Sugar Transporter STP7 Specificity for l-Arabinose and d ... · STP7 mediates the uptake of the pentoses L-arabinose and D-Xyl. In addition, our transport analyses revealed that almost

Sugar Transporter STP7 Specificity for L-Arabinose andD-Xylose Contrasts with the Typical Hexose TransportersSTP8 and STP12

Theresa Rottmann, Franz Klebl, Sabine Schneider, Dominik Kischka, David Rüscher, Norbert Sauer, andRuth Stadler1

Molecular Plant Physiology, University Erlangen-Nürnberg, 91058 Erlangen, Germany

ORCID IDs: 0000-0003-3242-6674 (T.R.); 0000-0003-4750-1155 (D.K.); 0000-0003-4357-2079 (N.S.); 0000-0002-3103-6343 (R.S.).

The controlled distribution of sugars between assimilate-exporting source tissues and sugar-consuming sink tissues is a key element forplant growth and development. Monosaccharide transporters of the SUGAR TRANSPORT PROTEIN (STP) family contribute to theuptake of sugars into sink cells. Here, we report on the characterization of STP7, STP8, and STP12, three previously uncharacterizedmembers of this family in Arabidopsis (Arabidopsis thaliana). Heterologous expression in yeast (Saccharomyces cerevisiae) revealed thatSTP8 and STP12 catalyze the high-affinity proton-dependent uptake of glucose and also accept galactose and mannose. STP12additionally transports xylose. STP8 and STP12 are highly expressed in reproductive organs, where their protein products mightcontribute to sugar uptake into the pollen tube and the embryo sac. stp8.1 and stp12.1 T-DNA insertion lines developed normally,which may point toward functional redundancy with other STPs. In contrast to all other STPs, STP7 does not transport hexoses but isspecific for the pentoses L-arabinose and D-xylose. STP7-promoter-reporter gene plants showed an expression of STP7 especially intissues with high cell wall turnover, indicating that STP7 might contribute to the uptake and recycling of cell wall sugars. Uptakeanalyses with radioactive L-arabinose revealed that 11 other STPs are able to transport L-arabinose with high affinity. Hence, functionalredundancy might explain the missing-mutant phenotype of two stp7 T-DNA insertion lines. Together, these data complete thecharacterization of the STP family and present the STPs as new L-arabinose transporters for potential biotechnological applications.

The first monosaccharide transporter gene of higherplants, STP1 (SUGAR TRANSPORT PROTEIN1), wasidentified almost three decades ago (Sauer et al., 1990).In the following years, the completion of the Arabi-dopsis (Arabidopsis thaliana) genome revealed the exis-tence of 13 additional genes homologous to STP1(Truernit et al., 1996, 1999; Büttner et al., 2000). STP1 toSTP14 are members of the Arabidopsis MONOSAC-CHARIDE TRANSPORTER (MST)-like superfamily.The MST-like family includes 53 monosaccharidetransporters, divided into seven individual subfamilies(Büttner, 2007). The detailed characterization of up tonow 10 STPs makes this family the best characterizedsubgroup of MSTs. Each of the STPs shows a uniqueexpression pattern, mainly in sink tissues or in sym-plastically isolated cells, except for STP3 and STP14,which are expressed in source leaves (Büttner et al.,

2000; Poschet et al., 2010). Sink tissues like roots, mer-istems, young leaves, and reproductive tissues dependon the delivery of Suc from photosynthetic source tis-sues. The source-to-sink transport of Suc occurs via thephloem. Symplastic connections of sieve elements toadjacent cells of the sink tissues allow efficient phloemunloading and further distribution via plasmodesmata(Patrick, 1997; Imlau et al., 1999; Turgeon and Wolf,2009). However, in some tissues, phloem unloadinginvolves an apoplasmic step (i.e. Suc is unloaded intothe apoplast, and from there it is actively taken up intothe sink cells by Suc transporters; Sauer, 2007). Extra-cellular Suc also can be cleaved to Glc and Fru by in-vertases (Sturm, 1999). In this case, STPs take upmonosaccharides to provide sink cells with sugars(Sauer, 2007). Furthermore, sugar uptake from theapoplast is essential for symplastically isolated cells likepollen (Scott et al., 2004), guard cells (Palevitz andHepler, 1985), cells of the inner integument of the seedcoat, the endosperm and the embryo (Stadler et al.,2005), as well as egg cells (Werner et al., 2011). Hence,these cell types express at least one or mostly evenmoresugar transporter genes. For example, at least six STPgenes are expressed in pollen tubes (Truernit et al.,1996, 1999; Schneidereit et al., 2003, 2005; Scholz-Starkeet al., 2003; Rottmann et al., 2016). Each STP transports aunique combination of the monosaccharides Glc, Gal,Man, Xyl, and Rib with different affinities (Büttner,2010), and some of the respective genes are regulated bythese substrates (Price et al., 2004; Büttner, 2010;

1 Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Ruth Stadler ([email protected]).

R.S. conceived the research plans and supervised the experiments;T.R. performed most of the experiments; D.K. and D.R. providedassistance to T.R.; T.R., R.S., and F.K. designed the experiments andanalyzed the data; S.S. performed the IC measurements; T.R. wrotethe article with contributions from R.S. and F.K.; N.S. supervised andcomplemented the writing.

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Cordoba et al., 2015; Rottmann et al., 2016). The ex-pression of some STPs is regulated further by diurnalrhythms (Stadler et al., 2003; Büttner, 2010; Poschetet al., 2010), pathogens (Truernit et al., 1996; Fotopouloset al., 2003; Nørholm et al., 2006; Lemonnier et al., 2014),or abiotic stresses (Yamada et al., 2011), and the trans-port activity of some STPs might be regulated byphosphorylation (Nørholm et al., 2006; Yamada et al.,2016). The distinct expression patterns and tight regu-lation of STPs as well as the specific transport charac-teristics of the respective proteins allow a fine-tunedsugar supply specifically adjusted to the type of sinktissue, developmental stage, metabolic state, and envi-ronmental conditions.The uptake of monosaccharides for the nutrition of

sink cells seems to be the main function of STPs.However, especially for the wound- and pathogen-induced STPs, an additional function has been pro-posed: the transporters could be responsible for keepingthe apoplast free of sugars and, thus, for depriving theapoplastically growing pathogens of their nutrition re-source (Lemonnier et al., 2014; Morkunas and Ratajczak,2014; Dodds and Lagudah, 2016). For long-distancetransport, mainly Suc is released form the source cellsvia SWEET transporters prior to its loading into thephloem and is unloaded from the phloem to the apoplastin sink tissues. Therefore, Suc is one of the most abun-dant sugars in the apoplast. The gene for the Suc trans-porter SUC3 is up-regulated in wounded tissues (Meyeret al., 2004), indicating that this transporter is responsiblefor Suc removal from the apoplast during infection andwounding. But it also has been reported that the elicitorflg22 induces the cell wall invertase Atßfruct1 and themonosaccharide transporter gene STP13 (Yamada et al.,2016). Atßfruct1 also is up-regulated together with STP4after infection with the biotrophic fungus Erysiphecichoracearum (Fotopoulos et al., 2003). Furthermore,pathogens also induce genes for host cell wall invertasesto break down Suc molecules (Fotopoulos et al., 2003).Thus, the interplay of SUCs, invertases, and STPs en-sures an efficient removal of metabolizable sugars fromthe apoplast.Wounding or pathogen attack as well as cell wall

modifications in the course of defense reactions includethe breakdown and turnover of cell wall polysacchar-ides, oligosaccharides, and glycoproteins, leading to therelease of their monosaccharide subunits. Inside thecells, these sugars can be recycled via sugar-specifickinases to sugar-1-phosphates, and a nonspecific UDP-sugar pyrophosphorylase (USP) converts them to UDP-sugars that can be reused for the synthesis of cell wallcomponents (Geserick and Tenhaken, 2013). In Arabi-dopsis, Glc, Rha, Gal, Xyl, arabinose, GalUA, and GlcAare the major sugar constituents of the cell wall(Zablackis et al., 1995), indicating that all of them haveto be taken up into the cells by transport proteins. AllSTPs characterized so far are able to transport Glc, andmost of them also exhibit sufficient transport rates forGal and Xyl (Büttner and Sauer, 2000; Poschet et al.,2010; Rottmann et al., 2016). L-Arabinose is, after D-Xyl,

the most widespread pentose sugar in our biosphere(Verho et al., 2011), but few STPs have been tested fortheir capacity to transport this sugar. STP1 transportedvery low amounts of L-arabinose when studied inXenopus laevis oocytes (Boorer et al., 1994), and STP2has been shown to transport L-arabinose with a Kmvalue of 4.5 6 2.2 mM, which is almost 10-fold higherthan its Km value for Gal (Subtil and Boles, 2011).

In this article, we describe the detailed analysis of theso far uncharacterizedmembers of the Arabidopsis STPfamily STP5, STP7, STP8, and STP12, which are allexpressed in sink tissues. The transport properties ofthe encoded proteins were analyzed in baker’s yeast,revealing that STP5 might be a nonfunctional protein,whereas STP8 and STP12 are hexose symporters andSTP7 mediates the uptake of the pentoses L-arabinoseand D-Xyl. In addition, our transport analyses revealedthat almost all STPs accept L-arabinose as an additionalsubstrate. T-DNA insertion lines for STP7, STP8, andSTP12 were characterized but did not show any phe-notypical differences compared with wild-type plants.Potential physiological functions for the three transportproteins are discussed.

RESULTS

Sequence Analysis of STPs

Sequence analysis of the Arabidopsis genome sug-gests that the STP monosaccharide transporter familyconsists of 14 members. Up to now, 10 STPs (STP1–STP4, STP6, STP9–STP11, STP13, and STP14) have beenstudied in detail (Büttner, 2010, and refs. therein;Poschet et al., 2010; Rottmann et al., 2016). To furtherinvestigate the four uncharacterized STPs, their codingsequences (CDSs) were amplified by RT-PCR on totalRNA from young siliques (STP5), siliques (STP7), pol-len tubes (STP8), or flowers (STP12) according to thepredicted expression patterns (Winter et al., 2007). Acomparison with the genomic sequences verified thepredicted exon/intron structure of each gene. Fivepossible intron positions are known in the STP genefamily (Büttner et al., 2000). STP5, STP8, and STP12,with four exons and three introns at the highly con-served positions 1, 2, and 5, share the most commonexon/intron structure of STPs. STP7 only exhibits threeexons and two introns at positions 1 and 2 (Table I). Theencoded STP proteins comprise between 502 and513 amino acids, their calculated molecular massesrange from 53.4 to 56.1 kD, and their isoelectric pointslie between 6.8 and 8.4 (Table I). STP7 includes two andSTP12 includes one Asn-X-Ser consensus sequences forpotential N-glycosylation, which are all localizedwithin transmembrane domains or face the cytosol and,therefore, are most likely not glycosylated. Hydropathyanalyses predicted 12 transmembrane domains for allfour proteins, an additional common feature to all STPs.The STP family evolved by several tandem and seg-mental gene duplication events (Johnson and Thomas,2007). STP5 branches very early and shows only a low

Plant Physiol. Vol. 176, 2018 2331

New Members of the Arabidopsis STP Family

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similarity to the other STPs. Its closest relative is STP3,with 51% identical and 73% similar amino acids. STP7shows the highest similarity to STP13 and STP14, with61%/78% and 59%/78% identical/similar amino acids,respectively. STP12 is very closely related to STP1 (79%identity and 90% similarity), as is STP8 to STP6 (87%identity and 95% similarity).

Analysis of STP Expression in Different Tissues

To verify the expression patterns of STP5, STP7,STP8, and STP12 that are predicted from microarraydata, RT-PCRs were performed on RNA preparationsfrom in vitro-grown pollen tubes, whole open flowers,leaves, stems, roots, whole seedlings, and siliques. APCR reaction with ACTIN2-specific primers served as acontrol for the presence of intact cDNA in each sample.As shown in Figure 1, PCR products with STP7- andSTP12-specific primers could be obtained from all tis-sues tested except for pollen tubes, indicating broadexpression patterns for the two genes. In contrast, STP5could only be amplified from silique and whole-seedling cDNA, indicating that STP5 expression is re-stricted to these tissues. The PCR with primers specificfor STP8 yielded bands only with cDNAs from flowersand especially from pollen tubes, suggesting that STP8also is expressed exclusively in floral tissues.

Functional Characterization of STP5, STP7, STP8, andSTP12 by Heterologous Expression of Their cDNAsin Yeast

To investigate the transport properties of the encodedproteins, STP5, STP7, STP8, and STP12were expressedin the hexose transport- and invertase-deficient Sac-charomyces cerevisiae strain CSY4000 (Rottmann et al.,2016). To this end, the CDS of all four STPs were am-plified by PCR and cloned into the yeast expressionvector NEV-N (Sauer and Stolz, 1994). The forwardprimers attached 15 bp of the STP1 59 untranslated re-gion (UTR) in front of the start codon, as this sequence isreported to optimize the expression of plant genes inS. cerevisiae (Stadler et al., 1995).

As shown in Figure 2A, the yeast strains expressingSTP8 (TRY1015) or STP12 (TRY1013) regained theability to take up [14C]Glc, whereas yeast transformed

with an STP antisense construct did not take up Glc.The strains TRY1015 and TRY1013 were used to de-termine the Km values, pH optima, and substratespecificities of both transporters. The Km values of STP8and STP12 for Glc were 24.8 6 2.1 and 17.4 6 0.9 mM(Fig. 2B), respectively, which is in the range of Kmvalues of other STP transporters. The maximum uptakerates (Vmax) also were comparable to those of otherSTPs, with Vmax = 37.5 6 3.3 mmol h21 mL21 packedcells (STP8) and 126 6 8.5 mmol h21 mL21 packed cells(STP12). Both proteins transported Glc best at low pHvalues (Fig. 2C). The possible uptake of other sugarswas analyzed by competitive inhibition experiments(i.e. measuring the uptake of [14C]Glc in the presence ofa 10-fold excess of nonradioactive sugars; Fig. 2D).Nonradioactive Rib and Suc did not reduce the uptakeof [14C]Glc via STP8 and STP12. However, the addition

Table I. Overview of general sequence features of the putative monosaccharide transporter genes STP5,STP7, STP8, and STP12 from Arabidopsis

The coding sequences of STP genes can be interrupted by introns at five conserved positions (position 1,2, 3, 4, or 5). The intron positions of each gene are indicated. Calculations of molecular mass and iso-electric points (IEP) were performed using the isoelectric point calculator (Kozlowski, 2016).

Gene Locus Name Exon/Intron Structure Amino Acids Molecular Mass (kD) IEP

STP5 At1g34580 1-2- - -5 506 54.37 8.4STP7 At4g02050 1-2- - - - 513 55.83 7.9STP8 At5g26250 1-2- - -5 507 56.12 6.8STP12 At4g21480 1-2- - -5 502 55.57 8.4

Figure 1. RT-PCR analyses of STP expression in different tissues. TotalRNApreparations from in vitro-germinated pollen tubes, whole flowers,leaves, stems, roots, whole seedlings, and siliques were tested for STP5,STP7, STP8, and STP12 expression with primers specific for the re-spective gene. Arrows indicate the sizes of PCR products derived fromreverse-transcribed mRNA (white) or genomic DNA (black). The pres-ence of cDNA in each sample was confirmed with ACTIN2-specificprimers.

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of Gal and Man interfered with Glc transport, indicat-ing that these hexoses might be additional substrates ofboth STP8 and STP12. In the STP12-expressing yeastcells, Gal and Man reduced [14C]Glc accumulation to24% and 36%, respectively, which is similar to the in-hibitory effect of Glc when it is added in the nonra-dioactive form. This suggests that STP12 mighttransports the hexoses Glc, Gal, and Man at similarrates. In contrast, [14C]Glc uptake still reached 55% or64% of the control in STP8-expressing cells when Gal or

Man was added, indicating that STP8 prefers Glc as asubstrate over Man or Gal. Nonradioactive Fru showedonly a slight interference with Glc uptake via STP8 andSTP12 and, therefore, is probably transported only atminor rates by both transporters. Xyl is an additionalsubstrate for STP12 but not for STP8, as it reduces [14C]Glc uptake via STP12 to 53% but does not influenceGlc transport activities of STP8. Furthermore, Glcuptake via both transporters decreased significantlyin the presence of CCCP (Fig. 2D), an uncoupler of

Figure 2. Characterization of STP8 and STP12 transport properties in baker’s yeast. A, Uptake of [14C]Glc into yeast strainsexpressing STP8 (black circles) or STP12 (gray circles) per mL of packed cells (p.c.) at an initial outside concentration of 100 mM

Glc at pH 5.5. A strain expressing STP12 in the antisense orientation was used as a negative control (triangles). B, Uptake rates forincreasing concentrations of [14C]Glc were determined for the calculation of Km values for D-Glc uptake of the STP8- and STP12-expressing yeast strains according to Lineweaver-Burk. The plot represents mean values and SD of at least three biological rep-licates for each sugar concentration. C, Uptake rates of STP8 and STP12 for [14C]Glc at different pH values at an initial outsideconcentration of 20 mM Glc. D, Determination of substrate specificity and sensitivity to uncouplers of STP8 and STP12. Thetransport activity of STP8 and STP12 for different sugars was determined by competitive inhibition of [14C]Glc uptake (10 mM

initial outside concentration) in the presence of nonradioactive sugars in 10-fold excess at pH 5.5. The addition of 100 mM coldGlc was used as a control. The uncoupler carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) was added to a final concen-tration of 50 mM. Data represent means and SE of three independent biological replicates. *, P# 0.05; **, P# 0.01; and ***, P#

0.001 by Student’s t test.





transmembrane proton gradients. This demonstratesthat STP8 and STP12 transport activity is driven by aproton gradient across the plasmamembrane, as shownpreviously for other STPs (Sauer et al., 1990; Truernitet al., 1996, 1999; Büttner et al., 2000; Scholz-Starkeet al., 2003; Schneidereit et al., 2005; Rottmann et al.,2016). In contrast to STP8 and STP12, the expression ofSTP5 or STP7 in CSY400 did not restore the ability ofthe resulting strains TRY1019 (STP5; Supplemental Fig.S1A) and TRY1021 (STP7; Fig. 3A, dashed line) to takeup [14C]Glc. In growth analyses on medium containingindividual carbon sources, TRY1019 showed no differ-ences in colony formation compared with the negativecontrol TRY1020 expressing STP5 in the antisense ori-entation. This was shown for 21 analyzed hexoses,pentoses, disaccharides, polysaccharides, and sugaralcohols, indicating that none of them is a substrate ofSTP5. A GFP-STP5 fusion localized to the plasmamembrane in yeast cells (Supplemental Fig. S1B), ex-cluding mistargeting of the plant protein in the heter-ologous expression system as a reason for the lack oftransport activity. This indicates that STP5 might in-deed be a pseudogene with a nonfunctional proteinproduct, as was already suggested by Büttner (2007).

Growth tests with Gal as a sole carbon source indi-cated the uptake of this sugar into yeast cells expressingSTP7. This could be confirmed by radioactive uptakemeasurements with [14C]Gal (Fig. 3A). However, theuptake proceeded quite slowly, and the Km value ofSTP7 for Gal uptake was determined to be only 28 mM(Supplemental Fig. S1C). In our search for a bettersubstrate, we observed that STP7-expressing yeast cellsaccumulated radioactive D-Xyl and L-arabinose to evenhigher levels (Fig. 3A). Detailed uptake measurementswith TRY1021 showed that the Km value of STP7 forL-arabinose transport is 290 6 30 mM and that Vmaxreaches 13.8 6 0.76 mmol h21 mL21 (Fig. 3B). The pHoptimum curve for this transport process peaks at pH4 (Fig. 3C). Together with the inhibition of transportupon the addition of the proton gradient uncouplerCCCP (Fig. 3D), this indicates that STP7 is a monosac-charide/H+ symporter, like all other STPs. The uptakeof radioactive L-arabinose via STP7 is inhibited by anexcess of nonradioactive L-arabinose or D-Xyl but notby D-arabinose (Fig. 3D). This suggests that STP7 candistinguish between the two enantiomers of arabinoseand transports only the naturally occurring L-isomerand the structurally closely related D-Xyl. This alsocould be seen in growth tests on agar plates, wherethe growth of STP7-expressing yeast cells was re-duced only on L-arabinose but not on D-arabinose(Supplemental Fig. S4B), although both sugars inhibityeast growth when taken up into the cells. All othersubstances tested, including various hexoses, pentoses,the disaccharide Suc, and even the trisaccharide raffi-nose, reduced the uptake rate of radioactive L-arabinosewhen added in nonradioactive form (Fig. 3D). How-ever, as those substrates showed no influence on yeastcolony formation in a growth analysis, it is more likelythat they bind only to the substrate pocket of STP7 but

are not transported across the membrane. This is fur-ther supported by the fact that an excess of Glc reducedarabinose uptake, but in a direct uptake measurement,[14C]Glc was not transported into the cells (Fig. 3A).Taken together, these results indicate that STP8 andSTP12 are energy-dependent, high-affinity hexose/H+

symporters, whereas STP7 is an H+ symporter for thepentoses L-arabinose and D-Xyl.

Reporter Gene Analysis of STP7, STP8, andSTP12 Expression

To further examine the expression patterns of thefunctional genes STP7, STP8, and STP12, transgenicpSTP:STPg-GFP and pSTP:STPg-GUS Arabidopsisplants were generated. Plants driving reporter geneexpression from a 1,391-bp promoter fragment of STP7were obtained by Agrobacterium tumefaciens-mediatedtransformation of Columbia-0 (Col-0) wild-type plantswith the vectors pTR247 (GUS) or pTR248 (GFP). Of theresulting Basta-resistant plants, at least seven inde-pendent lines were analyzed.

Strong GUS staining could be observed in roots andespecially root tips of 3-d-old seedlings (Fig. 4A). In2-week-old seedlings, GUS expression in roots was re-stricted to the tip of the main root and to the lateral roots(Fig. 4B), with a dark blue staining in lateral root pri-mordia (Fig. 4E). Additionally, the entire leaves (Fig. 4C),including trichomes (Fig. 4D) and the stipules (Fig. 4F),were dark blue. GUS expression in flowers increasedduringflower development (Fig. 4G). Youngflowerswithshort filaments (stage 12; Smyth et al., 1990) showed GUSstaining only in their sepals (Fig. 4H). In flowers withemerged petals (early stage 13) prior to pollination, theanthers and the stigma were stained blue (Fig. 4I) in ad-dition. The staining in these tissues was increased even inopen pollinated flowers of stage 14 (Fig. 4J). A strong bluestaining seemed to originate from mature pollen grains.This could be confirmed by staining of wild-type pistilspollinated with pSTP7:STP7g-GUS pollen (Fig. 4K). In-terestingly, the outgrowing pollen tubes in this semiin vivo experiment did not show GUS activity (Fig. 4K).Thisfinding is consistentwith the absence of STP7mRNAin pollen tubes shown by RT-PCR (Fig. 1). Further GUSstaining was observed in the nectaries of open flowersand developing siliques (Fig. 4L). The high expression ofSTP7 in roots was confirmed by the strong GFP fluores-cence in roots of plants expressing STP7-GFP under thecontrol of the native promoter (Fig. 4, M and N). GFPfluorescence also could be detected in mature pollengrains (Fig. 4O) but not in growing pollen tubes. No GFPfluorescence was detected in green parts of the plants,probably because of the strong autofluorescence of chlo-rophyll.

STP8 reporter plants were obtained by the transfor-mation of wild-type plants with the vectors pTR156(GUS) and pTR157 (GFP) containing a 1,979-bp frag-ment of the STP8 promoter and the whole genomicsequence of STP8 upstream of the GUS or GFP


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sequence. In nonfloral tissues, GUS staining was ob-served only in leaves (Fig. 5, A and B), where it wasrestricted to a patchy pattern along the vasculature,which might indicate an expression in myrosin cells(Shirakawa et al., 2016). No GUS staining was observedin any of the other vegetative tissues. In inflorescences,STP8-GUS expression became visible at first in devel-oping pollen grains of stage 12 flowers (Fig. 5, C andD).Prior to pollination, GUS staining was additionallydetected in the ovules (Fig. 5E). In pollinated pistils,blue staining was observed in pollen grains and ovulesas well as in the stigma and in the transmitting tract

(Fig. 5F). The intense GUS activity in pollen tubesgrown semi in vivo (Fig. 5, G and H) indicates that theGUS staining in stigma and the transmitting tractoriginated from the growing pollen tubes and not fromthe maternal tissue itself. This hypothesis was con-firmed by the analysis of plants expressing GFP as areporter gene. GFP fluorescence in the pistil could beobserved only in ovules and not in the stigma or thetransmitting tissue (Fig. 5I). Additionally, pollen tubesof the same plant showed aweakGFP fluorescence (Fig.5M). Detailed imaging demonstrated that the expres-sion of STP8-GFP in ovules was restricted to the central

Figure 3. Characterization of STP7 transport activity in transgenic baker’s yeast. A, Uptake of 14C-sugars into yeast strainTRY1021 expressing STP7 per mL of packed cells (p.c.) at pH 5.5. Initial outside concentrations of radioactive L-arabinose, D-Xyl,Gal, and Glc were 100 mM. B, Uptake rates for increasing concentrations of [14C]L-arabinose were determined for the calculationof theKm value for L-arabinose uptake of the STP7-expressing yeast strain according to Lineweaver-Burk. The plot represents meanvalues of at least three biological replicates for each sugar concentration including SD. C, Uptake rate of [14C]L-arabinose intoTRY1021 at different pH values with an initial outside concentration of 250 mM L-arabinose. D, Determination of the substratespecificity and sensitivity to uncouplers of STP7. The binding capacity of STP7 for different sugars was determined by competitiveinhibition of [14C]L-arabinose uptake (250 mM initial outside concentration) in the presence of nonradioactive sugars in 10-foldexcess. The addition of 2.5mM cold L-arabinose was used as a control. The uncoupler CCCPwas added to a final concentration of50 mM. Data represent means and SE of three independent biological replicates. *, P# 0.05 and ***, P# 0.001 by Student’s t test.





Figure 4. STP7 promoter activity and subcellular localization of STP7. A to L, Histochemical detection of GUS activity in Ara-bidopsis Col-0 expressing a pSTP7:STP7g-GUS fusion construct. A, Three-day-old seedling. B, Fourteen-day-old seedling withGUS staining in the lateral roots and the tip of the main root (arrowhead). C, Source leaf with trichomes. D, Trichomes at highermagnification. E, Main root with an emerging lateral root. F, Stipules of a 2-week-old seedling. G, Inflorescence with flowers ofdifferent developmental stages. H, Stage 12 flower bud. All flower stages 1 to 20 are according to Smyth et al. (1990).


Rottmann et al.



cell (Fig. 5, J–L). As the C-terminal fusion of GFP toSTP8 led to the localization of the fusion protein in theendomembrane system, the images shown in Figure 5were taken with new reporter plants harboring apSTP8:GFP-STP8g construct after transformation withthe vector pTR239.For STP12 reporter plants, the 1,129-bp fragment

between the STP12 start ATG and the stop codon of thenext upstream gene [At4g21490; NAD(P)H DEHY-DROGENASE B3] was used as a promoter. However,the nine analyzed lines all showed different expressionpatterns of STP12-GUS, which became especially ob-vious in seedlings (Supplemental Fig. S2). This indi-cated that the chosen promoter sequence was too short

and the expression of the construct, therefore, depen-ded greatly on the regulatory setting of the insertionlocus. Hence, a longer promoter fragment of 3,972 bpupstream of the start ATG of STP12was used for a newreporter gene construct. This fragment included thegenomic sequence of the upstream gene At4g21490with all introns. Of the plants transformed with thenew constructs pSTP12long:STP12g-GUS (pTR221) andpSTP12long:STP12g-GFP (pTR222), all analyzed linesshowed a consistent expression pattern. In seedlings,GUS staining was observed only in stipules (Fig. 6, Aand B) and emerging lateral roots (Fig. 6A), where itwas restricted to the base of the root primordia (Fig.6C). Flowers showed blue staining in ovules within the

Figure 4. (Continued.)I, Unpollinated flower in early stage 13. J, Pollinated stage 14 flower with strong GUS signal in pollen grains. K, Pollen tubesgrown semi in vivo on a wild-type stigma. L, Nectaries at the base of a young silique. M to O, Detection of GFP fluorescence(green) in pSTP7:STP7g-GFP reporter plants. M, Young lateral root; GFP channel with bright-field overlay. N, Single opticalsection of epidermis cells in a lateral root. O, Anther with mature pollen grains. Chlorophyll autofluorescence is given in red.P and Q, Single optical section (P) and maximum projection (Q) of a mesophyll protoplast expressing GFP-STP7c under thecontrol of the 35S promoter. Bars = 250 mm in A, 2.5 mm in B and C, 500 mm in D and G to J, 100 mm in E, K, L, and O, 50 mm inF, and 10 mm in M, N, P, and Q.

Figure 5. STP8 promoter activity in pSTP8:STP8g-reporter plants and subcellular localization of STP8. A to H, Histochemicaldetection of GUS activity in Arabidopsis Col-0 expressing a pSTP8:STP8g-GUS construct. A, Seven-day-old seedling with GUSstaining in the vicinity of the vascular tissue. B, Source leaf with a patchyGUS pattern along the vasculature. C, Inflorescencewithflowers of different developmental stages. D, Stage 12 flower primordium with developing pollen grains. E, Unpollinated flower.F, Pollinated flower. The arrowhead indicates pollen tubes growing through the ovary. G, Pollen tubes grown semi in vivo througha wild-type stigma. H, Pollen tubes at higher magnification. I to M, Detection of GFP fluorescence (green) in pSTP8:GFP-STP8greporter plants. I, Peeled ovary with GFP fluorescence in the ovules. Chlorophyll autofluorescence is given in red. J, Single sectionof the GFP-marked central cell in an isolated ovule with bright-field overlay. K, Maximum projection of an excised ovule stainedwith propidium iodide. L, Single section of a central cell in highermagnification.M, Tip of a pollen tube grown semi in vivo. N andO, Single optical section (N) and maximum projection (O) of a mesophyll protoplast expressing GFP-STP8c under the control ofthe 35S promoter. Bars = 1 mm in A and B, 2.5 mm in C, 500 mm in D to F, 50 mm in G and I, and 10 mm in H and J to O.






ovaries (Fig. 6, D and E), which was already detectableprior to pollination (Fig. 6F). The staining was evenmore intense and extended to the transmitting tissue inpistils, which had been opened prior to staining (Fig.6H). This staining pattern persisted in young siliques(Fig. 6G). As pSTP12long:STP12g-GUS pollen germi-nated semi in vivo on wild-type pistils did not showGUS expression (Fig. 6I), the GUS activity in thetransmitting tract did not originate from pollen tubesgrowing through but from the transmitting tissue itself.This finding is consistent with the result of the RT-PCR(Fig. 1), where also no STP12 transcripts were detectedin pollen tubes. The strong expression of STP12 inovules was confirmed by pSTP12long:STP12g-GFP lines,where GFP could be detected in ovules only (Fig. 6J).Optical sections (Fig. 6, K and M) and overlay projec-tions (Fig. 6L) of excised ovules revealed the restrictionof STP12-GFP expression to the outer layer of the innerintegument. STP12-GFP fluorescence persisted duringthe first days of embryo development (Fig. 6N).

Optical sections of STP7-GFP roots (Fig. 4N), STP8-GFP central cells (Fig. 5L), and integumental cells ofSTP12-GFP lines (Fig. 6M) already suggested that allthree proteins are localized in the plasma membrane.To further analyze their subcellular localization, STP-GFP and GFP-STP fusion constructs were expressed inArabidopsis protoplasts under the control of the 35Spromoter. In single optical sections (Fig. 4P) and max-imum projections (Fig. 4Q), the GFP-STP7 fusion pro-tein labeled the plasma membrane. STP7-GFP also waslocalized in the plasma membrane, with some proteinsremaining in the endomembrane system (SupplementalFig. S3, A and B). The GFP-STP8 fusion protein clearlylocalized in the plasma membrane (Fig. 5, N and O),whereas the fusion of GFP to the C terminus ofSTP8 greatly interfered with protein localization(Supplemental Fig. S3, C and D). Both GFP-STP12 andSTP12-GFP localized to the plasmamembrane (Fig. 6, Oand P; Supplemental Fig. S3, E and F), indicating that allthree STPs are plasma membrane proteins.

Characterization of stp8 and stp12 T-DNA Insertion Lines

To further analyze the physiological roles of STP8and STP12 in reproduction and lateral root develop-ment, Arabidopsis T-DNA insertion lines for the re-spective genes were characterized. Sequencing of themutant allele of Wiscseq_DsLox504H01 (Woody et al.,2007) showed that this line carries an insertion in theSTP8 gene 247 bp after the start codon at the end of thefirst intron (Fig. 7A). SALK_07655C (Alonso et al., 2003)is a T-DNA insertion line for STP12, and sequencingidentified the exact position of the insertion to be1,146 bp after the start codon in the middle of the thirdexon (Fig. 7D). As no other mutant lines for those STPshave been named so far, we refer to these lines as stp8.1and stp12.1, respectively. Homozygous stp8.1 andstp12.1 mutant plants were identified by PCR (Fig. 7, Band E). RT-PCR analyses with total mRNA of

homozygous stp8.1 and stp12.1 plants confirmed thecomplete loss of the respective full-length transcripts incomparison with wild-type plants (Fig. 7, C and F).Truncated mRNAs from the regions upstream of theinsertions could be detected in both mutants. However,in both cases, a possible translation of the partial mRNAwould lead to a truncated and, therefore, probablynonfunctional protein lacking several of the predictedtransmembrane helices, which are necessary to form afunctional hexose transporter. Downstream of the in-sertion in stp8.1, nomRNA fragment could be amplifiedby RT-PCR (Fig. 7C), whereas the insertion in stp12.1allows transcription of a downstream fragment (Fig.7F). Homozygous stp8.1 and stp12.1 lines were ana-lyzedwith respect to seed production, as ovules expressboth STP8 and STP12, indicating that both transportproteins might be involved in sugar accumulation indeveloping seeds. However, the mutants showed nosignificant differences in number or size of seeds com-pared with the wild type (Fig. 7, G and H). Further-more, the determination of soluble carbohydratecontents by ion chromatography revealed no signifi-cant decrease in inositol, Glc, and Suc accumulation instp8.1 or stp12.1 mutant seeds (Fig. 7I). No differencecould be observed in stp8.1 and stp12.1 seed germina-tion rates and time compared with the wild type (datanot shown). To directly compare the fertility of wild-type and stp8.1 ovules, a cross-pollination assay wasperformed. Pistils of a heterozygous STP8/stp8.1 plantwere pollinated with wild-type pollen. The descendantgeneration showed a 50:50 segregation ratio of hetero-zygous and wild-type plants (Fig. 7J), indicating thatwild-type and mutant alleles are inherited equally andthat the lack of STP8 in the central cell does not interferewith the fertility of the ovule. Even though STP8 isadditionally expressed in pollen tubes, in vitro pollengrowth assays revealed no significant difference inpollen tube length of stp8.1 pollen compared with thewild type (Fig. 7K). Reporter gene lines indicated strongexpression of STP12 in developing lateral roots andweaker expression in root tips. However, no differencesbetween stp12.1 and the wild type were detected con-cerning the length of the main root (Fig. 7L) and thenumber of lateral roots (Fig. 7M) on growth mediumcontaining Suc, Glc, or no sugar. As reported previ-ously (Rottmann et al., 2016), STP10 also is stronglyexpressed in young lateral roots, and the knockout ofSTP10 does not reduce lateral root formation. Thismight point to a redundant function of STP10 andSTP12 in lateral roots. Therefore, double knockoutplants homozygous for stp10.1 and stp12.1 were gen-erated and analyzed for lateral root formation, but thedouble knockouts produced asmany lateral roots as thewild type and both single knockout lines (Fig. 7M).

Characterization of stp7 T-DNA Insertion Lines

The transport activity of STP7 for L-arabinose indi-cates that this transporter might be involved in the


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Figure 6. STP12 promoter activity and subcellular localization of STP12. A to I, Histochemical detection of GUS activity inArabidopsis Col-0 expressing a pSTP12long:STP12g-GUS construct. A, Seven-day-old seedling with GUS staining in the lateralroots (arrowhead) and the stipules (arrow). B, Leaf base with stipules at higher magnification. C, Emerging lateral root with GUSstaining at the proximal end. D, Inflorescence with flowers of different developmental stages. E, Pollinated flower with GUSstaining in the ovules. F, Unpollinated flower. G, Young silique. H, Peeled ovary with GUS staining in ovules and the transmitting

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uptake of free arabinose released during the degra-dation of cell wall glycoproteins into the cells forrecycling. To further study this possible function, twoT-DNA insertion lines of STP7 were analyzed. Forboth lines, GK-157F08 and GK-253B12 (Kleinboeltinget al., 2012), which are referred to as stp7.1 and stp7.2,respectively, homozygous plants were identified byPCR (Fig. 8B) and the exact positions of the insertionswere determined by sequencing. Line stp7.1 carries aninsertion 680 bp after the start codon in the third exon.The insertion of stp7.2 lies in the same exon, but1,069 bp downstream of the start ATG (Fig. 8A).RT-PCR with RNA preparations from young siliquesconfirmed that both lines are real knockout lineshaving no full-length transcripts of STP7 (Fig. 8C).Transcripts of STP7 upstream and downstream of theinsertion sites could be amplified from both mutantlines (Fig. 8C), but as the insertions lie in the middle ofthe CDS, translation of those transcripts would lead totruncated and, therefore, probably nonfunctionalproteins. Plants of the homozygous stp7.1 and stp7.2lines were analyzed with regard to roots and pollen, asthose tissues/cells showed the strongest expression ofSTP7 in the reporter gene analysis. No differencescompared with the wild type could be detected con-cerning the length of the main root (Fig. 8D) and thenumber of lateral roots (Fig. 8E). Pollen tube growthin vitro also was not altered in stp7.1 and stp7.2 incomparison with the wild type (Fig. 8F). The mutantswere self-fertile and produced viable seeds in the samequantity as the wild type (Fig. 8G), indicating that theknockout of STP7 does not interfere with plant de-velopment. A missing-mutant phenotype of stp7knockout plants does not contradict the hypothesisthat STP7 might be involved in the recycling of L-arabinose, as also the ara1-1 mutation of the arabino-kinase, which is essential for arabinose recycling, doesnot lead to a phenotype under normal growth condi-tions (Dolezal and Cobbett, 1991). However, the ara1-1 plants are sensitive to arabinose and fail to grow on10 mM L-arabinose medium because of misinterpre-tation of the arabinose accumulation within the cells(Behmüller et al., 2016). To analyze whether STP7 isthe main transporter for arabinose uptake in therecycling process, stp7.2 and ara1-1 were crossed.However, the resulting stp7.2/ara1-1 double knockoutplants were not able to grow on 10 mM L-arabinose(Fig. 9A), indicating that the plants still take up arab-inose. This suggests that, despite the high expression

of STP7 in roots, the STP7 protein is not the onlytransporter involved in arabinose uptake from the rootapoplast into the cells.

L-Arabinose Transport Activity of Other STPs

The persistent arabinose sensitivity of stp7.2/ara1-1 double knockout plants led to the question of whichother transporters of the STP family might additionallycontribute to L-arabinose transport. To analyze this, theCDS of all STPs were amplified, cloned into the yeastvector NEV-N, and expressed heterologously in theyeast strain CSY4000, leading to the strains listed inSupplemental Table S4. For better comparability, allforward primers used for the amplifications attached15 bp of the STP1 59 UTR in front of the start codons,which should optimize the expression of the plantgenes in yeast cells. The STP-expressing yeast strainswere used for uptakemeasurements with radioactive L-arabinose in different concentrations. As shown inFigure 9C, almost all strains were able to accumulateradioactive L-arabinose, indicating that almost all STPsaccept L-arabinose as an additional substrate. Besidesthe probably nonfunctional STP5, only STP3 was notable to mediate L-arabinose uptake. The functionality ofSTP3 in CSY4000was verified by uptakemeasurementswith radioactive Glc (data not shown). Yeast strainswith STP6, STP8, STP11, or STP13 only took up minoramounts of L-arabinose even at high initial outsideconcentrations. The highest uptake rates were observedfor strains with STP1, STP4, and STP12. Interestingly, ina competitive inhibition experiment, the uptake of [14C]Glc via STP12 was reduced by the addition of both L-arabinose and D-arabinose, indicating that, in contrastto STP7, this transporter did not distinguish betweenthe two enantiomers (Supplemental Fig. S4A). This wasfurther confirmed by comparative growth tests onplates containing 50 mM either L- or D-arabinose. L-Arabinose inhibited the growth of both STP7- andSTP12-expressing yeasts, whereas D-arabinose onlyreduced the growth of yeasts expressing STP12(Supplemental Fig. S4B). The fact that the uptake ratesof STP4 and STP14 did not rise further with increasingconcentrations of L-arabinose indicated that theirtransport capacity was already saturated at thelowest tested concentration of 50 mM L-arabinose. Thiswas confirmed by determination of their Km values forL-arabinose transport (Fig. 9B). The resulting Km valuesof 33.4 6 5 mM for STP4 and 17.6 6 3.8 mM for STP14

Figure 6. (Continued.)tract. I, Pollen tubes grown semi in vivo through a wild-type stigma. J to N, Detection of GFP fluorescence (green) in pSTP12long:STP12g-GFP reporter plants. J, Peeled ovaries with GFP fluorescence in the ovules. Chlorophyll autofluorescence is given in red.K, Single section of an excised ovule stained with propidium iodide (red). L, Maximum projection of K. M, Single section of theovule integument at higher magnification. N, Single section and bright-field overlay of a developing seed 3 d after pollinationstained with propidium iodide (red). O and P, Single optical section (O) and maximum projection (P) of a mesophyll protoplastexpressingGFP-STP12c under the control of the 35S promoter. Bars = 1 mm in A and D, 50 mm in B, C, I, J, and N, 500 mm in E toG, 100 mm in H, and 10 mm in K to M, O, and P.


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Figure 7. Characterization of the stp8.1 (Wiscseq_DsLox504H01) and stp12.1 (SALK_07655C) T-DNA insertion lines. A and D,Genomic organization of STP8 (A) and STP12 (D). Exon regions containing coding sequences (gray bars) are numbered; intronsand UTRs are shown as black lines. Arrows indicate the primers used in B, C, E, and F. The positions of the T-DNA insertions aremarked. LB, Left border; RB, right border. B and E, PCR products obtained from genomic DNA preparations of Col-0 and ho-mozygous stp8.1 (B) or stp12.1 (E) plants with primer combinations for the detection of the respective wild-type allele (WT) and





were quite similar, but the maximum uptake rate ofSTP4 (Vmax = 113.7 6 8.5 mmol h21 mL21 packed cells)was almost 10-fold higher than that of STP14 (Vmax =13.2 6 1 mmol h21 mL21 packed cells).

DISCUSSION

This article elucidates the tissue specificity and ki-netic properties of STP7, STP8, and STP12, three pre-viously uncharacterized members of the Arabidopsismonosaccharide transporter family.

Substrate Specificities, Kinetics, and SubcellularLocalizations of STP7, STP8, and STP12

Analyses of the transport activities in yeast demon-strated that STP8 and STP12 are high-affinity, energy-dependent H+/monosaccharide symporters that acceptGlc, Gal, Man, and Fru as substrates, similar to mostother STPs. STP12 additionally mediated the uptake ofXyl, like STP1 to STP4, STP11, and STP13 (Büttner,2010). The Km values of STP8 and STP12 for Glc liewithin themicromolar range (24.86 2.1 and 17.46 0.9mM,respectively) and are comparable to the values mea-sured for most other STPs characterized so far (Saueret al., 1990; Truernit et al., 1996, 1999; Schneidereitet al., 2003, 2005; Scholz-Starke et al., 2003; Rottmannet al., 2016), with the exception of STP3, which has alower affinity for Glc (Büttner et al., 2000). It was al-ready known that STP7 does not transport Glc(Büttner, 2010), but surprisingly, STP7 was able tomediate L-arabinose uptake, with a Km value of 290 630 mM, and, furthermore, transported D-Xyl. In com-parison with this uptake activity, the very slow uptakeof Gal via STP7 is negligible. This indicates that, incontrast to all other STPs, STP7 does not transporthexoses but is specific for the pentoses L-arabinose andD-Xyl. L-Arabinose is a C3 epimer of D-Xyl and is struc-turallymore similar to D-Xyl than to D-arabinose. Thismay

explain why D-arabinose is not transported by STP7.Arabinose transport via STPs has been reported for STP1(Boorer et al., 1994) and STP2 (Subtil and Boles, 2011), butthe main substrates of both transporters are hexoses, andthey only transport L-arabinose atminor rates. STP7 seemsto have a quite unusual substrate-binding specificity, assome structurally very diverse molecules (trisaccharidesand sugar alcohols) competed with L-arabinose uptakewhen added in 10-fold excess. As none of these com-poundswas taken up into the cells, it is likely that they canbind to the outer substrate pocket of STP7 but do not passthe specificity filter for final transport across the mem-brane. The observed inhibitory effect of the uncouplerCCCP indicated that STP7, STP8, and STP12 use the en-ergy of the proton gradient across the plasma membraneand work as proton symporters like all other STPs. Yeastcells expressing STP5 transported none of the 21 analyzedhexoses, pentoses, disaccharides, polysaccharides, andsugar alcohols.As STP5-GFP localized to the yeast plasmamembrane, targeting to internal membranes or insuffi-cient transcription and translation can be ruled out asreasons for the missing uptake activity. This leaves thepossibilities that STP5 (1) catalyzes the uptake of so faruncharacterized substrates, (2) acts as an exporter, or (3) isnonfunctional. It is not unusual that large gene families ofmembers with partly redundant functions contain one ormore pseudogenes (Sauer et al., 2004; Schneider et al.,2007). The identification of 17 nucleotide substitutions,two of which cause amino acid exchanges, and a partialdeletion of the third intron of the genomic STP5 sequenceof the C24 ecotype compared with Col-0 (Büttner, 2010)further support the pseudogene theory.However, the factthat STP5 branches very early in the phylogenetic tree ofSTPs and only shows a low similarity to other STPs(Johnson and Thomas, 2007) supports the hypothesis thatit may transport a substrate quite different from those ofother STPs.

The expression of fusion constructs withGFP showeda clear localization of STP7, STP8, and STP12 in the

Figure 7. (Continued.)the mutant alleles (m). For primer combinations, see Supplemental Table S5. C and F, RT-PCR analyses of pollen tube RNAobtained from a homozygous stp8.1 mutant plant and of flower mRNA from a homozygous stp12.1 plant. stp8.1 and stp12.1mRNAs as well as corresponding mRNAs of wild-type plants were used as templates in RT-PCR with primers amplifying the STPsequences traversing, upstream of, or downstream of the insertion. Wild-type genomic DNAwas used as a control for genomiccontamination. PCR with ACTIN2-specific primers confirmed the presence of intact cDNA. For primer combinations, seeSupplemental Table S6. G to M, Phenotypic analyses of stp8.1 and stp12.1 mutant plants. G, Average number of seeds per si-lique6 SD after self-pollination of wild-type, stp8.1, and stp12.1 plants; n$ 50 siliques per genotype. H, Average area of matureseeds6 SD of homozygous stp8.1 and stp12.1 plants and their respective wild types; n$ 160 seeds per genotype. I, Comparisonof myoinositol, Glc, and Suc contents in ethanol extracts from mature seeds of stp8.1, stp12.1, and their respective wild-typeplants. Extracts of dry seeds of at least three plants per genotypewere analyzed by ion chromatography and normalized to the freshweight (FW) of the samples. J, Genotypes regarding STP8 in the F1 descendants of a cross-pollination experiment with hetero-zygous stp8.1/STP8 pistils and pollen from awild-type plant. Bars represent mean values6 SE of the percentage of each genotypein the F1 generation of four independent crossings (n = 78 in total). K, Lengths of stp8.1 and wild-type pollen tubes germinatedin vitro for 6 h. Mean values 6 SE of three biological replicates are shown (n . 250 for each genotype in each experiment).L, Means6 SE of the main root length of 12-d-old stp12.1 andwild-type seedlings onMurashige and Skoog (MS) mediumwithoutsugars (MS-0), with 2% (w/v) Glc (MS-Gluc), or with 2% (w/v) Suc (MS-Suc); n. 20 for each sample.M,Number of lateral roots of12-d-old stp10.1, stp12.1, stp10.1/stp12.1 double knockout, and wild-type seedlings on MS medium without sugars; n . 20 foreach sample.


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plasma membrane of Arabidopsis protoplasts, which isthe reported subcellular localization of all characterizedSTPs (Büttner, 2010). Obviously, no STP is targeted tothe tonoplast, andmonosaccharide transport across thismembrane is mediated by proteins of the Vacuolar GlcTransporter and Tonoplast Monosaccharide Trans-porter subfamilies (Büttner, 2007).

STP8 May Contribute to Sugar Uptake into Cells of theMyrosinase-Glucosinolate System

Promoter-reporter gene analyses demonstrated STP8expression predominantly in pollen grains, pollentubes, and ovules andweaker in cells along the vasculartissue of leaves. The only other STPs expressed in leavesare STP3 (Büttner et al., 2000) and STP14 (Poschet et al.,

2010). The cells marked by STP8-GUS in leaves mightrepresent myrosin idioblasts, which, in Arabidopsis,are localized in the phloem parenchyma or the adjacentglucosinolate-containing cells (Andréasson et al., 2001).Both cell types are part of the myrosinase-glucosinolatesystem that is regarded as a defense system againstgeneralist herbivores (Rask et al., 2000). Myrosin cellsstore large amounts of b-thioglucoside glucohydrolase(TGG) in their vacuoles. Tissue disruption destroys thecell membranes and, thereby, brings myrosinase intocontact with glucosinolates of neighboring cells,resulting in the production of a variety of toxic degra-dation products (Wittstock and Halkier, 2002). The twoisoforms of TGG in Arabidopsis show an expres-sion pattern very similar to STP8 when fused to GUS(Barth and Jander, 2006). As the synthesis of largeamounts of enzymes or glucosinolates requires a lot of

Figure 8. Characterization of stp7 T-DNA insertion lines. A, Genomic organization of STP7. Exon regions containing codingsequences (gray bars) are numbered; introns and UTRs are shown as black lines. The positions of the T-DNA insertions in stp7.1(GK-157F08) and stp7.2 (GK-253B12) in the third exon are indicated. Arrows indicate positions of the primers used for PCRshown in B and C. LB, Left border; RB, right border. B, PCR products obtained from genomic DNA preparations of Col-0 andhomozygous stp7.1 and stp7.2 plants with primers for the detection of the wild-type alleles (WT) and for the mutant alleles (m).For primer combinations, see Supplemental Table S5. C, PCR analyses of cDNAs derived from flower RNA of homozygous stp7.1and stp7.2mutants and of awild-type plant with primers amplifying the STP7 sequence traversing, upstream of, or downstream ofthe insertions. Wild-type genomic DNA was used as a control for genomic contamination. PCR with ACTIN2-specific primersconfirmed the presence of intact cDNA. For primer combinations, see Supplemental Table S6. D toG, Phenotypic analyses of stp7mutant plants. D, Length ofmain roots of 11-d-old stp7.1 and stp7.2 seedlings in comparisonwith thewild type onMS-0medium.Means of three biological replicates6 SD are shown; n. 30 for each genotype. E, Average number6 SD of lateral roots of 11-d-old stp7.1, stp7.2, andwild-type seedlings onMSmediumwithout sugar; n. 30 for each sample. F, Lengths of stp7.1, stp7.2, andwild-type pollen tubes germinated in vitro for 6 h onmedium containing 250 or 200mM Suc.Mean values6 SE of three biologicalreplicates are shown (n . 250 for each genotype in each experiment). G, Average number of seeds per silique 6 SD after self-pollination of wild-type, stp7.1, and stp7.2 plants; n $ 30 siliques per genotype.







metabolic energy, the expression of STP8 as an importerfor sugars could contribute to the sustenance of myr-osin idioblasts or glucosinolate cells.

Sugar Uptake into Reproductive Cells via STP8 and STP12

The strongest expression of STP8 was observed inflowers in both the male and female gametophytes. Aspollen tubes grow very rapidly, they probably have toimport carbohydrates from the apoplast to cover theirenergy demand. Furthermore, their cell wall is com-posed mainly of callose, which is synthesized fromUDP-Glc and, therefore, enforces the need for carbo-hydrate uptake via sugar transporters (Chen and Kim,

2009). In addition to STP8, it has already been shownthat STP2, STP4, STP6, STP9, STP10, and STP11 areexpressed exclusively or preferentially in the male ga-metophyte (Truernit et al., 1996, 1999; Schneidereitet al., 2003, 2005; Scholz-Starke et al., 2003; Rottmannet al., 2016), which indicates a high functional redun-dancy. This redundancy also might be the explanationfor the absence of an obvious phenotype regardingpollen tube growth in the stp8.1 knockout plants. In-terestingly, although many STPs are expressed in pol-len, they all show different temporal expressionpatterns. The male-specific expression of STP8-GUSfirst became visible shortly before anthesis and per-sisted during pollen germination and tube growth,which is different from all other STPs (Truernit et al.,

Figure 9. Analysis of stp7.2/ara1-1 double mutants and L-arabinose transport activity of different STPs. A, L-Arabinose treatmentof wild-type Col-0, ara1-1, stp7.2, and ara1-1/stp7.2 double knockout plants. Plants were grown on MS plates with 10 mM L-arabinose for 18 d. Plants grown on 10 mM Suc served as a control. B, Uptake rates of STP4- or STP14-expressing yeast strains atincreasing concentrations of [14C]L-arabinosewere determined for the calculation of theKm values according to Lineweaver-Burk.The plot represents mean values of at least three biological replicates for each sugar concentration including SD. C, L-Arabinoseuptake rates of yeast strains expressing different STPs at distinct initial outside concentrations of arabinose at pH 5.5. Ayeast strainexpressing STP2 in the antisense orientation was used as a negative control (neg.). p.c., Packed cells.


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1996, 1999; Schneidereit et al., 2003, 2005; Scholz-Starkeet al., 2003; Rottmann et al., 2016). In contrast to STP4,STP6, STP9, STP10, and STP11, STP8 is present in stage12 flowers, when pollen grains are almost mature, andunderwent pollen mitosis (Sanders et al., 1999). In thisstage, pollen grains probably do not take up sugars forstorage any longer, indicating that the STP8 protein inpollen grains might rather be a prearrangement forimmediate sugar uptake during the initiation of pollengermination. Expression during pollen developmentand in pollen tubes also has been reported for AtSUC1,encoding a Suc transport protein. AtSUC1was reportedto be necessary for efficient pollen germination, as suc1knockout pollen displayed reduced fertility in segre-gation analyses (Stadler et al., 1999; Sivitz et al., 2008).In addition to AtSUC1, at least four other Suc trans-porter genes (Meyer et al., 2004; Qin et al., 2009; Leydonet al., 2013, 2014) and the gene for CELL WALL IN-VERTASE2 (cwINV2;Hirsche et al., 2009) are expressedin pollen tubes. The transmitting tissue probably pro-vides the growing pollen tubes with carbohydrates,most likely in the form of Suc that is unloaded from thephloem. Suc could either be taken up directly into thepollen tubes via SUCs or cleaved by cwINV2 intomonosaccharides that are imported via STPs. Theoverlapping expression of at least six high-affinity butlow-capacity monosaccharide transporters and fivelow-affinity but highly efficient Suc transporters inpollen tubes might ensure that the growing pollen tubeis, in any case, provided with enough energy to com-plete its journey and fertilize the egg cell.The expression of STP8 in central cells and of STP12

in the inner integument fits well into the model of en-ergy supply to ovules. Whereas ovule primordia aresymplastically connected to the phloem, no functionalplasmodesmata are present between inner and outerintegument in mature ovules and phloem tracers arenot able to enter the ovule. The outer integumentfunctions as a symplastic extension of the funicularphloem, and Suc delivered via the phloem can movefreelywithin this seed coat cell layer (Stadler et al., 2005;Werner et al., 2011). The Suc efflux carrier SWEET15,which is located in the outer integument, is likely re-sponsible for mediating Suc efflux from the outer in-tegument into the apoplast toward the innerintegument (Chen et al., 2015). After cleavage by cellwall invertases, the resultingmonosaccharides could beimported into the inner integument by STP12. Micro-array data indicate that, in addition to STP12, alsoSTP1, STP5, STP7, and STP14 are expressed in the seedcoat (Büttner, 2010; Chen et al., 2015). However, STP5,STP7, and STP14 do not transport Glc, and the expres-sion of STP1 is quite low. stp12 knockout plants showedno obvious phenotype regarding seed production,sugar concentration in seeds, or seed germination, in-dicating that STP1 or the gene of another Glc-transporting STP might be induced to functionallycomplement the missing STP12. The next gaps insymplastic connectivity lie between the inner integu-ment, embryo sac, and endosperm. SWEET11 may be

involved in the release of Suc from the inner integu-ment, and SWEET12 appears to play a role in thetransport of Suc out of cells at the micropylar end of theseed coat (Chen et al., 2015). The cells of the embryo sacneed to import sugar via transport proteins. The strongexpression of STP8 in the central cell indicates that therespective transporter might be responsible for sugaruptake into this cell type of the embryo sac. The centralcell also might transfer the sugar to the symplasticallyconnected antipodals (Mansfield et al., 1991). However,the egg cell and the synergids are not connected to thecentral cell via plasmodesmata, and no sugar trans-porter gene has been reported to be expressed in thesecells. Therefore, it is not known whether these cells dotake up sugars prior to fertilization at all or if they feedon storage components imported earlier. As theknockout of STP8 does not interfere with ovule fertility,the uptake of Glc into the mature central cell is eithernot obligatory or the loss of STP8 can be complementedby another sugar transporter. STP8 expression ceasessoon after fertilization, whereas STP12 is expressed upto 3 d after fertilization. This is in line with the obser-vation that, during early stages of seed development,the embryo contains a high level of hexoses, whereas inlater stages, the embryo is fed mainly with Suc by thetransporters SUC3 and SUC5 (Baud et al., 2002, 2005;Meyer et al., 2004). Taken together, STP8 and STP12probably contribute to sugar transport into symplasti-cally isolated cells of the ovules. Analyses of double/multiple stp and suc knockout plants will be necessaryto elucidate the physiological roles of both transportertypes for seed development.

STP12 May Be Involved in Sugar Supply to DevelopingLateral Roots

Reporter gene analysis revealed that STP12 also isexpressed at the base of emerging lateral roots. Incontrast to the expression in the inner integument, thisexpression was only detectable with the more sensitiveGUS system but not in GFP reporter plants, indicatingthat STP12 expression in roots is lower compared withthat in ovules. Both the integument and roots are pho-tosynthetically inactive tissues and rely on the import ofsugars; hence, STP12 expression is sink specific, as isreported for most STPs (Büttner, 2010). Besides STP12,other STPs are expressed in root tissues like vasculature(STP1 and STP13) and root tips (STP4 and STP7;Truernit et al., 1996; Sherson et al., 2000; Schofield et al.,2009; Büttner, 2010; Cordoba et al., 2015). STP10 showsan expression pattern very similar to STP12, with a highexpression level in lateral root primordia (Rottmannet al., 2016), indicating that STP10 and STP12 mighthave at least partly redundant functions. However,stp12 single as well as stp10/12 double knockout plantsdid not exhibit any alterations in lateral root growth. Aslateral roots are fast growing but photosyntheticallyinactive, they rely on sugar supply from the phloem,especially as roots are reported to store almost no starch





(Tsai et al., 2009). Sugar unloading from the phloem inroots has been suggested to occur mainly via the sym-plastic distribution of Suc (Oparka et al., 1994). How-ever, the expression of CELL WALL INVERTASE1(Büttner, 2010) and of genes for at least five hexose-transporting STPs indicates that Suc also might beexported into the apoplast, cleaved into monosaccha-rides, and taken up by STPs. This combination ofsymplastic and apoplastic sugar transport could ensurea sufficient provision of nutrients to fast-growing tis-sues. It has been proven in maize (Zea mays) that sym-plasmic diffusion of Suc alone cannot satisfy the carbondemands of the primary root tip (Bret-Harte and Silk,1994). Hofmann et al. (2009) could show, based onmicroarray and quantitative PCR data, that STP12 isup-regulated in root syncytia induced by the nematodeHeterodera schachtii and that the STP12 transporter ap-pears to play a major role in hexose import into thesefeeding structures. Together with the fact that the ex-pression of many STPs is regulated by their substrates(Price et al., 2004; Cordoba et al., 2015; Rottmann et al.,2016), diurnal rhythms (Stadler et al., 2003; Büttner,2010; Poschet et al., 2010), wounding (Truernit et al.,1996), pathogens (Stadler et al., 2003; Büttner, 2010;Poschet et al., 2010), or abiotic stresses (Yamada et al.,2011), this indicates that the expression of STPs is highlyflexible and can be adjusted to external circumstances.

STP7 Is a Transporter for the Cell Wall Pentoses D-Xyland L-Arabinose

Analysis of pSTP7:STP7g-GUS plants revealed theexpression of the STP7 gene in roots of 3-d-old seed-lings, root tips and lateral roots of older seedlings,leaves with trichomes and stipules, sepals, nectaries,stigmata, and pollen grains. Comparison with the lesssensitive GFP reporter system indicated that root tips,lateral roots, and pollen are the sites of strongest STP7expression. Hence, STP7 expression is not sink specific,but the common feature of almost all tissues showingSTP7 expression is that they are in a process of devel-opment. Growth and developmental processes inplants are usually accompanied by cell wall turnover.This involves the degradation and recycling of cell wallpolysaccharides, oligosaccharides, and glycoproteins.The sugars Glc, Rha, Gal, Xyl, arabinose, GalUA, andGlcA are the major sugar constituents of the cell wall,with L-arabinose accounting for 5% to 10% of cell wallsaccharides in Arabidopsis (Zablackis et al., 1995). Ithas been reported that L-arabinose-containing mole-cules undergo hydrolysis by glycoside hydrolases andthat the resulting free L-arabinose can be recycled toUDP-L-arabinose that can be reused for cell wall com-ponent synthesis. This recycling reaction is catalyzed bythe arabinokinase ARA1 and the UDP-sugar pyro-phosphorylase USP (Geserick and Tenhaken, 2013). Asboth enzymes are localized in the cytosol, the recyclingof L-arabinose from the cell wall requires uptake fromthe apoplast into the cell. This step could be mediated

by STP7, which is located in the plasma membrane andis able to transport L-arabinose. Furthermore, STP7 alsocould contribute to the recycling of Xyl, as this is an-other important cell wall sugar and an additional sub-strate of STP7. The expression of STP7 in tissues that donot undergo significant cell wall turnover, like fullydeveloped leaves, could contribute to the removal ofsugars from the apoplast, which is discussed as animportant defense mechanism against apoplasticpathogens by depriving them of potential nutrients(Lemonnier et al., 2014; Dodds and Lagudah, 2016). Italso has been reported that, under certain circum-stances, such as prolonged darkness, sugar starvation,or leaf senescence, cell wall sugars are remobilized bythe activity of glycosyl hydrolases and, thus, can bereimported into the cell for further metabolism (Leeet al., 2007; Poschet et al., 2010).

The high expression of STP7 in pollen is in line withthe assumption that the arabinose salvage pathwayplays an important role in developing pollen and con-tributes to a portion of the pectic arabinan of the pollencell wall: First, the rice (Oryza sativa) L-arabinokinaseCAP1 has been shown to be necessary for the normaldevelopment of pollen (Ueda et al., 2013). Additionally,knockdown of USP influences pollen development(Schnurr et al., 2006; Kotake et al., 2007; Geserick andTenhaken, 2013) as well as the knockdown of UAM3 inrice, where the UAM3 protein is necessary for theconversion of UDP-Ara in the furanose form to UDP-Ara in the pyranose form (Sumiyoshi et al., 2015). Incontrast, knockout of STP7 did not interfere with pollenfertility. Similarly, the stp7.1 and stp7.2 knockout linesdid not show differences in root development com-pared with the wild type. However, it has been repor-ted that, for other tissues, L-arabinose recycling is notessential, as there also exists a de novo synthesis path-way (Dolezal and Cobbett, 1991). To test whether STP7is the main L-arabinose transporter in Arabidopsis, thestp7.2 knockout line was crossed with the ara1-1 mu-tant. The ara1-1 mutation leads to a developmental ar-rest of plants grown on 10 mM L-arabinose as a result ofhigh intracellular L-arabinose levels (Behmüller et al.,2016). Additional mutation of the main L-arabinoseuptake transporter should lead to at least partial com-plementation of the ara1-1 phenotype. However,stp7.2/ara1-1 double knockout plants were not able togrow on L-arabinose-containing medium, suggestingthat they still take up L-arabinose and that STP7 is notthe only L-arabinose transporter in roots. This could beconfirmed by analyses of the L-arabinose uptake ac-tivities of all STPs. With the exception of STP3 andSTP5, all STPs mediated the uptake of at least minoramounts of L-arabinose. Taking into account that, be-sides STP7, also STP1, STP4, STP10, STP12, and STP13are localized in roots and they all transport L-arabinose,functional redundancy may be the explanation forpersistent L-arabinose uptake and sensitivity in stp7.2/ara1-1 double knockout plants. The detailed analysis ofSTP4 and STP14 L-arabinose uptake revealed that theyboth have similar Km values but quite different


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maximum uptake rates. With respect to all 12 L-arabinose-transporting STPs, this could mean also thatL-arabinose uptake can be precisely regulated and ad-justed to every tissue by the combination of expressedSTPs.The Km values of STP4 and STP14 for L-arabinose

(33.4 6 5 and 17.6 6 3.8 mM, respectively) are the lowestfor all eukaryotic L-arabinose transporters known so far.This makes the members of the STP family also interest-ing candidates for expression in yeast cells designedfor ethanol production from lignocellulosic biomass.Depending on plant species, this raw material containsvarying amounts of Glc, Gal, Man, Xyl, and arabinose(Lee, 1997). S. cerevisiae is the most commonly selectedmicrobe for biomass fermentation because of its highethanol productivity and tolerance for inhibitors presentin biomass hydrolysates, as well as the fact that it isamenable to genetic engineering (Li et al., 2015). Wild-type S. cerevisiae is unable to utilize L-arabinose and D-Xyl as fermentative substrates, but heterologous pentoseutilization pathways from pentose-assimilating orga-nisms have been introduced (Subtil and Boles, 2011).However, the efficient uptake of pentose sugars into yeastcells is still a limiting factor for the cofermentation of sugarmixtures, as found in biomass hydrolysates. The endog-enous yeast transporters ScGal2, ScHXT9, and ScHXT10only transport minor amounts of arabinose, and its up-take is inhibited in sugar mixtures containing Glc or Gal(Subtil and Boles, 2011). This also is the case for mostother arabinose transporters from naturally arabinose-fermenting fungus species that were tested in baker’syeast, such as KmAXT1 from Kluyveromyces marxianus orPgAxt1p fromPichia guilliermondii (Knoshaug et al., 2015).Bacterial arabinose transporters do not support the up-take of sugars into yeast cells, as most of them are nottargeted correctly to the plasma membrane (Subtil andBoles, 2011). The use of members of the STP family couldrule out some of these drawbacks: they are localized in theyeast plasma membrane and mediate the uptake ofarabinosewith high affinity anddiverse velocities. STP7 ispentose specific, additionally excluding the inhibition ofarabinose and Xyl uptake by an excess of Glc in sugarmixtures, as is typical for lignocellulosic biomass.

MATERIALS AND METHODS

Strains, Growth Conditions, and Genotyping

Arabidopsis (Arabidopsis thaliana ecotype Col-0) was grown under long-dayconditions (16 h of light/8 h of dark) at 22°C and 60% relative humidity or in thegreenhouse in potting soil. Plants used for the generation of protoplasts weregrown under a short-day regime (8 h of light/16 h of dark). For the analysis ofseedlings or roots, seeds were cultivated on MS plates (Murashige and Skoog,1962). The T-DNA insertion lines stp7.1 (GK-157F08; Kleinboelting et al., 2012),stp7.2 (GK-253B12; Kleinboelting et al., 2012), stp8.1 (Wiscseq_DsLox504H01;Woody et al., 2007), and stp12.1 (SALK_07655C;Alonso et al., 2003)were obtainedfrom the Nottingham Stock Centre (http://arabidopsis.info/). The ara1-1mutantline (Dolezal and Cobbett, 1991) was kindly provided by Raimund Tenhaken(Division Plant Physiology, University of Salzburg). The primers used for geno-typing are listed in Supplemental Table S5. Segregation analysis of the stp8.1 allelewas performed by PCR-based genotyping with the same primer pairs. Geno-typing of the ara1-1 point mutant was done by modified mismatch amplification

mutation assay coupled with melt analysis (Melt-MAMA; Birdsell et al., 2012)with forward primers ending either with the base of the mutant or the wild-typeallele and a common reverse primer. Instead of separating wild-type and mutantPCRproducts bymelting temperature, PCR formutant andwild-type alleleswerecarried out in two separate reaction tubes and analyzed on a 2% agarose gel. Thepositions of the T-DNA insertions were determined by the sequencing of PCRproducts obtained from mutant genomic DNA preparations with the respectiveprimer pairs (Supplemental Table S5). Arabidopsis was transformed viafloral dipwith Agrobacterium tumefaciens strain GV3101 (Holsters et al., 1980; Clough andBent, 1998). Escherichia coli strain DH5a (Hanahan, 1983) was used for all cloningsteps. Heterologous expression analyses were performed in Saccharomyces cer-evisiae strain CSY4000 (Rottmann et al., 2016).

RNA Isolation and RT-PCR

Total RNA was isolated from different Arabidopsis tissues with TRIzol re-agent (Invitrogen). For RNA isolation from pollen tubes, pollen of about30 flowers was germinated in vitro on a cellulosic membrane for at least 5 h(Rodriguez-Enriquez et al., 2013). Pollen tubes were collected by vortex mixingthe membrane in 500 mL of TRIzol. The QuantiTect Reverse Transkription Kit(Qiagen) was used for the reverse transcription reaction. PCR for the detectionof STP5, STP7, STP8, and STP12 transcripts were carried out with the primerpairs listed in Supplemental Tables S1 and S6. A PCR with primers for ACTIN2was performed as a positive control.

Cloning of Reporter Gene Constructs for STP7, STP8,and STP12

For the pSTP7:STP7g-reporter plants, a 2,934-bp fragment including the ge-nomic sequence of STP7 and 1,391 bp upstream of the start ATG was amplifiedwith the primer pair STP7-1391f+CACC and STP7c+1539r (Supplemental TableS2), cloned into pENTR/D-TOPO (Invitrogen), and inserted upstream of theGUS- or GFP-nos terminator box by LR reaction in pBASTA-GUS or pBASTA-GFP (Rottmann et al., 2016), yielding plasmids pTR247 and pTR248, respectively.For reporter plants expressing GUS or GFP fusions of STP8 under the control ofthe native promoter, a 2,903-bp fragment was amplified with primers STP8g-1979f+CACC and STP8g+920r (Supplemental Table S2) and cloned intopENTR/D-TOPO (Invitrogen). The second part of STP8g was amplified with theprimer pair STP8g+840f and STP8g+1921r+A+AscI. The resulting 1,081-bp frag-ment was subcloned into pCR-Blunt II-TOPO (Thermo Scientific) and inserteddownstream of the first part of STP8 via the internal MfeI site and the attachedAscI site. The complete pSTP8:STP8g sequence was finally cloned into pBASTA-GUS or pBASTA-GFP by LR reaction, yielding plasmids pTR156 and pTR157,respectively. To generate the construct pSTP8:GFP-STP8g, the 35S promoter ofpMDC43 was substituted by a 1,979-bp fragment of the STP8 promoter regionamplified with primers STP8g-1797f+SbfI and STP8g-1r+KpnI (SupplementalTable S2) via the attached SbfI and KpnI sites. The genomic sequence of STP8wasamplified with STP8c+1f+CACC and STP8c+1524r and inserted downstream ofGFP via LR reaction. For the pSTP12:STP12g-reporter constructs with the shortpromoter, a 3,095-bp fragment was amplified with the primers STP12g-1129f+CACC and STP12g+1962r and cloned into pENTR/D-TOPO, yielding vectorpTR150. For the constructs with the extended promoter, a 3,667-bp fragmentupstream of the STP12 CDS was amplified with primers STP12g-3972f+NotI andSTP12g-314r and ligated into pTR150 via the NotI site of the pENTR/D-TOPObackbone and the MfeI site internal to the STP12 promoter sequence. BothpSTP12long:STP12g and pSTP12short:STP12g were then brought into pBASTA-GUS and pBASTA-GFP via LR reaction, leading to plasmids pTR158 (GUS)and pTR159 (GFP)with the short promoter and pTR221 (GUS) and pTR222 (GFP)with the extended promoter.

For the subcellular localization of STP7, STP8, and STP12, fusion constructswith GFP under the control of the 35S promoter were generated. The codingsequences of the STPs were amplified from constructs pTR215 (STP7), pTR164(STP8), or pTR162 (STP12; see below) with the primer pairs listed inSupplemental Table S3. For STPc-GFP fusion constructs, the reverse primerslacking the stop codon were used, and for GFP-STPc fusions, the reverseprimers were 3 bp longer to include the stop codons. Both PCR fragments ofSTP7were cloned into pJET1.2/blunt (Thermo Scientific) and then inserted intothe NcoI site of pSS87 (Schneider et al., 2012) for C-terminal and pCS120(Dotzauer et al., 2010) for N-terminal GFP, yielding plasmids pTR250 andpTR251, respectively. PCR products of STP8 and STP12 were ligated intoTOPO/pENTR (Invitrogen) and then inserted into pMDC83 (Curtis andGrossniklaus, 2003) for STP-GFP or pMDC43 (Curtis and Grossniklaus, 2003)




http://arabidopsis.info/











for GFP-STP, yielding plasmids pTR176 (STP8c-pMDC83), pTR177 (STP8-pMDC43), pTR178 (STP12c-pMDC43), and pTR179 (STP12c-pMDC83).

Isolation and Transformation of Protoplasts

Leaf mesophyll protoplasts were generated from Col-0 plants as described(Drechsel et al., 2011) and transformed via the polyethylene glycol method(Abel and Theologis, 1994). Transformed protoplasts were incubated for ap-proximately 40 h at 22°C in the dark prior to microscopic analysis.

Microscopy

Protoplasts, GFP-reporter plants, and GFP-expressing yeast cells were analyzedon aLeicaTCSSPII confocal laser scanningmicroscope (LeicaMicrosystems) using a488-nm argon laser for excitation and processed with Leica Confocal Software 2.5.Detection windows ranged from 497 to 526 nm for GFP, from 589 to 684 nm forpropidium iodide, and from 682 to 730 nm for chlorophyll autofluorescence. Imagesof GUS plants were taken with a stereomicroscope (Leica MZFLIII; Leica Micro-systems) or amicroscope (Zeiss Axioskop; Carl Zeiss). Imageswere processed usingthe analySIS Doku 3.2 software (Soft Imaging System).

Functional Characterization of STPs by HeterologousExpression in Baker’s Yeast

The CDS of STPs were amplified from cDNA preparations from differenttissues (Supplemental Table S4) with the primer pairs listed in SupplementalTable S4 that introduced a NotI site on both sides of the PCR products as well asthe sequence 59-AAGCTTGTAAAAGAA-39 (part of the STP1 59 UTR; Stadleret al., 1995) upstream of the start codon. Due to low expression levels, the CDS ofSTP2, STP3, and STP5 had to be amplified in two overlapping fragments each (forprimers, see Supplemental Table S4), which were then assembled via an internalBamHI (STP2) or MluI (STP3 and STP5) site. All complete CDS fragments wereligated into theNotI site of the vector NEV-N (Sauer and Stolz, 1994), yielding theplasmids listed in Supplemental Table S4. STP5c was additionally amplifiedwithprimers attaching an NcoI site to both ends of the fragment and ligated intoplasmid NEV-CGFP (see below). The constructs were then used for lithiumacetate-mediated transformation (Soni et al., 1993) of S. cerevisiae CSY4000(Rottmann et al., 2016), yielding the strains listed in Supplemental Table S4. Forgrowth tests and uptake experiments, yeast strains were precultured in maltose-casamino acids medium (0.67% [w/v] yeast nitrogen base, 1% [w/v] casaminoacids, 0.01% [w/v] Trp, and 2% [w/v] maltose) to anOD600 of 1. For growth tests,yeasts were washed and then diluted to an OD600 of 2 in water. Eight-fold serialdilutions were spotted onmedium containing 0.01% (w/v) Trp and 10 mM of thesugar/sugar alcohol to be tested as sole carbon source or 10 mM maltose as acontrol. Plates were incubated at 29°C for at least 5 d. Transport tests with 14C-labeled sugars were performed as described (Sauer and Stadler, 1993). In uptakeexperiments for the determination of Km values, pH dependency, and substratespecificity, yeasts were incubated with the 14C-labeled sugars for 5 min to keepuptake in the linear range.

Generation of the Yeast Expression Vector NEV-CGFP

For the analysis of plant protein localization in yeast cells, a new expressionvector was generated that allows the insertion of a CDS upstream of GFP viaNcoI. To this end, GFP released from pSO35s (Klepek et al., 2005) by digestionwithNcoI/PciI was inserted into theNcoI site of NEV-Nco (Nieberl et al., 2017),leading to the new plasmid NEV-CGFP.

Pollen Germination Assays

In vitro and semi in vivo pollen germination for RNA extraction, growthanalysis, and reporter gene detection were performed as described (Rottmannet al., 2016). Pollen tube length was measured with a self-written half automaticprogram in Python (Python Software Foundation) and plotted with Matplotlib(Hunter, 2007), which also was used for all other graphs.

Analysis of Carbohydrate Content

Preparation of plant material and determination of carbohydrate content viaion chromatography were performed as described by Schneider et al. (2008).

Accession Numbers

The Arabidopsis Genome Initiative accession numbers for the genes used inthis study are as follows: At1g34580 (STP5), At4g02050 (STP7), At5g26250(STP8), At4g21480 (STP12), and At4g16130 (ARA1).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Analyses of STP5 and STP7 expressing baker’syeast.

Supplemental Figure S2. Analyses of pSTP12short:STP12g-GUS plants.

Supplemental Figure S3. Confocal images of the subcellular localization ofSTP7-GFP, STP8-GFP, and STP12-GFP in Arabidopsis protoplasts.

Supplemental Figure S4. Analysis of arabinose transport activity ofSTP12-expressing S. cerevisiae.

Supplemental Table S1. List of primers used for the detection of STPtranscripts in different tissues by RT-PCR.

Supplemental Table S2. List of primers used for the generation of pSTP:STPg-GUS and pSTP:STPg-GFP reporter lines.

Supplemental Table S3. List of primers used for the generation of STPc-GFP and GFP-STPc fusion constructs for expression in protoplasts.

Supplemental Table S4. List of primers and templates used for the ampli-fication of STPs for the generation of constructs for heterologous expres-sion in baker’s yeast.

Supplemental Table S5. Primers used for PCR-based genotyping of theT-DNA insertion lines stp7.1, stp7.2, stp8.1, stp10.1, and stp12.1 and of thepoint mutant ara1-1.

Supplemental Table S6. Primers used for the detection of STP transcriptsin the T-DNA insertion lines stp7.1, stp7.2, stp8.1, stp10.1, and stp12.1 byRT-PCR.

ACKNOWLEDGMENTS

We thank Carola Schroeder for excellent experimental help and RaimundTenhaken for providing the Arabidopsis mutant ara1-1.

Received October 17, 2017; accepted December 29, 2017; published January 8,2018.

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Sugar Transporter STP7 Specificity for l-Arabinose and d ... · STP7 mediates the uptake of the pentoses L-arabinose and D-Xyl. In addition, our transport analyses revealed that almost