SWEET17, a Facilitative Transporter,Mediates Fructose Transport across the Tonoplast of Arabidopsis Roots and Leaves

Fructose (Fru) is a major storage form of sugars found in vacuoles, yet the molecular regulation of vacuolar Fru transport is poorly studied. Although SWEET17 (for SUGARS WILL EVENTUALLY BE EXPORTED TRANSPORTERS17) has been characterized as a vacuolar Fru exporter in leaves, its expression in leaves is low. Here, RNA analysis and SWEET17-β-glucuronidase/-GREEN FLUORESCENT PROTEIN fusions expressed in Arabidopsis (Arabidopsis thaliana) reveal that SWEET17 is highly expressed in the cortex of roots and localizes to the tonoplast of root cells. Expression of SWEET17 in roots was inducible by Fru and darkness, treatments that activate accumulation and release of vacuolar Fru, respectively. Mutation and ectopic expression of SWEET17 led to increased and decreased root growth in the presence of Fru, respectively. Overexpression of SWEET17 specifically reduced the Fru content in leaves by 80% during cold stress. These results intimate that SWEET17 functions as a Fru-specific uniporter on the root tonoplast. Vacuoles overexpressing SWEET17 showed increased ¹⁴C]Fru uptake compared with the wild type. SWEET17-mediated Fru uptake was insensitive to ATP or treatment with NH₄Cl or carbonyl cyanide m-chlorophenyl hydrazone, indicating that SWEET17 functions as an energy-independent facilitative carrier. The Arabidopsis genome contains a close paralog of SWEET17 in clade IV, SWEET16. The predominant expression of SWEET16 in root vacuoles and reduced root growth of mutants under Fru excess indicate that SWEET16 also functions as a vacuolar transporter in roots. We propose that in addition to a role in leaves, SWEET17 plays a key role in facilitating bidirectional Fru transport across the tonoplast of roots in response to metabolic demand to maintain cytosolic Fru homeostasis.Sugars are main energy sources for generating ATP, major precursors to various storage carbohydrates as well as key signaling molecules important for normal growth in higher plants (Rolland et al., 2006). Depending on the metabolic demand, sugars are translocated over long distances or stored locally. SWEET (for SUGARS WILL EVENTUALLY BE EXPORTED TRANSPORTERS) and SUC/SUT (for Sucrose transporter/Sugar transporter)-type transporters are responsible for transfer of Suc from the phloem parenchyma into the sieve element companion cell complex for long-distance translocation (Riesmeier et al., 1992; Sauer, 2007; Kühn and Grof, 2010; Chen et al., 2012). Suc or hexoses derived from Suc hydrolysis in the cell wall are then taken up into sink cells by SUT (Braun and Slewinski, 2009) or monosaccharide transporters, such as sugar transporter1 (Sauer et al., 1990; Pego and Smeekens, 2000; Sherson et al., 2003). Alternatively, sugars are thought to move between cells via plasmodesmata (Voitsekhovskaja et al., 2006; Ayre, 2011). Major sugar storage pools within plant cells are soluble sugars stored in the vacuole, starch in plastids, and lipids in oil bodies.Vacuoles, which can account for approximately 90% of the cell volume (Winter et al., 1993), play central roles in temporary and long-term storage of soluble sugars (Martinoia et al., 2007; Etxeberria et al., 2012). Some agriculturally important crops such as sugar beet (Beta vulgaris; Leigh, 1984; Getz and Klein, 1995), citrus (Citrus spp.; Echeverria and Valich, 1988), sugarcane (Saccharum officinarum; Thom et al., 1982), and carrot (Daucus carota; Keller, 1988) can store considerable amounts (>10% of plant dry weight) of Suc, Glc, or Fru in vacuoles of the storage parenchyma. Due to a high capacity of vacuoles for storing sugars, vacuolar sugars can serve as an important carbohydrate source during energy starvation, e.g. after starch has been exhausted (Echeverria and Valich, 1988), as well as for the production of other compounds (e.g. osmoprotectants). Sugars are known to regulate photosynthesis; therefore, the release of sugars from vacuoles could be important for modulating photosynthesis (Kaiser and Heber, 1984). Moreover, vacuole-derived sugars are commercially used to produce biofuels, such as ethanol, from sugarcane. Knowledge of the key transporters involved in sugar exchange between the vacuole and cytoplasm is thus relevant in the context of bioenergy (Grennan and Gragg, 2009).To facilitate the exchange of sugars across the tonoplast, plant vacuoles are equipped with a multitude of transporters (Neuhaus, 2007; Etxeberria et al., 2012; Martinoia et al., 2012) comprising both facilitated diffusion and active transport systems of vacuolar sugars (Martinoia et al., 2000). Typically, Suc is actively imported into vacuoles by tonoplast monosaccharide transporter (AtTMT1/AtTMT2; Schulz et al., 2011) and exported by the SUT4 family (AtSUC4, OsSUT2; Eom et al., 2011; Payyavula et al., 2011; Schulz et al., 2011). Two H⁺-dependent sugar antiporters, the vacuolar Glc transporter (AtVGT1; Aluri and Büttner, 2007) and AtTMT1 (Wormit et al., 2006), mediate Glc uptake across the tonoplast to promote carbohydrate accumulation in Arabidopsis (Arabidopsis thaliana). The Early Responsive to Dehydration-Like6 protein has been shown to export vacuolar Glc into the cytosol (Poschet et al., 2011), likely via an energy-independent diffusion mechanism (Yamada et al., 2010). Defects in these vacuolar sugar transporters alter carbohydrate partitioning and allocation and inhibit plant growth and seed yield (Aluri and Büttner, 2007; Wingenter et al., 2010; Eom et al., 2011; Poschet et al., 2011).In contrast to numerous studies on vacuolar transport of Suc and Glc, limited efforts have been devoted to the molecular mechanism of vacuolar Fru transport even though Fru is predominantly located in vacuoles (Martinoia et al., 1987; Voitsekhovskaja et al., 2006; Tohge et al., 2011). Vacuolar Fru is important for the regulation of turgor pressure (Pontis, 1989), antioxidative defense (Bogdanović et al., 2008), and signal transduction during early seedling development (Cho and Yoo, 2011; Li et al., 2011). Thus, control of Fru transport across the tonoplast is thought to be important for plant growth and development. One vacuolar Glc transporter from the Arabidopsis monosaccharide transporter family, VGT1, has been reported to mediate low-affinity Fru uptake when expressed in yeast (Saccharomyces cerevisiae) vacuoles (Aluri and Büttner, 2007). Yet, the high vacuolar uptake activity to Fru intimates the existence of additional high-capacity Fru-specific vacuolar transporters (Thom et al., 1982). Recently, quantitative mapping of a quantitative trait locus for Fru content of leaves led to the identification of the Fru-specific vacuolar transporter SWEET17 (Chardon et al., 2013).SWEET17 belongs to the recently identified SWEET (PFAM:PF03083) super family, which contains 17 members in Arabidopsis and 21 in rice (Oryza sativa; Chen et al., 2010; Frommer et al., 2013; Xuan et al., 2013). Based on homology with 27% to 80% amino acid identity, plant SWEET proteins were grouped into four subclades (Chen et al., 2010). Analysis of GFP fusions indicated that most SWEET transporters are plasma membrane localized. Transport assays using radiotracers in Xenopus laevis oocytes and sugar nanosensors in mammalian cells showed that they function as largely pH-independent low-affinity uniporters with both uptake and efflux activity (Chen et al., 2010, 2012). In particular, clade I and II SWEETs transport monosaccharides and clade III SWEETs transport disaccharides, mainly Suc (Chen et al., 2010, 2012). Mutant phenotypes and developmental expression of several SWEET transporters support important roles in sugar translocation between organs. The clade III SWEETs, in particular SWEET11 and 12, mediate the key step of Suc efflux from phloem parenchyma cells for phloem translocation (Chen et al., 2012). Moreover, SWEETs are coopted by pathogens, likely to provide energy resources and carbon at the site of infection (Chen et al., 2010). Mutations of SWEET8/Ruptured pollen grain1 in Arabidopsis, and RNA inhibition of OsSWEET11 (also called Os8N3 or Xa13) in rice, and petunia (Petunia hybrida) NEC1 resulted in male sterility (Ge et al., 2001; Yang et al., 2006; Guan et al., 2008), possibly caused by inhibiting the Glc supply to developing pollen (Guan et al., 2008). Interestingly, two members, SWEET16 and SWEET17, of the family localize to the tonoplast (Chardon et al., 2013; Klemens et al., 2013). Allelic variation or mutations that affect SWEET17 expression caused Fru accumulation in Arabidopsis leaves, indicating that it plays a key role in exporting Fru from leaf vacuoles (Chardon et al., 2013). A more recent study demonstrated that SWEET16 also functions as a vacuolar sugar transporter (Klemens et al., 2013). Surprisingly, however, SWEET17 expression in mature leaves was comparatively low (Chardon et al., 2013), which leads us to ask whether SWEET17 could mainly function in other tissues under specific developmental or environmental conditions. Although Arabidopsis SWEET17 has been shown to transport Fru in a heterologous system where it accumulated in part at the plasma membrane (Chardon et al., 2013), the biochemical properties of SWEET17 were still elusive. SWEET16 and SWEET17 from Arabidopsis belong to the clade IV SWEETs. Whether clade IV proteins both transport vacuolar sugars in planta deserves further studies.Here, we used GUS/GFP fusions to reveal the root-dominant expression and vacuolar localization of the SWEET17 protein in vivo and its regulation by Fru levels. Phenotypes of mutants and overexpressors were consistent with a role of SWEET17 in bidirectional Fru transport across root vacuoles. The uniport feature of SWEET17 transport was further confirmed using isolated mesophyll vacuoles. Similarly, SWEET16 is also shown to function in vacuolar sugar transport in roots. Our work, performed in parallel to the two other studies (Chardon et al., 2013; Klemens et al., 2013), provides direct evidence for Fru uniport by SWEET17 and presents functional analyses to uncover important roles of these vacuolar transporters in maintaining intracellular Fru homeostasis in roots.