependence of the transporter encoded by this cDNA were determined by expression in baker's yeast Saccharomyces cerevisiae. The PmSUC2 protein catalyses the transport of sucrose into transgenic yeast cells. Invertase null mutants of yeast expressing PmSUC2 accumulate sucrose more than 200-fold. This transport was sensitive to uncouplers or SH-group inhibitors. Plasma membranes from yeast cells expressing the PmSUC2 protein were purified and fused to proteoliposomes containing cytochrome-c-oxidase. In this system sucrose is accumulated only when proton motive force is generated, indicating that PmSUC2 is a sucrose-H+ symporter. The apparent molecular weight of the PmSUC2 protein is 35 kDa on 10% SDS-polyacrylamide gels. The presented data strongly support the theory of phloem loading from the apoplastic space by a sucrose-H+ symporter.

An important, most likely essential step for the long distance transport of sucrose in higher plants is the energy-dependent, uncoupler-sensitive loading into phloem cells via a sucrose-H+ symporter. This paper describes functional expression in Saccharomyces cerevisiae of two cDNAs encoding energy-dependent sucrose transporters from the plasma membrane of Arabidopsis thaliana, SUC1 and SUC2. Yeast cells transformed with vectors allowing expression of either SUC1 or SUC2 under the control of the promoter of the yeast plasma membrane ATPase gene (PMA1) transport sucrose, and to a lesser extent also maltose, across their plasma membranes in an energy-dependent manner. The KM-values for sucrose transport are 0.50 mM and 0.77 mM, respectively, and transport by both proteins is strongly inhibited by uncouplers such as carbonyl cyanide m-chlorophenylhydrazone (CCCP) and dinitrophenol (DNP), or SH-group inhibitors. The VMAX but not the KM-values of sucrose transport depend on the energy status of transgenic yeast cells. The two proteins exhibit different patterns of pH dependence with SUC1 being much more active at neutral and slightly acidic pH values than SUC2. The proteins share 78% identical amino acids, their apparent molecular weights are 54.9 kDa and 54.5 kDA, respectively, and both proteins contain 12 putative transmembrane helices. A modified SUC1-His6 cDNA encoding a histidine tag at the SUC1 C-terminus was also expressed in S. cerevisiae. The tagged protein is fully active and is shown to migrate at an apparent molecular weight of 45 kDa on 10% SDS-polyacrylamide gels.