SWEET17, a Facilitative Transporter, Mediates …SWEET17, a Facilitative Transporter, Mediates...

SWEET17, a Facilitative Transporter, Mediates FructoseTransport across the Tonoplast of ArabidopsisRoots and Leaves1[W][OPEN]

Woei-Jiun Guo*, Reka Nagy2, Hsin-Yi Chen, Stefanie Pfrunder, Ya-Chi Yu, Diana Santelia,Wolf B. Frommer*, and Enrico Martinoia

Institute of Tropical Plant Sciences, National Cheng Kung University, Tainan City 7013, Taiwan (W.-J.G., H.-Y.C.,Y.-C.Y.); Department of Plant Biology, Carnegie Institution for Science, Stanford, California 94305 (W.-J.G., W.B.F.);and Institute of Plant Biology, University of Zürich, CH–8008 Zurich, Switzerland (R.N., S.P., D.S., E.M.)

Fructose (Fru) is a major storage form of sugars found in vacuoles, yet the molecular regulation of vacuolar Fru transport is poorlystudied. Although SWEET17 (for SUGARS WILL EVENTUALLY BE EXPORTED TRANSPORTERS17) has been characterized as avacuolar Fru exporter in leaves, its expression in leaves is low. Here, RNA analysis and SWEET17-b-glucuronidase/-GREENFLUORESCENT PROTEIN fusions expressed in Arabidopsis (Arabidopsis thaliana) reveal that SWEET17 is highly expressed inthe 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 toincreased and decreased root growth in the presence of Fru, respectively. Overexpression of SWEET17 specifically reduced the Frucontent in leaves by 80% during cold stress. These results intimate that SWEET17 functions as a Fru-specific uniporter on the roottonoplast. Vacuoles overexpressing SWEET17 showed increased [14C]Fru uptake compared with the wild type. SWEET17-mediatedFru uptake was insensitive to ATP or treatment with NH4Cl or carbonyl cyanide m-chlorophenyl hydrazone, indicating thatSWEET17 functions as an energy-independent facilitative carrier. The Arabidopsis genome contains a close paralog of SWEET17in clade IV, SWEET16. The predominant expression of SWEET16 in root vacuoles and reduced root growth of mutants under Fruexcess 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 tomaintain cytosolic Fru homeostasis.

Sugars are main energy sources for generating ATP,major precursors to various storage carbohydrates aswell as key signaling molecules important for normalgrowth in higher plants (Rolland et al., 2006). Depending

on the metabolic demand, sugars are translocated overlong distances or stored locally. SWEET (for SUGARSWILL EVENTUALLY BE EXPORTED TRANSPORTERS)and SUC/SUT (for Sucrose transporter/Sugar trans-porter)-type transporters are responsible for transfer ofSuc from the phloem parenchyma into the sieve elementcompanion cell complex for long-distance translocation(Riesmeier et al., 1992; Sauer, 2007; Kühn and Grof,2010; Chen et al., 2012). Suc or hexoses derived fromSuc hydrolysis in the cell wall are then taken up into sinkcells by SUT (Braun and Slewinski, 2009) or monosac-charide transporters, such as sugar transporter1 (Saueret al., 1990; Pego and Smeekens, 2000; Sherson et al., 2003).Alternatively, sugars are thought to move between cellsvia plasmodesmata (Voitsekhovskaja et al., 2006; Ayre,2011). Major sugar storage pools within plant cells aresoluble 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 rolesin temporary and long-term storage of soluble sugars(Martinoia et al., 2007; Etxeberria et al., 2012). Someagriculturally important crops such as sugar beet (Betavulgaris; Leigh, 1984; Getz and Klein, 1995), citrus (Citrusspp.; Echeverria and Valich, 1988), sugarcane (Saccharumofficinarum; Thom et al., 1982), and carrot (Daucus carota;Keller, 1988) can store considerable amounts (.10% of

1 This work was supported by the Department of Energy (grantno. DE–FG02–04ER15542 to W.B.F.), the National Science Council,Taiwan (grant no. NSC–101–2313–B–006–001–MY3 to W.-J.G.), theSwiss National Foundation (grant no. Nr3100A0–117790/1 to E.M.),and a fellowship from the Swiss National Foundation Marie Heim-Voegtlin (grant_PMPDP3_139645 to D.S.).

2 Present address: Promega, Wallisellenstrasse 55, 8600 Duben-dorf, 8006 Zurich, Switzerland.

* Address correspondence to [email protected] [email protected].

The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Woei-Jiun Guo ([email protected]).

W.-J.G., R.N., and H.-Y.C. performedmost of the experiments; S.P.and Y.-C.Y. provided technical assistance to R.N. and H.-Y.C.; D.S.performed carbohydrate analysis; W.-J.G. generated all constructsand transgenic plants; W.-J.G., R.N., W.B.F., and E.M. designed ex-periments; all authors analyzed data. W.-J.G., W.B.F., and E.M. con-ceived of the project and wrote the paper (with input from D.S.).

[W] The online version of this article contains Web-only data.[OPEN] Articles can be viewed online without a subscription.www.plantphysiol.org/cgi/doi/10.1104/pp.113.232751

Plant Physiology�, February 2014, Vol. 164, pp. 777–789, www.plantphysiol.org � 2013 American Society of Plant Biologists. All Rights Reserved. 777 www.plantphysiol.orgon April 17, 2020 - Published by Downloaded from

Copyright © 2014 American Society of Plant Biologists. All rights reserved.

mailto:[email protected]


http://www.plantphysiol.org


http://www.plantphysiol.org/cgi/doi/10.1104/pp.113.232751


plant dry weight) of Suc, Glc, or Fru in vacuoles of thestorage parenchyma. Due to a high capacity of vacuolesfor storing sugars, vacuolar sugars can serve as an im-portant carbohydrate source during energy starvation,e.g. after starch has been exhausted (Echeverria andValich, 1988), as well as for the production of othercompounds (e.g. osmoprotectants). Sugars are knownto regulate photosynthesis; therefore, the release of sugarsfrom vacuoles could be important for modulatingphotosynthesis (Kaiser and Heber, 1984). Moreover,vacuole-derived sugars are commercially used to producebiofuels, such as ethanol, from sugarcane. Knowledge ofthe key transporters involved in sugar exchange betweenthe vacuole and cytoplasm is thus relevant in the contextof bioenergy (Grennan and Gragg, 2009).

To facilitate the exchange of sugars across the tono-plast, plant vacuoles are equipped with a multitude oftransporters (Neuhaus, 2007; Etxeberria et al., 2012;Martinoia et al., 2012) comprising both facilitated dif-fusion and active transport systems of vacuolar sugars(Martinoia et al., 2000). Typically, Suc is actively im-ported into vacuoles by tonoplast monosaccharidetransporter (AtTMT1/AtTMT2; Schulz et al., 2011) andexported by the SUT4 family (AtSUC4, OsSUT2; Eomet al., 2011; Payyavula et al., 2011; Schulz et al., 2011).Two H+-dependent sugar antiporters, the vacuolar Glctransporter (AtVGT1; Aluri and Büttner, 2007) andAtTMT1 (Wormit et al., 2006), mediate Glc uptakeacross the tonoplast to promote carbohydrate accumula-tion in Arabidopsis (Arabidopsis thaliana). The Early Re-sponsive to Dehydration-Like6 protein has been shownto export vacuolar Glc into the cytosol (Poschet et al.,2011), likely via an energy-independent diffusionmechanism (Yamada et al., 2010). Defects in these vac-uolar sugar transporters alter carbohydrate partitioningand allocation and inhibit plant growth and seed yield(Aluri and Büttner, 2007; Wingenter et al., 2010; Eomet al., 2011; Poschet et al., 2011).

In contrast to numerous studies on vacuolar transportof Suc and Glc, limited efforts have been devoted to themolecular mechanism of vacuolar Fru transport eventhough Fru is predominantly located in vacuoles(Martinoia et al., 1987; Voitsekhovskaja et al., 2006; Tohgeet al., 2011). Vacuolar Fru is important for the regulationof turgor pressure (Pontis, 1989), antioxidative defense(Bogdanovi�c et al., 2008), and signal transduction duringearly seedling development (Cho and Yoo, 2011; Li et al.,2011). Thus, control of Fru transport across the tonoplastis thought to be important for plant growth and devel-opment. One vacuolar Glc transporter from the Arabi-dopsis monosaccharide transporter family, VGT1, hasbeen reported to mediate low-affinity Fru uptake whenexpressed in yeast (Saccharomyces cerevisiae) vacuoles(Aluri and Büttner, 2007). Yet, the high vacuolar uptakeactivity to Fru intimates the existence of additional high-capacity Fru-specific vacuolar transporters (Thom et al.,1982). Recently, quantitative mapping of a quantitativetrait locus for Fru content of leaves led to the identifi-cation 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 mem-bers in Arabidopsis and 21 in rice (Oryza sativa; Chenet al., 2010; Frommer et al., 2013; Xuan et al., 2013). Basedon homology with 27% to 80% amino acid identity, plantSWEET proteins were grouped into four subclades (Chenet al., 2010). Analysis of GFP fusions indicated that mostSWEET transporters are plasma membrane localized.Transport assays using radiotracers in Xenopus laevisoocytes and sugar nanosensors in mammalian cellsshowed that they function as largely pH-independentlow-affinity uniporters with both uptake and effluxactivity (Chen et al., 2010, 2012). In particular, clade Iand II SWEETs transport monosaccharides and cladeIII SWEETs transport disaccharides, mainly Suc (Chenet al., 2010, 2012). Mutant phenotypes and developmentalexpression of several SWEET transporters support im-portant roles in sugar translocation between organs. Theclade III SWEETs, in particular SWEET11 and 12, mediatethe key step of Suc efflux from phloem parenchyma cellsfor phloem translocation (Chen et al., 2012). Moreover,SWEETs are coopted by pathogens, likely to provideenergy resources and carbon at the site of infection(Chen et al., 2010). Mutations of SWEET8/Rupturedpollen grain1 in Arabidopsis, and RNA inhibition ofOsSWEET11 (also called Os8N3 or Xa13) in rice, andpetunia (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 develop-ing pollen (Guan et al., 2008). Interestingly, two members,SWEET16 and SWEET17, of the family localize to thetonoplast (Chardon et al., 2013; Klemens et al., 2013).Allelic variation or mutations that affect SWEET17expression caused Fru accumulation in Arabidopsisleaves, indicating that it plays a key role in exportingFru from leaf vacuoles (Chardon et al., 2013). A morerecent study demonstrated that SWEET16 also functionsas a vacuolar sugar transporter (Klemens et al., 2013).Surprisingly, however, SWEET17 expression in matureleaves was comparatively low (Chardon et al., 2013),which leads us to ask whether SWEET17 could mainlyfunction in other tissues under specific developmentalor environmental conditions. Although ArabidopsisSWEET17 has been shown to transport Fru in a het-erologous system where it accumulated in part at theplasma membrane (Chardon et al., 2013), the biochemi-cal properties of SWEET17 were still elusive. SWEET16and SWEET17 from Arabidopsis belong to the clade IVSWEETs. Whether clade IV proteins both transportvacuolar sugars in planta deserves further studies.

Here, we used GUS/GFP fusions to reveal the root-dominant expression and vacuolar localization of theSWEET17 protein in vivo and its regulation by Frulevels. Phenotypes of mutants and overexpressors wereconsistent with a role of SWEET17 in bidirectional Frutransport across root vacuoles. The uniport feature ofSWEET17 transport was further confirmed using isolatedmesophyll vacuoles. Similarly, SWEET16 is also shown tofunction in vacuolar sugar transport in roots. Our work,performed in parallel to the two other studies (Chardon

778 Plant Physiol. Vol. 164, 2014

Guo et al.

www.plantphysiol.orgon April 17, 2020 - Published by Downloaded from Copyright © 2014 American Society of Plant Biologists. All rights reserved.


et al., 2013; Klemens et al., 2013), provides direct evidencefor Fru uniport by SWEET17 and presents functionalanalyses to uncover important roles of these vacuolartransporters in maintaining intracellular Fru homeostasisin roots.

RESULTS

SWEET17 Proteins Are Highly Expressed in Roots

A very recent report had indicated that SWEET17(At4g15920) functions as a Fru exporter in leaf vacuoles.However, SWEET17 expression appeared to be very lowin leaves (Chardon et al., 2013), indicating that SWEET17may predominantly function in sink organs other thanleaves. A quantitative reverse transcription (qRT)-PCRanalysis revealed that SWEET17 mRNA was expressedto high levels in roots of 2-week-old seedlings (Fig. 1). Insoil-grown mature plants, some aerial organs, i.e. stems,flowers, and siliques, also accumulated high levels ofSWEET17 transcripts. By contrast, expression of SWEET17was comparatively low in both young and matureleaves (Fig. 1). The high levels of SWEET17 transcriptsin roots observed here correlatedwell with the steady-stateexpression profile from the Arabidopsis eFP Browser(Supplemental Fig. S1A; Winter et al., 2007) and theTranslatome database (polysome-bound mRNA;Mustroph et al., 2009; Supplemental Fig. S1B). Becausesteady-state mRNA levels do not necessarily reflectprotein abundance (Krügel and Kühn, 2013), transla-tional fusions were analyzed. We generated transgenicArabidopsis plants expressing a C-terminal translationalGUS gene fusion of SWEET17 driven by the nativeSWEET17 promoter (SWEET17-GUS). In particular, the

full length of SWEET17 gene containing all introns wasused to observe the abundance and localization of theprotein in planta. In 7-d-old transgenic seedlings,SWEET17-GUS fusion proteins were mainly found incotyledons and roots (Fig. 2A). A similar expression pat-tern was also observed in 2-week-old seedlings (Fig. 2B),where, however, GUS activity was much reduced inaerial tissues. The expression pattern of SWEET17proteins was also consistent with the expression patternanalyzed by plants expressing the GUS reporter drivenby the SWEET17 promoter (Supplemental Fig. S1C). Inroots, SWEET17 was predominantly expressed in roottips (Fig. 2C) and mature regions of roots (Fig. 2D),while only low expression was observed in the elonga-tion zone of roots (Fig. 2C). Three independent reporterlines showed comparable patterns of GUS staining (datanot shown). Hand sections of mature roots histochemi-cally stained for GUS activity further demonstrated thatSWEET17 predominantly accumulated in the root cortex(Fig. 2E). The cell type-specific expression was com-parable with that of root array data from the Arabi-dopsis eFP Browser (Supplemental Fig. S2, A and B)and the Translatome database (Supplemental Fig. S2C).In soil-grown mature plants, expression of SWEET17-GUS was consistently observed to be high in roots andlow in aerial tissues, such as leaves, stems, and flowers(Supplemental Fig. S3A). When the reaction time ofGUS staining was doubled (to 4 h), low and patchyexpression of the SWEET17-GUS fusion proteins wasobserved in mature leaves (Fig. 2F). After extendedstaining, some GUS activity was also observed in thevascular tissues of flowers (Fig. 2G) and the bottom ofsiliques (Fig. 2H) but not in seeds (Fig. 2I).

Expression of SWEET17 Is Regulated by Fru in Roots

Because expression of some sugar transporters ismodulated by sugar levels (Williams et al., 2000), weinvestigated if sugars would affect the spatial patternof expression of SWEET17. Transgenic Arabidopsisseedlings expressing SWEET17-GUS fusion proteins weregrown on media supplemented with 1% (w/v) Suc for5 d then transferred to media without or with 1% (w/v)Suc, Glc, or Fru for 2 d. Histochemical staining for GUSactivity showed that SWEET17 accumulation was similarin the presence of Suc or Glc (Supplemental Fig. S3B) buthighly induced by 1% (w/v) Fru in the elongation zone ofroots (Fig. 2, J and K). To test whether SWEET17 ex-pression responds to a low intracellular sugar status,seedlings expressing SWEET17-GUS fusion proteinswere grown on media supplemented with 1% (w/v) Sucunder 16-h daylength for 5 d then transferred to sugar-free media in the dark for an additional 2 d. Extendeddark periods lead to sugar starvation (Usadel et al.,2008). Interestingly, accumulation of SWEET17-GUSfusion proteins in the elongation zone of roots wasalso highly induced in darkness (Fig. 2, J and L). The sameFru- and sugar starvation-inducible patterns in roots wereobserved at the transcriptional level using SWEET17

Figure 1. Developmental expression profile of the SWEET17 gene inArabidopsis. Total RNA was isolated from roots (R) and shoots (S) of2-week-old seedlings or young leaves (YL), mature leaves (ML), stems(St), flowers (FL), and siliques (Si) of 7- to 8-week-old soil-grown plants.cDNA was used for qRT-PCR with specific primers for SWEET16 andSWEET17. The y axis indicates the relative expression level normalizedto an internal control, Actin2. The results are means 6 SE from threeindependent biological repeats.

Plant Physiol. Vol. 164, 2014 779

Fructose-Specific Transporter on the Root Tonoplast


http://www.plantphysiol.org/cgi/content/full/pp.113.232751/DC1








promoter:GUS fusions (Supplemental Fig. S3C). Weakinduction of SWEET17-GUS expression in leaves wasalso observed upon exposure to darkness or by com-bining darkness with cold stress (Supplemental Fig. S4).These observations are consistent with a predicted roleof SWEET17 as a Fru uniporter for uptake of excesscytosolic Fru or for release of the stored Fru across thevacuolar membrane to maintain Fru homeostasis.

SWEET17 Proteins in Root Vacuoles

To investigate whether SWEET17 is targeted to thetonoplast also in roots, we generated transgenic Arabi-dopsis plants expressing a C-terminal translational GFPfusion of SWEET17 (SWEET17-GFP) driven by the nativeSWEET17 promoter. To allow possible transcriptionalregulation, the full-length genomic SWEET17 gene wasused. Confocal images of intact roots from homozygoustransformants revealed that the fluorescence derivedfrom SWEET17-GFP fusion proteins was predominantlypresent at the tonoplast of root tip cells (Fig. 3A). Vacuolarlocalization was evidenced by fluorescence surroundingsmall premature vacuoles located inside the plasmamembrane that also stained with FM4-64 (Fig. 3, B and C;Wayne, 2009). A tonoplast localization was also observedin mature regions of roots as shown by fluorescencelining the inner side of the nucleus and plasma mem-brane (Fig. 3, D–F; Wayne, 2009). The fluorescencederived from SWEET17-GFP fusion proteins in the cyto-sol or at the plasma membrane was absent. As expected,tonoplast-specific localization of SWEET17-GFP was alsoobserved in mesophyll protoplasts (Supplemental Fig. S5).Despite overall low levels of fluorescence in leaf tissues,

SWEET17-GFP fluorescence in Arabidopsis protoplastswas clearly lining chloroplast autofluorescence towardthe vacuole (Supplemental Fig. S5A) and not detected atthe plasma membrane (Supplemental Fig. S5B). Theseobservations may indicate that SWEET17 locates to andfunctions predominantly at the tonoplast of roots.

Import Activity of SWEET17 Confers Tolerance toFru Inhibition

High levels of cytosolic Fru inhibit root growth andarrest seedling development (Bhagyalakshmi et al., 2004;Cho and Yoo, 2011). If SWEET17 could act as a vacuolarimporter to store excess Fru in roots, as suggested byexpression patterns (Figs. 2 and 3), altered expression ofSWEET17 could affect intracellular Fru partitioning andthereby affect the sensitivity of roots to Fru toxicity. Totest this hypothesis, Arabidopsis knockout mutantssweet17-1 and sweet17-2 with transfer DNA (T-DNA)insertions in the SWEET17 gene were obtained. Whilethe relative root growth of the segregating wild type(ecotype Columbia; Col-TDNA) and mutants (sweet17-1and sweet17-2) was similar when grown on media withor without 1% (w/v) Suc, root growth of sweet17-1 andsweet17-2 seedlings was significantly more sensitive to1% (w/v) to 2% (w/v) Fru compared with the wildtype (Fig. 4A). SWEET17 thus appears to contribute, atleast partially, to Fru tolerance in roots. We thereforetested whether SWEET17 is also sufficient for Fru toler-ance. We generated transgenic Arabidopsis plants over-expressing the full-length genomic SWEET17 gene drivenby the constitutive Cauliflower mosaic virus 35S promoter.In 7-d-old seedlings, reverse transcription (RT)-PCR

Figure 2. Expression patterns of SWEET17in Arabidopsis. Histochemical analysis ofGUS activity in transgenic Arabidopsisexpressing SWEET17-GUS fusion proteinsdriven by the SWEET17 native promoter.A, Seven-day-old seedlings. B, Two-week-old plate-grown plants. C and D, Root tipand mature zone of 7-d-old seedlings.E, Hand section of D. F, Mature rosetteleaf. G, Flower. H, Silique. I, Seed. Effectsof Fru or darkness on the expression ofSWEET17-GUS are also demonstrated.Five-day-old seedlings were treatedwith no sugar (J), 1% (w/v) Fru (K), andno sugar in the dark (L) for 2 d, and thenthe histochemical staining was per-formed. The reaction time for GUSstaining was 2 h for seedlings and allroot tissues (A–E and J–L) and 4 h forleaves, flowers, siliques, and seeds (F–I).Bars = 1 mm (A, B, F, and G), 0.5 mm(H), and 100 mm (C–E and I–L).


Guo et al.








analysis showed that levels of SWEET17 transcripts inhomozygous overexpressing lines 35S:SWEET17-1,35S:SWEET17-6, and 35S:SWEET17-2 were highly in-creased compared with plants transformed with theempty vector (Col-Vector; Supplemental Fig. S6A). Incontrast to the increased sensitivity of root growth insweet17 mutants (Fig. 4A), three independent over-expressor lines (35S:SWEET17-1, 35S:SWEET17-6, and35S:SWEET17-2) showed enhanced tolerance comparedwith wild-type plants (Col-Vector-1 and Col-Vector-2)in the presence of 0.1% to 1% (w/v) Fru (Fig. 4B). Excesssugars are known to inhibit seed germination (Dekkerset al., 2004). To examine if SWEET17 also contributes toFru translocation during early seedling development,we compared germination rates of mutants andoverexpressors subjected to excess Fru. However, after2 to 4 d of incubation, we did not observe significantdifferences between the overexpressor or mutant linesand the wild type (Supplemental Fig. S6, B and C; datanot shown).

Overexpression of SWEET17 Decreases Fru Accumulationin Leaves

The induction of SWEET17 expression by energystarvation (Fig. 2, J and L) indicates that SWEET17 maybe able not only to import, but also to export Fru storedin the vacuole into the cytosol when there is a metabolicdemand. To examine how the increased export activityof SWEET17 proteins affects the capacity of vacuoles tostore sugars, we determined the sugar content of leavesof SWEET17 overexpressors under standard growthconditions and in response to cold stress (4°C) for1 week. Cold stress had been shown to induce Fruaccumulation in vacuoles by 2- to 10-fold within 24 h(Wormit et al., 2006). Under standard conditions, nodramatic differences in leaf sugar levels were observedwhen comparing the wild types and overexpressors(Supplemental Fig. S7A). However, under cold stressconditions, the Fru content of leaves of 35S:SWEET17-1

overexpressor plants was significantly reduced by80% compared with the wild type transformed withthe empty vector (Col-Vector; Fig. 5). No significantdifferences in levels of Glc, Suc, and other sugarswere observed in cold-stressed overexpressors (Fig. 5;Supplemental Fig. S7B). These results provide func-tional evidence that SWEET17 is involved in vacuolarFru export in planta.

Direct Evidence for a Role of SWEET17 as a Low-AffinityTonoplast Fru Uniporter

The sensitivity of root growth to Fru inhibition insweet17 mutants (Fig. 4A) and the decreased Fru con-tent in cold-stressed SWEET17 overexpressors (Fig. 5)indicated that SWEET17 mediates bidirectional Fru-specific transport in vivo. To directly measure the trans-port properties of SWEET17, leaf vacuoles were isolatedfrom overexpressor plants grown under standardconditions and used to perform time course uptake assayswith [14C]Suc, [14C]Glc, and [14C]Fru. Vacuoles isolatedfrom 35S:SWEET17-1 and 35S:SWEET17-6 mesophyllprotoplasts exhibited enhanced time-dependent Fruuptake activity comparedwith those isolated from controlleaves (Col-Vector; Fig. 6A). By comparison, the uptakeactivities for Glc or Suc were not significantly increased(Supplemental Fig. S8). These results show that SWEET17can import Fru specifically into vacuoles along a con-centration gradient in vivo.

To confirm if SWEET17 acts as a uniporter as indicatedfrom phenotypes (Figs. 4 and 5), we examined the effectof ATP, the uncoupler NH4Cl (5 mM;Wormit et al., 2006),or a protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP, 10 mM; Eom et al., 2011), on Fruuptake activity mediated by SWEET17 (Fig. 6B). After anincubation time of 20 min in the presence of 0.2 mM [14C]Fru and Mg-ATP, vacuoles from plants overexpressingSWEET17 had taken up 2.5 times more Fru comparedwith those expressing the empty vector (Fig. 6B). Theabsence of ATP as well as treatments of NH4Cl or CCCP,

Figure 3. Tonoplast localization of theSWEET17-GFP fusion protein in Arabidopsisroots. Fluorescence of GFP fusion proteins in7-d-old seedlings was imaged by confocalmicroscopy. A to C, root tips. D to F, matureroots. The GFP fluorescence images (A andD), the FM4-64 staining (6 mM for 5 min) tolabel the plasma membrane (B and E), andthe merged images (C and F) of the samecells are shown. The arrowheads point to theSWEET17-GFP-labeled vacuole membraneslining the inside of the nucleus. Bar = 10 mm.










which disrupt the preexisting proton gradient, had nosignificant impact on transport activity (Fig. 6B).

To investigate the affinity of the vacuolar Fru trans-porter, we performed a concentration-dependent uptakeexperiment. Saturation of Fru uptake was not observedat concentrations up to 20 mM that were used here (Fig.6C). The estimated Km and Vmax values of SWEET17activity were 25.6 mM and 479.8 pmol Fru mL vacuole–1

min–1, respectively, demonstrating that SWEET17 is alow-affinity Fru transporter.

Both Clade IV SWEETs Function as Vacuolar Transportersin Roots

The Arabidopsis genome contains a close homologof SWEET17 in clade IV, named SWEET16. In a recentstudy, SWEET16 (At3g16690) was also shown to function

as a vacuolar sugar facilitator in vascular parenchymacells (Klemens et al., 2013). However, the expression ofSWEET16, measured using a promoter-GUS fusion, isrelatively low in most tissues, including roots (Klemenset al., 2013). Because SWEET16 and SWEET17 proteinsshare 70% amino acid identity (Supplemental Fig. S9A),we suspected that SWEET16 may play a partially re-dundant function with SWEET17 in roots under cer-tain environmental stimuli. To address this hypothesis,we performed similar experiments as for SWEET17.Interestingly, the qRT-PCR analysis showed predomi-nant expression of SWEET16 mRNA in roots comparedwith all aerial organs, such as leaves, stems, and flowers,in both young seedlings and mature plants (Fig. 1). Theexpression pattern was consistent with the develop-mental expression profile from the Arabidopsis eFPBrowser (Supplemental Fig. S1A) and the Translatomedatabase (Supplemental Fig. S1B). To investigate ifSWEET17 and SWEET16 are the only SWEET geneshighly expressed in roots, we performed qRT-PCR toanalyze expression levels of all 17 Arabidopsis SWEETgenes in roots. Under the condition tested, SWEET17and SWEET16 genes, which are part of clade IV (Chenet al., 2010), were the most highly expressed SWEETgenes; a few other SWEET genes were expressed at lowlevels in roots (Fig. 7). The pattern was consistent withthe expression profile derived from the AtGeneExpressdatabase (Supplemental Fig. S9B).

To analyze the tissue-specific expression of SWEET16proteins, we generated transgenic Arabidopsis expressingC-terminal translational fusions to GUS and GFP underthe control of the native SWEET16 promoter (SWEET16-GUS/GFP), again using the full-length genomic SWEET16gene. Similar to SWEET17, both SWEET16-GUS fusionproteins and promoter activity were mainly detected inroots (Supplemental Fig. S10, A and B). SWEET16 fusions

Figure 4. Altered expression of SWEET17 affects sensitivity of rootgrowth to excess Fru. Arabidopsis seeds were germinated and grown onmedia supplemented with 1% (w/v) Suc for 5 d, and then seedlings weretransferred to media containing various concentrations of Fru (w/v).Relative growth of primary roots was measured after 6 d of treatments.A, Comparison of root growth in two independent sweet17mutant linesand the wild type that was identified from the segregating mutant pop-ulation (Col-TDNA). B, Comparison of root growth in three independentSWEET17 overexpressor lines (35S:SWEET17-1, 35S:SWEET17-6, and35S:SWEET17-2) and the wild type that was transformed with theempty vector (Col-Vector-1 and Col-Vector-2). The data presented aremeans 6 SE (n = 8). Significant differences from the wild type were deter-mined by Student’s t test indicated by asterisks (*P , 0.05, **P , 0.01).

Figure 5. Ectopic expression of SWEET17 reduces Fru accumulation inleaves under cold stress conditions. Sugar contents in leaves of trans-genic Arabidopsis expressing a 35S:SWEET17 transgene were shown.Samples were harvested from 6-week-old soil-grown plants treatedwith cold stress (4˚C) for 1 week. Results are means 6 SE (n = 9).Significant differences from the wild type transformed with the emptyvector (Col-Vector) or identified from the segregating mutant popula-tion (Col-TDNA) were determined by Student’s t test indicated by as-terisks (**P , 0.01).


Guo et al.








accumulated predominantly in the cortex of matureroots (Supplemental Fig. S10, C and D). The cell type-specific expression was consistent with the ArabidopsiseFP Browser (Supplemental Fig. S2, A and B) and theTranslatome database (Supplemental Fig. S2C). Confo-cal images of intact roots in homozygous transformedseedlings clearly demonstrated that the fluorescence ofSWEET16-GFP fusion proteins localized to the vacuolarmembrane in roots (Supplemental Fig. S10, E–G). Tofunctionally characterize SWEET16 in planta, we obtainedArabidopsis sweet16 T-DNA insertion knockout mutants(Supplemental Fig. S11A) and generated a sweet17-1/sweet16-1 double mutant (Supplemental Fig. S11B) aswell as SWEET16 overexpressor lines (SupplementalFig. S11C). Vacuoles isolated from sweet17-1/sweet16-1showed a marked decrease in Fru uptake activity(Supplemental Fig. S12A). Although overexpression ofSWEET16 in leaves did not increase the Fru transportactivity (Supplemental Fig. S12B), the Fru content inleaves was significantly decreased in response to coldstress (Supplemental Fig. S7B) compared with standardconditions (Supplemental Fig. S7A). These results implythat SWEET16 is probably involved in Fru transport inplanta, although, under the conditions tested, to a muchlower degree than SWEET17. Consistent with this obser-vation, root growth of sweet16-1 and sweet16-2 seedlingswas significantly more sensitive to 0.5% to 2% (w/v) ofFru than the wild type (Fig. 8A). However, comparedwith sweet16 single mutants, the sweet17-1/sweet16-1double mutant did not show enhanced sensitivity to ex-cess Fru. Overexpression of SWEET16 significantly en-hanced root growth at 0.5% to 2% (w/v) of Fru (Fig. 8B).

DISCUSSION

SWEET17 Function at the Root Tonoplast

Tight regulation of sugar compartmentation betweenthe cytosol and vacuole is important for proper plantdevelopment (Martinoia et al., 2000). A quantitative trait

Figure 7. Expression of Arabidopsis SWEET genes in roots. Total RNAwasisolated from roots of 23-d-old liquid-cultured seedlings, and the cDNAwasused for qRT-PCR with specific primers for each SWEET gene. The y axisindicates the relative expression level normalized to the internal controlActin2. The results are means6 SE from four independent biological repeats.

Figure 6. The transport activity of Arabidopsis SWEET17 in vivo.A, Time course of Fru uptake into mesophyll vacuoles isolated fromplants expressing the empty vector (Col-Vector) or the 35S:SWEET17transgene. Vacuoles were incubated with 0.2 mM of [14C]-labeled Fru.Results are means 6 SE (n = 3–4). Significant differences from vacuolesexpressing the vector were determined. B, Effector analysis of Fruuptake into vacuoles isolated from plants expressing the empty vectoror the 35:SWEET17 transgene. The uptake level under treatments of noATP, 5 mM NH4Cl, and 10 mM CCCP was determined. Results aremeans 6 SE (n = 3–4). Significant differences from vacuoles expressingthe 35:SWEET17 and incubated with 4 mM ATP were determined.C, Kinetic analysis of Fru uptake activity in vacuoles isolated fromplants overexpressing SWEET17. Results are means 6 SD (n = 4). InB and C, the uptake level was determined after 3 to 20 min of incu-bation in 0.2 mM (B) or indicated (C) [14C]-labeled Fru. Significantdifferences were determined by Student’s t test indicated by asterisks(**P , 0.01).

















loci analysis identified SWEET17 as a key player incontrolling leaf Fru content and provided evidencethat SWEET17 localizes to the vacuole. However,based on promoter-GUS fusions, SWEET17 expressionwas restricted to the main vein of the leaf and wasfound only in the basal part of the leaf (Chardon et al.,2013). Using qRT-PCR analysis and a whole-geneSWEET17-GUS translational fusion, we show thatSWEET17 is highly expressed in roots and confirm thatit is only present at low levels in leaves at all stagesanalyzed (Figs. 1 and 2; Supplemental Fig. S3A). Thetrend correlated well with results from microarray andtranslatome databases (Supplemental Fig. S1, A andB). In contrast to high levels of steady-state SWEET17transcripts detected in stems, flowers, and siliques(Fig. 1; Supplemental Fig. S1A), only very low levels ofSWEET17 proteins (analyzed by a GUS fusion) wereobserved in those tissues (Fig. 2, F–H; Supplemental

Fig. S3A). Organ-specific posttranslational regulationmay modulate SWEET17 function, as been observedfor other sugar transporters (Krügel and Kühn, 2013).The discrepancy between our observations using awhole-gene construct and the previous report using apromoter-GUS construct (Chardon et al., 2013) may bedue to intragenic regulatory elements within introns orexons that regulate gene expression. Intron contribu-tion to expression has been widely observed and wasalso reported for the Arabidopsis Suc transportersSUC1 and SUC9 (Sivitz et al., 2007) as well as somenutrient transporters such as Ammonium transporter1(Yuan et al., 2007) and Nitrate transporter2 (Laugier et al.,2012). Further studies will be required to uncover theregulatory mechanism. Nevertheless, these observationspoint out that expression of SWEET17 is tightly regulatedto cope with the dynamics of cellular sugar homeo-stasis (Fig. 2, J–L; Supplemental Figs. S3C and S4). Thetonoplast-specific localization of SWEET17-GFP in Arabi-dopsis roots and leaves (Fig. 3; Supplemental Fig. S5)provided clear evidence for its role on vacuoles assuggested by transient ectopic expression of SWEET17complementary DNA (cDNA)-GFP fusions in proto-plasts (Chardon et al., 2013). Taken together, these ob-servations point to a major role of SWEET17 on the roottonoplast.

Accumulation of SWEET17 is predominantly foundin the cortex cells of mature roots (Fig. 2, D and E;Supplemental Fig. S2, B and C). The cortex cells mainlyserve as a storage pool of carbohydrates and metabolitesrelated to biotic and abiotic stresses (Hassan andMathesius, 2012). In some plant species, the prime lo-cation of pathogen or symbiotic bacterial infection inroots occurs through the cortex (Czajkowski et al., 2010).A previous study had shown that OsSWEET11 ex-pressed in rice leaves can be hijacked by pathogens toexport sugars for their energy supply during infection(Chen et al., 2010). Several SWEET family members arealso highly induced by pathogen infection. WhetherSWEET17 is involved in releasing stored vacuolarsugars in root cortex cells during interaction with rootmicroorganisms will be a very interesting aspect toinvestigate in the future.

SWEET17 Regulates Fru Homeostasis in Roots

Given that most of Fru is stored in vacuoles(Voitsekhovskaja et al., 2006; Tohge et al., 2011), vacuolarSWEET17 is probably involved in importing excesscytosolic Fru into vacuoles for storage in roots aftersugars are unloaded from the phloem stream and hy-drolyzed by invertases. Several observations supportthis notion. In the presence of externally supplied Fru,which activates Fru accumulation in vacuoles, expressionof SWEET17 was highly induced in the elongation zoneof roots (Fig. 1, J and K), where active sugar uptake fromthe outside of roots occurs and most of Fru accumulates(Jones and Darrah, 1996). Root growth was signifi-cantly reduced in sweet17 mutant plants under excess

Figure 8. Altered expression of SWEET16 affects sensitivity of rootgrowth to excess Fru. Arabidopsis seeds were germinated and grown onmedia supplemented with 1% (w/v) Suc for 5 d, and then seedlings weretransferred to media containing various concentrations of Fru (w/v).Relative growth of primary roots were measured after 6 d of treatments.A, Comparison of root growth in two independent sweet16mutant linesand the wild type that was identified from the segregating mutant pop-ulation (Col-TDNA). B, Comparison of root growth in two independentSWEET16 overexpressor lines (35S:SWEET16-2 and 35S:SWEET16-3)and the wild type that was transformed with the empty vector (Col-Vector-1 and Col-Vector-2). The data presented are means 6 SE (n = 8).Significant differences from the wild type were determined by Student’s ttest indicated by asterisks (*P , 0.05 and **P , 0.01).


Guo et al.












Fru (Fig. 4A), probably caused by a limited allocationof Fru into vacuoles leading to a higher cytosolic Fruconcentration (Cho and Yoo, 2011). The inhibitory ef-fect on root growth could be alleviated by ectopicoverexpression of SWEET17 (Fig. 4B). We could alsodemonstrate vacuolar import activity of SWEET17using vacuole transport assays (Fig. 6A). These importfeatures of SWEET17 led us to conclude that, physio-logically, SWEET17 mediates Fru uptake into vacuolesfor storage in response to a high concentration of cy-tosolic Fru.Fru stored in root vacuoles may serve as an important

source of energy for actively growing cells (Echeverriaand Valich, 1988; Etxeberria et al., 2012), such as thosein the elongation region where newly formed cells arequickly expanding and accelerate maturation of the or-ganelles. Interestingly, upon 48 h incubation in darkness,which leads to intracellular energy depletion and triggersthe release of stored sugars (Usadel et al., 2008), expres-sion of SWEET17 is up-regulated in the root elongationzone (Fig. 2, J and L). The induction in the dark suggeststhat SWEET17 also can function in Fru export from thevacuole to meet energy requirement in planta. Theexporter activity of SWEET17 became evident underconditions in which high levels of Fru accumulate invacuoles, i.e. under cold stress (Kaplan et al., 2004; Wormitet al., 2006). The reduced Fru accumulation by ectopicexpression of SWEET17 demonstrated that SWEET17can passively export Fru out of vacuoles along a con-centration gradient in vivo as observed in a transportassay performed in a heterologous system (Chardon et al.,2013). Despite the distinct reduction of Fru contents inleaves (Fig. 5), we did not observe phenotypic differencesbetween overexpressors and wild-type plants (data notshown). It is possible that other compensatory mech-anisms such as organic compounds and sugar alcoholsare also induced during the osmotic adjustment to sustaincold tolerance (Sanghera et al., 2011).Despite the fact that SWEET17 protein levels were ex-

tremely low in leaves (Fig. 2F; Supplemental Figs. S3Aand S5), the Fru content of leaves was dramaticallyincreased in leaves of sweet17 mutants under normalconditions (Chardon et al., 2013). The reduced Fru up-take activity of leaf-derived vacuoles from sweet17-1/sweet16-1 (Supplemental Fig. S12A) demonstrates thatdespite low levels of the protein, SWEET17 in leafvacuoles is likely sufficient. Alternatively, the reducedimport activity of Fru in roots of sweet17 mutants mayreduce their storage capacity and sink strength, which, inturn, can reduce Suc unloading and ultimately increasesugar or carbohydrate accumulation in leaf mesophyllcells (Ayre, 2011).

SWEET17 Acts as a Bidirectional Fru-Specific Facilitator

Transport assays using isolated leaf mesophyll vac-uoles verified that SWEET17 is an important vacuolarFru-specific transporter (Fig. 6; Supplemental Fig. S12,A and B). Although we cannot exclude the possibility

that SWEET17 exhibits a minor transport activity forother hexoses or disaccharides, such activities would bevery low compared with Fru (Supplemental Fig. S8).Furthermore, excess Suc or Glc did not affect the ex-pression level of SWEET17-GUS (Supplemental Fig. S3B).When sugar accumulation is induced by cold stress(Wingenter et al., 2010), only the concentration of Fruwas significantly affected in leaves of SWEET17 over-expressors (Fig. 5; Supplemental Fig. S7B) and in vacuoleslacking SWEET17 (Chardon et al., 2013). Thus, unlikeArabidopsis vacuolar TMTs (Schulz et al., 2011), VGT1(Aluri and Büttner, 2007), and ERD Six-like1 (Yamadaet al., 2010), which may act as general tonoplastichexose transporters (Wormit et al., 2006), SWEET17exhibits Fru-specific transport functions in vivo and isprobably regulated by Fru signaling. Moreover, insensi-tivity of the transport activity to ATP and treatments withNH4Cl or CCCP is consistent with a proton-independentbidirectional facilitator activity (Fig. 6B). This finding isalso consistent with our functional assays (Figs. 4 and 5)and previous observations performed in a heterologousoocyte expression system (Chardon et al., 2013). Theuniport feature appears similar to that of other plasmamembrane SWEET members (Chen et al., 2010, 2012).One common property of all SWEETs described is thehigh apparent Km value ranging from 9 mM for SWEET1(to Glc; Chen et al., 2010) to 71 mM for SWEET12 (to Suc;Chen et al., 2012). Similarly, the high Km value at 25.6 mM

determined here for SWEET17 (Fig. 6C) suggests thatvacuolar SWEETs share this property with those of theplasma membrane. An even higher Km of 102 mM wasreported for the Arabidopsis vacuolar monosaccharidefacilitator ERD Six-like1 (Yamada et al., 2010). A relativelower Km for Fru transport compared with Glc may re-flect the preference of many plants to accumulate moreGlc than Fru.

Clade IV SWEETs Function as Vacuolar Transporters

SWEET17 has a very close homolog, SWEET16 (Sup-plemental Fig. S9A), which has recently been demon-strated to function as a vacuolar transporter as well(Klemens et al., 2013). Furthermore, using a promoter-GUS construct, it had been shown that SWEET16 ismainly expressed at very low levels in xylem parenchymacells in leaves and flower stalks (Klemens et al., 2013).However, similar to SWEET17, our expression analysisby qRT-PCR as well as by the GUS reporter showedthat SWEET16 is predominantly expressed in rootcortex cells at all stages (Fig. 1; Supplemental Fig. S10,A–D). Although our expression profiles are differentfrom the previous report (Klemens et al., 2013), all ourresults are well in line with data from public expressiondatabases (Supplemental Figs. S1 and S2). Moreover,localization of a stable translational GFP fusion clearlyassigned SWEET16 to the tonoplast in Arabidopsis roots(Supplemental Fig. S10, E–G), as shown by transientexpression in leaf protoplasts (Klemens et al., 2013).Therefore, we conclude that SWEET16, like SWEET17,



















mainly functions in roots. Compared with other SWEETs,SWEET16 and SWEET17 are the only two SWEETs foundto be highly expressed in roots (Fig. 7; SupplementalFig. S9B). These results imply that clade IV SWEETshave evolved from other SWEET clades to specificallyfunction on root vacuoles. This is in analogy with theAluminum-activated malate transporters or full-sizeATP-binding cassette transporter family in which oneclade (Aluminum-activated malate transporter) and onesubfamily C are also localized on the vacuolar membrane,while the others are localized on the plasma membrane(Kang et al., 2011).

From their expression patterns, we hypothesizedthat these two SWEETs would either function in acomplementary way, e.g. one transporting Glc, the otherFru, or be redundant. Mutations in both SWEET17 andSWEET16 dramatically reduced the Fru uptake activity ofvacuoles (Supplemental Fig. S12A). A reduced accu-mulation of Fru was also observed in a SWEET16overexpressor line under cold stress (SupplementalFig. S7B). However, in contrast to the previous studyperformed in an oocyte expression system (Klemenset al., 2013), we did not detect any transport activity ofSWEET16 for sugars (Supplemental Fig. S12B; data notshown). It is possible that the lower uptake activitymediated by SWEET16 (Km higher than 10 mM measuredin an oocyte expression system; Klemens et al., 2013),compared with SWEET17 (Km lower than 10 mM mea-sured in the same oocyte expression system; Chardonet al., 2013), on the tonoplast was below the detectionlimit due to a high background (Fig. 6A; SupplementalFig. S8). Nevertheless, similar to SWEET17, alteredsensitivity of the root growth to excess Fru was ob-served in sweet16 mutants and overexpressors (Fig. 8).However, a loss of function of both sweet16 andsweet17 double mutant did not further reduce the roottolerance to excess Fru compared with sweet16 singlemutant (Fig. 8A). These results indicate that SWEET16and SWEET17 may function in independent pathwaysby exchanging sugars across the tonoplast of root cells.Given that SWEET17 is expressed to a much higherlevel in roots (Fig. 1; Supplemental Fig. S1), it is likelythat SWEET17 is the dominant Fru transporter on theroot tonoplast.

In summary, our work provides functional characteri-zations of the vacuolar Fru-specific uniporter SWEET17.Based on these results, we propose that when photo-synthesis is very active, Suc is allocated from the leaf tothe root where parts of Suc are hydrolyzed to Glc andFru and is imported into vacuoles for storage by avariety of hexose transporters and by SWEET17, re-spectively. Depending on the metabolic state of rootcells, defects of SWEET17 will either reduce the vacu-olar loading or unloading. In this case, excess of Fru inthe cytosol will lead to growth retardation due to toxiceffects of Fru, while under energy-limiting conditions,the plant is not able to use all the carbohydrate reserves.During these processes, SWEET16 may also contributeto sugar compartmentation across the root vacuole in-dependently from SWEET17.

MATERIALS AND METHODS

Plant and Growth Conditions

Arabidopsis (Arabidopsis thaliana) plants and mutants used in this study are all inColumbia ecotype. Arabidopsis plants were grown with potting medium or solidagar media in a controlled growth room (22°C/18°C day/night temperature, 16-h-light/8-h-dark regime of approximately 100 mmol m–2 s–1 illumination). For treat-ments with excess sugars, surface sterilized seeds (30% [v/v] of concentrated bleachand 0.1% [v/v] triton) were stratified at 4°C in the dark for 3 d and sown on one-half-strength Murashige and Skoog (MS) solid media supplemented with 1% (w/v)Suc. After 5 d of growth, similar sized seedlings were transferred to one-half-strength MS media supplemented with various concentrations of Fru, Glc, or Sucas indicated. Expression patterns of reporter genes were observed after 2 d oftransfer. To analyze effects on root growth, lengths of primary roots were recordedafter 6 d of transfer, and the relative root growth was determined using NationalInstitutes of Health Image J 1.46v. For sugar content analysis, plants were grown inthe growth chamber under 8-h-light/16-h-dark regime for 6 weeks. For treatment ofcold stress, plants were grown for 5 weeks and subsequently transferred to a cooledgrowth chamber (4°C) for 1 week before analysis. For vacuole isolation, plants weregrown for 4 to 5 weeks in a growth chamber under 8-h-light/16-h-dark regime, thentransferred to low-light condition (40 mmolm–2 s–1) for 5 d before isolation. For liquidculture, seedlings were grown for 7 d on ameshwithMS solidmedia containing 1%(w/v) Suc. Plants were then transferred to MS liquid media containing 1% to 2%(w/v) Suc and cultured for 16 d before analysis. The MS or one-half-strength MSmedia used in this study included full or half strength of MS salt, respectively, 0.05%(w/v) MES, and 1.5% (w/v) agar for solid medium (adjust to pH 5.7 with KOH).

Generation of Plants Expressing GUS and GFPReporter Genes

For PSWEET17:GUS and PSWEET16:GUS transcriptional constructs, SWEET17 andSWEET16 promoter fragments containing the 59 untranslated region were am-plified from Arabidopsis genomic DNA with Phusion polymerase (New Eng-land Biolabs) and specific primers (5-PSWT17-BP and 3-PSWT17-BP for PSWEET17;5-PSWT16-BP and 3-PSWT16-BP for PSWEET16). The resulting 2,746 bp and 1,521fragments, respectively, were first cloned into pDONR221-f1 and then trans-ferred to a binary vector, pWUGW, that contains a GUS reporter gene usingGateway technology (Invitrogen). For SWEET17-GUS/GFP and SWEET16-GUS/GFP translational constructs, the promoter fragments were first ampli-fied from the pDONR221-f1 clones mentioned above and cloned into pGEM-TEasy (Promega). The promoter fragments were then cloned via PstI and KpnIsites into pMDC32-SWEET17 and pMDC32-SWEET16 constructs (refer to thesection “Overexpression of SWEET17 and SWEET16 in Arabidopsis“) to replacethe 35S promoter in those plasmids. The full PSWEET17-SWEET17 and PSWEET16-SWEET16 genomic fragments were then amplified using Phusion polymerase,cloned into pGEM-T Easy, digested, and subcloned into SacII and PstI sites ofpUTKan or pGTkan harboring the GUS or GFP reporter gene, respectively. Allconstructs were transformed into wild-type Arabidopsis plants using Agro-bacterium tumefaciens strain C58 PGV3850 and flower dipping method (Cloughand Bent, 1998). Transformants were identified on one-half-strength MSmediumcontaining 50 mg mL–1 kanamycin. For analysis of reporter gene expression,plants from six independent transgenic lines were examined for each construct.Patterns of gene expression were consistent within a construct, and represen-tative three homozygous lines were further analyzed in other experiments. Se-quences of primers are listed in Supplemental Table S1.

Histochemical Localization of GUS

Expression of transcriptional or translational fusions of the GUS reportergene was analyzed by histochemical staining. Transgenic plants or excisedplant tissues were stained at 37°C for 2 or 4 h as indicated using 5-bromo-4-chloro-3-indolyl-b-glucuronic acid solution (0.1 M NaH2PO4, 10 mM EDTA,0.5 mM each of potassium ferricyanide and potassium ferrocyanide, 0.1% [v/v]Triton X-100, and 0.25 mg ml–1 X-glucuronidecyclohexylamine salt). Afterstaining, tissues were cleared by replacing the staining solution with severalchanges of 70% (v/v) and 90% (v/v) ethanol as necessary.

Confocal Microscopy for GFP Observation

Fluorescence imaging of plants expressing SWEET17-GFP and SWEET16-GFPwas performed on a Carl Zeiss LSM780 confocal microscope (Instrument


Guo et al.













Development Center, National Cheng Kung University). Seedlings were incu-bated in 6 mM of FM4-64 (Invitrogen) for 5 min before imaging to visualize theplasma membrane. GFP was visualized by excitation with an argon laser at 488nm and spectral detector set between 500 and 545 nm for the emission. The redfluorescence of FM4-64 was visualized by excitation with an argon laser at 561nm and spectral detector set between 566 and 585 nm for the emission.

Identification of SWEET16 and SWEET17Insertion Mutants

The T-DNA insertion mutants for SWEET17 (sweet17-1 and sweet17-2;Chardon et al., 2013) and SWEET16 (sweet16-1 and sweet16-2) were obtainedfrom the Salk Institute Genomic Analysis Laboratory (http://signal.salk.edu/cgi-bin/tdnaexpress): SM_3_15143, as sweet16-1, and SM_3_30077, as sweet16-2.To confirm the homozygosity of T-DNA insertion lines, gene-specific primerswere used to examine wild-type alleles (swt17-1-RP and swt17-1-LP for sweet17-1;swt17-2-RP and SWT17-5-UTR for sweet17-2; swt16-1-RP and swt16-1-LP forsweet16-1; and swt16-2-RP and swt16-2-LP for sweet16-2). Locations of T-DNAinsertions in sweet16 mutants were confirmed by sequencing PCR products am-plified with gene-specific primers (swt16-1-RP and swt16-2-RP) and the corre-sponding T-DNA left border primer (Spm32). The positions were located at 32and 945 bp downstream of the translational start codon, corresponding to thefourth and the first exon in the sweet16-1 and sweet16-2 mutant, respectively(Supplemental Fig. S11A). To confirm the expression level, RT-PCR was per-formed with specific primers (SWT17-5-UTR and swt17-1-LP for SWEET17;SWT16-5-UTR and SWT16-3-UTR for SWEET16) to amplify the full-length ofcDNA. Sequences of primers were listed in Supplemental Table S1.

Overexpression of SWEET17 and SWEET16 in Arabidopsis

To express SWEET17 and SWEET16 under the control of the 35S promoter,full-length SWEET17/SWEET16 genomic sequences including all introns wereamplified using Phusion polymerase with gene-specific primers (SWT17-5-UTR-BP and SWT17-3-UTR-BP for SWEET17 and SWT16-5-UTR-BP and SWT16-3-UTR-BP for SWEET16). The resulting 2,856- and 2,119-bp fragments were clonedinto pDONR221-f1 and then transferred to a binary vector pMDC32 (Curtis andGrossniklaus, 2003) via Gateway technology. The resulting plasmids, pMDC32-SWEET17, pMDC32-SWEET16, and the corresponding empty vector pMDC32without the Gateway cassette, were transformed into wild-type Arabidopsisplants using the A. tumefaciens strain C58 PGV3850 and flower dipping method(Clough and Bent, 1998). Transformants were identified on one-half-strength MSmedium containing 25 mg mL–1 hygromycin B. Six single-copy T2 lines wereobtained, and three homozygous lines of highest expression were used in thisstudy. Sequences of primers were listed in Supplemental Table S1.

Quantitative PCR and RT-PCR Analysis

For aerial tissues in mature plants, total mRNA was isolated from leaves,stems, flowers, and siliques of 7- to 8-week-old soil-grown plants. For mRNAfrom young seedlings, shoots and roots of 2-week-old plants or 7-d-old wholeseedlings grown on one-half-strength MS agar were harvested for extraction. Toanalyze expression in mature roots, total mRNAwas isolated from roots of 23-d-old liquid-cultured seedlings. Total mRNA was isolated using TRIsure (Bioline)reagent or RNeasy Mini Kit (Qiagen) as instructed by the manufacture. Theresulting cDNA produced by Moloney murine leukemia virus (Qiagen) wasdiluted and used as the template and subjected to 25 to 30 cycles of PCR reaction(94°C for 30 s, 55°C for 30 s, and 72°C for 40 s) using Taq DNA polymerase and apair of gene-specific primers as indicated. Amplification of an Actin cDNA(Actin2, At3G18780) using Act2-F and Act2-R primers (Supplemental Table S1)was used to normalize results from different samples. For RT-PCR, sampleswere separated on a 1.5% (w/v) agarose gel. For real-time quantitative PCR, theamplification was performed using HotStart-IT SYBR Green qPCR Master Mix(USB) or KAPA SYBR FAST qPCR Kit (Kapa Biosystems) according to themanufacturer’s instructions on a 7300 PCR system (Applied Biosystems). Therelative expression level was determined by comparing with the expression ofActin2 (1,000 3 2–[CtSWEET–CtActin2]), where Ct represents the threshold cycle. Thegene-specific primers used in qRT-PCR for 17 Arabidopsis SWEET genes werelisted in Supplemental Table S1.

Extraction and Assay of Soluble Sugars

Ground, freeze-dried Arabidopsis rosette leaf material (approximately 0.5 gfresh weight) was extracted in 800 mL ice-cold 0.7 M perchloric acid for 5 min

with intermittent mixing, using a Mixer Mill (Retsch). All the subsequent stepswere carried out between 0°C and 4°C. After centrifugation (5 min, 5,000g,4°C), 600 mL of supernatant (soluble fraction) was adjusted to the pH between 5 to6 by adding 2 M KOH, 0.4 M MES, and 0.4 M KCl (approximately 0.4 mL mL–1

supernatant). Precipitated potassium perchlorate was removed by centrifugation(10 min, 10,000g, 4°C). For desalting, samples of the neutralized soluble fraction(250 mL) were applied to sequential 1.5-mL columns of Dowex 50 W and Dowex1 (Sigma-Aldrich). The neutral compounds were eluted with 5 mL of water,lyophilized, and redissolved in 200 mL of water. Soluble sugars (Glc, Suc, andFru) were separated using High-Performance Anion-Exchange Chromatog-raphy with Pulsed Amperometric Detection on a Dionex PA-20 column(Thermo Scientific), according to the following conditions: eluent A, 100 mM

NaOH; eluent B, 150 mM NaOH and 500 mM sodium acetate. At a flow rate of0.5 mL min–1, the gradient was as follows: 0 to 7 min, 100% (v/v) A and 0%(v/v) B; 7 to 26.5 min, a concave gradient to 20% (v/v) A and 80% (v/v) B(maltooligosaccharide elution); 26.5 to 32 min, hold at 20% (v/v) A and80% (v/v) B (column wash step); and 32 to 40 min, step to 100% (v/v) A and0% (v/v) B (column reequilibration). Peaks were identified by coelution withknown maltooligosaccharide standards. Peak areas were determined usingChromeleon software (Dionex, Thermo Scientific).

Vacuole Isolation from Arabidopsis Leaves

Vacuoles were isolated as described by Song et al. (2010) with some minormodifications. Firstly, to isolate mesophyll protoplasts, the abaxial epidermis ofmature healthy leaves was abraded using sandpaper (P500). The abraded leaveswere floated on medium A (0.5 M sorbitol, 1 mM CaCl2 2H2O, and 10 mM MES-KOH, pH 5.6) containing 1% (w/v) Cellulase R10 and 0.5% (w/v) MazerozymeR10 for 1.5 h at 30°C. The released protoplasts were collected by centrifugationfor 8 min at 400g (4°C) on a cushion of osmotically stabilized Percoll (100% [v/v]Percoll, 0.5 M sorbitol, 1 mM CaCl2 2H2O, and 10 mM MES, pH 6). The super-natant was aspired, and the sedimented protoplasts were resuspended in theresidual solution, which was completed to a final Percoll concentration of 40%(v/v) with the osmotically stabilized Percoll. A Percoll gradient was formed byoverlayering the suspended protoplasts with 3:7 (v/v) mix of Percoll (pH 7.2;500 mM sorbitol and 20 mM HEPES in Percoll 100% [v/v]) and medium B (400mM sorbitol, 30 mM potassium gluconate, and 20 mM HEPES, pH 7.2, adjustedwith imidazole), overlaid with medium B containing 1 mg mL–1 bovine serumalbumin (BSA) and 1 mM dithiothreitol (DTT) . After centrifugation for 8 min at250g (4°C), the protoplasts were recovered from the upper interphase and lysedwith the same volume of prewarmed (42°C) lysis medium (200 mM sorbitol, 20mM EDTA, 10 mM HEPES, pH 8.0, with KOH, 10% (w/v) Ficoll, 0.2 mg mL–

1 BSA, and 1 mM DTT) under 37°C for a maximum of 10 min. Vacuoles werepurified by overlaying 5 mL of the lysate with 5 mL of 1:1 fresh lysis medium/medium C (400 mM betaine, 30 mM potassium gluconate, 20 mM HEPES, pH 7.2,adjusted with imidazole, 1 mg mL–1 BSA, and 1 mM DTT) and 1 mL medium C.After centrifugation for 8 min at 1,300g (4°C), the vacuoles were recovered fromthe interface between the middle and upper layer. Microscopic analysis indi-cated that contamination with intact protoplasts was less than 3%.

Transport Analysis with Arabidopsis Vacuole

Transport experiments were performed using silicone oil centrifugationtechnique as described previously (Song et al., 2010). The Michaelis-Mentennonlinear least-square regression fits were calculated using the SSmicmenfunction without initial parameters within the nls function of R 2.14.0 (http://www.R-project.org).

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Organ-specific expression of ArabidopsisSWEET17 and SWEET16.

Supplemental Figure S2. Cell type-specific expression of ArabidopsisSWEET17 and SWEET16 genes in roots.

Supplemental Figure S3. Developmental expression of SWEET17 and itsregulation in response to the sugar status.

Supplemental Figure S4. Expression of SWEET17-GUS fusion proteins inshoots under normal conditions or in response to darkness and cold stress.









http://www.R-project.org

http://www.R-project.org






Supplemental Figure S5. Tonoplast localization of SWEET17-GFP fusionproteins in leaves.

Supplemental Figure S6. Functional characterization of SWEET17 over-expressing lines.

Supplemental Figure S7. The sugar composition in leaves of SWEET17and SWEET16 overexpressors.

Supplemental Figure S8. Analysis of sugar transport activity of vacuolesectopically expressing Arabidopsis SWEET17.

Supplemental Figure S9. Comparison of Arabidopsis clade IV SWEETgenes.

Supplemental Figure S10. Expression patterns of SWEET16-GUS/GFP fu-sion proteins in planta.

Supplemental Figure S11. Identification of SWEET16 mutants and over-expressing lines.

Supplemental Figure S12. Characterization of SWEET16 transport activityin planta.

Supplemental Table S1. Specific primers used in this study.

ACKNOWLEDGMENTS

We thank Dr. Bo Burla at University of Zürich for performing the curvefitting and Drs. Tong Seung Tseng and Kate Dreher at Carnegie Institution forScience for constructive comments on the manuscript.

Received November 18, 2013; accepted December 29, 2013; published Decem-ber 31, 2013.

LITERATURE CITED

Aluri S, Büttner M (2007) Identification and functional expression of theArabidopsis thaliana vacuolar glucose transporter 1 and its role in seedgermination and flowering. Proc Natl Acad Sci USA 104: 2537–2542

Ayre BG (2011) Membrane-transport systems for sucrose in relation towhole-plant carbon partitioning. Mol Plant 4: 377–394

Bhagyalakshmi N, Thimmaraju R, Narayan MS (2004) Various hexosesand di-hexoses differently influence growth, morphology and pigmentsynthesis in transformed root cultures of red beet (Beta vulgaris). PlantCell Tissue Organ Cult 78: 183–195

Bogdanovi�c J, Mojovi�c M, Milosavi�c N, Mitrovi�c A, Vucini�c Z, Spasojevi�cI (2008) Role of fructose in the adaptation of plants to cold-inducedoxidative stress. Eur Biophys J 37: 1241–1246

Braun DM, Slewinski TL (2009) Genetic control of carbon partitioning ingrasses: roles of sucrose transporters and tie-dyed loci in phloem load-ing. Plant Physiol 149: 71–81

Chardon F, Bedu M, Calenge F, Klemens PAW, Spinner L, Clement G,Chietera G, Léran S, Ferrand M, Lacombe B, et al (2013) Leaf fructosecontent is controlled by the vacuolar transporter SWEET17 in Arabi-dopsis. Curr Biol 23: 697–702

Chen LQ, Hou BH, Lalonde S, Takanaga H, Hartung ML, Qu XQ, GuoWJ, Kim JG, Underwood W, Chaudhuri B, et al (2010) Sugar trans-porters for intercellular exchange and nutrition of pathogens. Nature468: 527–532

Chen LQ, Qu XQ, Hou BH, Sosso D, Osorio S, Fernie AR, Frommer WB(2012) Sucrose efflux mediated by SWEET proteins as a key step forphloem transport. Science 335: 207–211

Cho YH, Yoo SD (2011) Signaling role of fructose mediated by FINS1/FBPin Arabidopsis thaliana. PLoS Genet 7: e1001263

Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16: 735–743

Curtis MD, Grossniklaus U (2003) A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol 133:462–469

Czajkowski R, de Boer WJ, Velvis H, van der Wolf JM (2010) Systemiccolonization of potato plants by a soilborne, green fluorescent protein-tagged strain of Dickeya sp. biovar 3. Phytopathology 100: 134–142

Dekkers BJ, Schuurmans JA, Smeekens SC (2004) Glucose delays seedgermination in Arabidopsis thaliana. Planta 218: 579–588

Echeverria E, Valich J (1988) Carbohydrate and enzyme distribution inprotoplasts from valencia orange juice sacs. Phytochemistry 27: 73–76

Eom JS, Cho JI, Reinders A, Lee SW, Yoo Y, Tuan PQ, Choi SB, Bang G, ParkYI, ChoMH, et al (2011) Impaired function of the tonoplast-localized sucrosetransporter in rice, OsSUT2, limits the transport of vacuolar reserve sucroseand affects plant growth. Plant Physiol 157: 109–119

Etxeberria E, Pozueta-Romero J, Gonzalez P (2012) In and out of the plantstorage vacuole. Plant Sci 190: 52–61

Frommer WB, Sosso D, Chen LQ (2013) SWEET glucoside transportersuperfamily. In CK Gordon, ed, Encyclopedia of Biophysics. Springer,New York, pp 2556–2558

Ge YX, Angenent GC, Dahlhaus E, Franken J, Peters J, Wullems GJ,Creemers-Molenaar J (2001) Partial silencing of the NEC1 gene resultsin early opening of anthers in Petunia hybrida. Mol Genet Genomics 265:414–423

Getz HP, Klein M (1995) Characteristics of sucrose transport and sucrose-induced H+ transport on the tonoplast of red beet (Beta vulgaris L.)storage tissue. Plant Physiol 107: 459–467

Grennan AK, Gragg J (2009) How sweet it is: identification of vacuolarsucrose transporters. Plant Physiol 150: 1109–1110

Guan YF, Huang XY, Zhu J, Gao JF, Zhang HX, Yang ZN (2008) RUP-TURED POLLEN GRAIN1, a member of the MtN3/saliva gene family, iscrucial for exine pattern formation and cell integrity of microspores inArabidopsis. Plant Physiol 147: 852–863

Hassan S, Mathesius U (2012) The role of flavonoids in root-rhizospheresignalling: opportunities and challenges for improving plant-microbeinteractions. J Exp Bot 63: 3429–3444

Jones DL, Darrah PR (1996) Re-sorption of organic compounds by roots ofZea mays L. and its consequences in the rhizosphere III. Characteristicsof sugar influx and efflux. Plant Soil 178: 153–160

Kaiser G, Heber U (1984) Sucrose transport into vacuoles isolated frombarley mesophyll protoplasts. Planta 161: 562–568

Kang J, Park J, Choi H, Burla B, Kretzschmar T, Lee Y, Martinoia E (2011) PlantABC transporters. The Arabidopsis Book 9: e0153, doi/10.1199/tab.0153

Kaplan F, Kopka J, Haskell DW, Zhao W, Schiller KC, Gatzke N, SungDY, Guy CL (2004) Exploring the temperature-stress metabolome ofArabidopsis. Plant Physiol 136: 4159–4168

Keller F (1988) A large-scale isolation of vacuoles from protoplasts ofmature carrot taproots. J Plant Physiol 132: 199–203

Klemens PA, Patzke K, Deitmer JW, Spinner L, Le Hir R, Bellini C, BeduM, Chardon F, Krapp A, Neuhaus HE (2013) Overexpression of thevacuolar sugar carrier AtSWEET16 modifies germination, growth, andstress tolerance in Arabidopsis. Plant Physiol 163: 1338–1352

Krügel U, Kühn C (2013) Post-translational regulation of sucrose trans-porters by direct protein-protein interactions. Front Plant Sci 4: 237

Kühn C, Grof CP (2010) Sucrose transporters of higher plants. Curr OpinPlant Biol 13: 288–298

Laugier E, Bouguyon E, Mauriès A, Tillard P, Gojon A, Lejay L (2012)Regulation of high-affinity nitrate uptake in roots of Arabidopsis de-pends predominantly on posttranscriptional control of the NRT2.1/NAR2.1 transport system. Plant Physiol 158: 1067–1078

Leigh R (1984) The role of the vacuole in the accumulation and mobiliza-tion of sucrose. Plant Growth Regul 2: 339–346

Li P, Wind JJ, Shi X, Zhang H, Hanson J, Smeekens SC, Teng S (2011)Fructose sensitivity is suppressed in Arabidopsis by the transcriptionfactor ANAC089 lacking the membrane-bound domain. Proc Natl AcadSci USA 108: 3436–3441

Martinoia E, Kaiser G, SchrammMJ, Heber U (1987) Sugar transport across theplasmalemma and the tonoplast of barley mesophyll protoplasts. Evidencefor different transport systems. J Plant Physiol 131: 467–478

Martinoia E, Maeshima M, Neuhaus HE (2007) Vacuolar transporters andtheir essential role in plant metabolism. J Exp Bot 58: 83–102

Martinoia E, Massonneau A, Frangne N (2000) Transport processes ofsolutes across the vacuolar membrane of higher plants. Plant CellPhysiol 41: 1175–1186

Martinoia E, Meyer S, De Angeli A, Nagy R (2012) Vacuolar transportersin their physiological context. Annu Rev Plant Biol 63: 183–213

Mustroph A, Zanetti ME, Jang CJ, Holtan HE, Repetti PP, Galbraith DW,Girke T, Bailey-Serres J (2009) Profiling translatomes of discrete cellpopulations resolves altered cellular priorities during hypoxia in Ara-bidopsis. Proc Natl Acad Sci USA 106: 18843–18848

Neuhaus HE (2007) Transport of primary metabolites across the plantvacuolar membrane. FEBS Lett 581: 2223–2226


Guo et al.











http://doi/10.1199/tab.0153


Payyavula RS, Tay KH, Tsai CJ, Harding SA (2011) The sucrose trans-porter family in Populus: the importance of a tonoplast PtaSUT4 to bio-mass and carbon partitioning. Plant J 65: 757–770

Pego JV, Smeekens SCM (2000) Plant fructokinases: a sweet family get-together. Trends Plant Sci 5: 531–536

Pontis HG (1989) Fructans and cold stress. J Plant Physiol 134: 148–150Poschet G, Hannich B, Raab S, Jungkunz I, Klemens PA, Krueger S, Wic

S, Neuhaus HE, Büttner M (2011) A novel Arabidopsis vacuolar glucoseexporter is involved in cellular sugar homeostasis and affects the com-position of seed storage compounds. Plant Physiol 157: 1664–1676

Riesmeier JW, Willmitzer L, Frommer WB (1992) Isolation and charac-terization of a sucrose carrier cDNA from spinach by functional ex-pression in yeast. EMBO J 11: 4705–4713

Rolland F, Baena-Gonzalez E, Sheen J (2006) Sugar sensing and signalingin plants: conserved and novel mechanisms. Annu Rev Plant Biol 57:675–709

Sanghera GS, Wani SH, Hussain W, Singh NB (2011) Engineering coldstress tolerance in crop plants. Curr Genomics 12: 30–43

Sauer N (2007) Molecular physiology of higher plant sucrose transporters.FEBS Lett 581: 2309–2317

Sauer N, Friedländer K, Gräml-Wicke U (1990) Primary structure, ge-nomic organization and heterologous expression of a glucose trans-porter from Arabidopsis thaliana. EMBO J 9: 3045–3050

Schulz A, Beyhl D, Marten I, Wormit A, Neuhaus E, Poschet G, BüttnerM, Schneider S, Sauer N, Hedrich R (2011) Proton-driven sucrosesymport and antiport are provided by the vacuolar transporters SUC4and TMT1/2. Plant J 68: 129–136

Sherson SM, Alford HL, Forbes SM, Wallace G, Smith SM (2003) Roles ofcell-wall invertases and monosaccharide transporters in the growth anddevelopment of Arabidopsis. J Exp Bot 54: 525–531

Sivitz AB, Reinders A, Johnson ME, Krentz AD, Grof CPL, Perroux JM,Ward JM (2007) Arabidopsis sucrose transporter AtSUC9: high-affinitytransport activity, intragenic control of expression, and early floweringmutant phenotype. Plant Physiol 143: 188–198

Song WY, Park J, Mendoza-Cózatl DG, Suter-Grotemeyer M, Shim D,Hörtensteiner S, Geisler M, Weder B, Rea PA, Rentsch D, et al (2010)Arsenic tolerance in Arabidopsis is mediated by two ABCC-type phy-tochelatin transporters. Proc Natl Acad Sci USA 107: 21187–21192

Thom M, Komor E, Maretzki A (1982) Vacuoles from sugarcane suspen-sion cultures. II. Characterization of sugar uptake. Plant Physiol 69:1320–1325

Tohge T, Ramos MS, Nunes-Nesi A, Mutwil M, Giavalisco P, SteinhauserD, Schellenberg M, Willmitzer L, Persson S, Martinoia E, et al (2011)

Toward the storage metabolome: profiling the barley vacuole. PlantPhysiol 157: 1469–1482

Usadel B, Bläsing OE, Gibon Y, Retzlaff K, Höhne M, Günther M, Stitt M(2008) Global transcript levels respond to small changes of the carbonstatus during progressive exhaustion of carbohydrates in Arabidopsisrosettes. Plant Physiol 146: 1834–1861

Voitsekhovskaja OV, Koroleva OA, Batashev DR, Knop C, Tomos AD,Gamalei YV, Heldt HW, Lohaus G (2006) Phloem loading in twoScrophulariaceae species. What can drive symplastic flow via plasmo-desmata? Plant Physiol 140: 383–395

Wayne RO (2009) The vacuole. In Plant Cell Biology: From Astronomy toZoology. Elsevier, Atlanta, pp 101–118

Williams LE, Lemoine R, Sauer N (2000) Sugar transporters in higherplants—a diversity of roles and complex regulation. Trends Plant Sci 5:283–290

Wingenter K, Schulz A, Wormit A, Wic S, Trentmann O, Hoermiller II,Heyer AG, Marten I, Hedrich R, Neuhaus HE (2010) Increased activityof the vacuolar monosaccharide transporter TMT1 alters cellular sugarpartitioning, sugar signaling, and seed yield in Arabidopsis. PlantPhysiol 154: 665–677

Winter D, Vinegar B, Nahal H, Ammar R, Wilson GV, Provart NJ (2007)An “Electronic Fluorescent Pictograph” browser for exploring and an-alyzing large-scale biological data sets. PLoS ONE 2: e718

Winter H, Robinson DG, Heldt HW (1993) Subcellular volumes and me-tabolite concentrations in barley leaves. Planta 191: 180–190

Wormit A, Trentmann O, Feifer I, Lohr C, Tjaden J, Meyer S, Schmidt U,Martinoia E, Neuhaus HE (2006) Molecular identification and phy-siological characterization of a novel monosaccharide transporterfrom Arabidopsis involved in vacuolar sugar transport. Plant Cell 18:3476–3490

Xuan YH, Hu YB, Chen LQ, Sosso D, Ducat DC, Hou BH, Frommer WB(2013) Functional role of oligomerization for bacterial and plant SWEETsugar transporter family. Proc Natl Acad Sci USA 110: E3685–E3694

Yamada K, Osakabe Y, Mizoi J, Nakashima K, Fujita Y, Shinozaki K,Yamaguchi-Shinozaki K (2010) Functional analysis of an Arabidopsisthaliana abiotic stress-inducible facilitated diffusion transporter formonosaccharides. J Biol Chem 285: 1138–1146

Yang B, Sugio A, White FF (2006) Os8N3 is a host disease-susceptibilitygene for bacterial blight of rice. Proc Natl Acad Sci USA 103: 10503–10508

Yuan L, Loqué D, Ye F, Frommer WB, von Wirén N (2007) Nitrogen-dependent posttranscriptional regulation of the ammonium trans-porter AtAMT1;1. Plant Physiol 143: 732–744





SWEET17, a Facilitative Transporter, Mediates …SWEET17, a Facilitative Transporter, Mediates...

Documents

Transcript of SWEET17, a Facilitative Transporter, Mediates …SWEET17, a Facilitative Transporter, Mediates...