The sugar transporter inventory of tomato: genome-wide identification and expression analysis.

Plant and Cell Physiology Advance Access published May 14, 2014

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The Sugar Transporter Inventory Of Tomato: Genome-Wide Identification And Expression Analysis.

Short title: Identification Of Tomato Sugar Transporters.

Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601 Japan

Mail: [email protected] Tel/Fax: +81 52789 4026

Subject areas: membrane and transport

Number of black and white figures: 2 Number of color figures: 6 Number of tables: 1 Supplemental Tables: 1

© The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For Permissions, please e-mail: [email protected]

Downloaded from http://pcp.oxfordjournals.org/ at Ohio State University on May 27, 2014

Prof K. Shiratake

The Sugar Transporter Inventory Of Tomato: Genome-Wide Identification And Expression Analysis.

Short title: Identification Of Tomato Sugar Transporters. 1, †

1, †

1

1

2

3

Stefan Reuscher , Masahito Akiyama , Tomohide Yasuda , Haruko Makino , Koh Aoki , Daisuke Shibata

1

Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601 Japan

2

Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Gakuen-cho, Sakai 599-8531

Japan 3

Kazusa DNA Research Institute, Kazusa-kamatari, Kisarazu 292-0818, Japan

†

Both authors contributed equally.

*

Corresponding author.

Abbrevations: AA amino acid, BLAST base local alignment search tool, ERD6 early response to dehydration 6, EMS ethylmethanesulfonate, EST expressed sequence tag, HT hexose transporter, INT inositol transporter, MST monosaccharide transporter, pGlcT plastidic glucose translocator, PM plasma membrane, PMT polyol monosaccharide transporter, SFP sugar facilitator protein, SOT sorbitol transporter, ST sugar transporters, STP sugar transporter protein, SUT sucrose transporter, TMD transmembrane domain, TMT tonoplast monosaccharide transporter, VGT vacuolar glucose transporter, VM vacuolar membrane

Footnotes: The nucleotide sequences reported in this paper have been submitted to the DNA Database of Japan (DDBJ) with the accession number: AB845639 to AB845668


1, *

and Katsuhiro Shiratake

Abstract

The mobility of sugars between source and sink tissues in plants depends on sugar transport proteins. Studying the corresponding genes allows the manipulation of the sink strength of developing fruits and thereby improve fruit quality for human consumption. Tomato (Solanum lycopersicum) is both, a major horticultural crop and a model for the development of fleshy fruits. In this article we provide a comprehensive inventory of tomato sugar transporters, including the SUCROSE TRANSPORTER family, the SUGAR

TRANSPORTER

PROTEIN

family,

the

SUGAR

FACILITATOR

PROTEIN

family,

the

POLYOL/MONOSACCHARIDE TRANSPORTER family, the INOSITOL TRANSPORTER family, the PLASTIDIC GLUCOSE TRANSLOCATOR family, the TONOPLAST MONOSACCHARIDE TRANSPORTER family and the VACUOLAR GLUCOSE TRANSPORTER family. EST sequencing and phylogenetic analyses established a

putatively encoding sugar transporters. The expression of 29 sugar transporter genes in vegetative tissues and during fruit development was analyzed. Several sugar transporter genes were expressed in a tissues- or developmental stage-specific manner. This information will be helpful to better understand source-to-sink movement of photoassimilates in tomato. Identification of fruit-specific sugar transporters might be a first step to find novel genes contributing to tomato fruit sugar accumulation.

Keywords

EST, Fruit development, Gene expression analysis, Sugar transporter, Tomato


nomenclature for all analyzed tomato sugar transporters. In total we identified 52 genes in tomato

List of table and figures

Table 1: Comprehensive nomenclature and feature list of 52 sugar transporters identified in tomato.

Figure 1: Phylogenetic analysis of tomato sugar transporters. Figure 2: Exon-Intron structure of 52 sugar transporters identified in tomato. Figure 3: Phylogenetic analysis of the SUCROSE TRANSPORTER family. Figure 4: Phylogenetic analysis of the SUGAR TRANSPORTER PROTEIN family. Figure 5: Phylogenetic analysis of the EARLY RESPONSE TO DEHYDRATION 6/SUGAR-PORTER FAMILY PROTEIN family. Figure 6: Phylogenetic analysis of the POLY/MONOSACCHARIDE TRANSPORTER family.

Figure 8: Expression analysis of selected tomato sugar transporters.


Figure 7: Phylogenetic analysis of five tomato sugar transporter protein families.

Introduction

In plants carbohydrates are used as a universal energy currency e.g. in the form of sucrose, as building blocks for cell walls, for signaling purposes, to maintain osmotic homeostasis under certain abiotic stress conditions and for various other purposes. Carbohydrates are also one of the most important sources of calories and taste in cultivated crops and are thus critical for both the nutritional and the commercial value of crops. Apart

sources of energy and as metabolites. Also two classes of carbohydrates related to sugars have complimentary roles in plants. Sugar alcohols (also called polyols) like sorbitol, mannitol or inositol and organic acids like malate or citrate have a variety of functions, often characteristic to a distinct clade of plants. Movement of sugars on the whole plant level is controlled by loading and unloading of transport tissues, thereby creating sink- and source organs (Bresinsky and Körner 2011). Typical sources are photosynthetically active tissues, but also storage organs, like tubers, bulbs or the starchy endosperm of graminaceous plants can act as source tissues. In contrast to this, sink tissues rely on the supply of photoassimilates from other parts of the plants. These tissues typically include the roots and leaves during early stages of development, but also developing buds, flowers and fruits are considered sink tissues. In the majority of plant species sucrose is the preferred long-distance transport form of carbohydrates in the phloem. Both, loading and unloading from phloem vessels or from companion cells require active transmembrane-transport of sugars (Turgeon and Wolf 2009). On the intracellular level sugars are transported between different compartments. Most notably, the vacuole serves as a storage organ for diverse carbohydrates (Shiratake and Martinoia 2007). The transport of sugars across the plasma membrane (PM) or the vacuolar membrane (VM) is mediated by transport proteins either utilizing active transport mechanisms or enabling passive diffusion at an accelerated rate. In the case of source-to-sink transport active loading and unloading processes are essential to create a sugar concentration gradient which drives the movement of water and solutes in the phloem. While the sucrose transporter family (SUCs or SUTs) is a rather small protein family with three members in tomato and nine members in Arabidopsis, the proteins that transport monosaccharides are a lot more diverse.


from sucrose, monosaccharides like glucose, fructose, galactose, mannose, or ribose play crucial roles as

Up to 53 members in Arabidopsis were reported (Doidy et al. 2012). They are separated in seven families, which can be found across the plant kingdom, including mosses. This suggests that the presence of these seven families represent an evolutionary ancient condition (Johnson et al. 2006). The currently recognized subfamilies are, in the sequence of descending number of members in tomato: the SUGAR TRANSPORTER PROTEIN family (STPs or MSTs for monosaccharide transporters or HT for hexose transporters), the SUGAR FACILITATOR PROTEIN family (SFPs) which is also called EARLY RESPONSE TO DEHYDRATION 6-like (ERD6-like) family, the POLYOL/MONOSACCHARIDE TRANSPORTER family (PMTs or PLTs), the INOSITOL TRANSPORTER FAMILY (INTs or ITRs), the TONOPLAST MONOSACCHARIDE TRANSPORTER family (TMTs), the PLASTIDIC

Members from these families have been identified in a large variety of plant species, including grape and rice, leading to an ambiguous nomenclature (Afoufa-Bastien et al. 2010; Johnson and Thomas 2007). If feasible, in this study we will follow the nomenclature used for Arabidopsis family members. While for some families (namely the SUCs and the STPs) comprehensive data (transport activity, expression data, subcellular localization, transgenic approaches) exists, many members from the smaller families are scarcely characterized, especially in non-model species. Each family will be described in greater detail in the Results and Discussion chapter, taking into account the newly gained information from tomato. All analyzed protein families belong to the major facilitator super family of proteins (MFS family) which is characterized by 12 transmembrane domains (TMDs). The novel SWEET family of sugar transporters was identified in 2010 and belongs to a different superfamily which is characterized by seven TMDs (Chen et al. 2010; Chen et al. 2012; Xuan et al. 2013). Because of these differences and their relative novelty SWEET transporter will not be included in this study, although there are 32 SWEET transporters encoded in the tomato genome.

Sugar accumulation during tomato fruit development Developing fruits are strong sinks for sugars and adequate supply from photosynthetic tissues is critical for accumulation of sugars during fruit development (Ho et al. 1982). Sugar supply for fruit metabolism and storage occurs via the phloem. After unloading from the phloem sugars are distributed within the fruit using both apoplastic and symplastic pathways (Patrick and Offler 1996; Ruan and Patrick 1995). Ripe fruits of commercial tomato variants contain mostly glucose and fructose at an equimolar concentration but next to no


GLUCOSE TRANSLOCATOR family (pGlcTs) and the VACUOLAR GLUCOSE TRANSPORTER family (VGTs).

sucrose (Klee and Giovannoni 2011). This is due to the combined activity of cell wall-bound and vacuolar invertases which also contribute significantly to fruit sink strength although their exact contribution is not fully understood. (Klann et al. 1996; Jin et al. 2009; Yelle et al. 1991; Zanor et al. 2009). While the transporters for the loading of sucrose into the phloem are identified, less is known about how photoassimilates from mesophyll cell reach the phloem or how export towards developing fruits is facilitated (Riesmeier et al. 1992; Riesmeier et al. 1994). Recently, transporters from the SWEET family were found to export sucrose to the leaf apoplast for subsequent loading into companion cells by SUT transporters (Chen et al. 2012). Uptake of hexoses from the fruit apoplast to the storage parenchyma cells is thought to be achieved

RNAi-mediated knock-down (Ruan et al. 1997; Dibley et al. 2005; McCurdy et al. 2010). Although organic acids are important for fruit metabolism and also contribute to the taste of tomato fruits relatively little is known about the factors that determine their concentration in ripe fruits. A number of metabolomics studies on the ripening of tomato fruit showed dynamic patterns of accumulation (Carrari et al. 2006; Carrari et al. 2007; Carrari and Fernie 2006; Luengwilai et al. 2010; Roessner-Tunali et al. 2003; Schauer et al. 2006). However, it is currently unknown how much de novo synthesis and import of organic acids contribute to the final content in ripe fruits.

A comprehensive transporter inventory. The genes characterized in aforementioned studies about tomato fruit development and sugar accumulation were discovered by reverse genetic methods or through homologous cDNA cloning. With the complete genome sequence of tomato available it became possible to use forward genetic methods to gain a more complete picture of the genes involved in sugar accumulation during tomato fruit ripening (Sato et al. 2012). A first step towards this goal is to create a comprehensive inventory of all putative sucrose and monosaccharide transporters encoded in the tomato genome. EST databases found at e.g. the Sol Genomics Network (http://www.solgenomics.net/) or TOMATOMICS (http://www.bioinf.mind.meiji.ac.jp /tomatomics/)(Aoki et al.

2010) can then be used to confirm the gene models predicted in the reference genome set and to obtain fulllength cDNAs for cloning. Also transcriptome data (at TOMATOMICS) and metabolomic data of Solanaceae species (KaPPA-View4 SOL at http://www.kpv.kazusa.or.jp/kpv4-sol/) is available and can be integrated into


by the hexose transporters LeHT1 and LeHT3 based on transport activity of fruit discs, expression patterns and

the gene inventory data. A dwarf variety of tomato, called ‘Micro-Tom’ is used as a model system for tomato genetics and physiology because of its small size and shortened life-cycle compared to commercial cultivars (Meissner et al. 1997). Ethylmethanesulfonate (EMS) mutant lines and gamma ray irradiation-induced mutant lines

of

‘Micro-Tom’

have

been

generated

and

are

available

from

TOMATOMA

(http://www.tomatoma.nbrp.jp/index.jsp) (Saito et al. 2011).

In this study we comprehensively analyzed the sugar transporter (ST) gene families in tomato. We identified 52 genes putatively encoding sugar transporter proteins in the tomato reference genome (Sato et al. 2012). Out

families. Within each family the AA sequences of putative STs from tomato were compared to characterized members of this family from other plant species. Since it is accepted that the expression of a gene points to a function at the place and time of expression we performed an expression analysis for selected STs that had EST evidence. By doing this we hoped to find genes with interesting expression patterns, with a special focus on fruit development.


of these 35 showed EST evidence. Using phylogenetic methods we assigned all putative ST genes to known

Results and Discussion Identification of putative sugar transporters BLAST searches of the translated tomato genome, using the AA sequences of characterized STs as queries, identified 52 loci putatively encoding full-length sugar transporters from diverse subfamilies (Table 1). From 35 of these loci at least one EST was available in public databases. Loci without EST evidence might only show expression under certain conditions or in specific tissues or might represent pseudogenes. All loci encoding AA sequences shorter than 100 AA were discarded, since they are very unlikely to encode functional transport proteins. Out of the 35 ESTs, four clones (annotated as STP9, SFP3, SFP5 and INT2) contained non-sugar

Sequencing of EST clones confirmed the predicted exon-intron structure in most cases. In the case of differences between predicted and experimentally found gene models the gene model determined by EST sequencing was used for further analysis. In the case of SlSTP16 the EST showed a 2 bp deletion (leading to a premature termination codon) compared to the reference genome. We assumed this deletion to be an artifact of EST cloning and used a corrected sequence for further analysis. Additionally, this deletion might be specific to Micro-Tom and is thus not found in the Heinz reference genome. A phylogenetic analysis using all putative tomato ST AA sequences assigned the identified STs to eight subfamilies (SUC/SUT, STP, SFP/ERD6-like, PMT/PLT, INT/ITR, pGlcT, TMT, VGT) which is in accordance with current literature (Fig. 1). The gene models of the STs analyzed in this study showed between no intron (STP family) and up to 17 introns (SFP family) (Fig. 2). The number of exons and their length was found to be conserved within the subfamilies, whereas the length of the introns was more variable. Hereafter, tomato ST subfamilies and their members will be discussed in comparison with current literature.

SUTs/SUCs In tomato three AA sequences were identified as belonging to the SUCROSE TRANSPORTER family (Fig. 3). All three putative tomato SUTs showed EST evidence. The proteins consist of 512 (SlSUT1), 605 (SlSUT2) and 501 AA (SlSUT4), respectively, forming 11 or 12 predicted TMDs. While SlSUT1 and 4 showed comparable exonintron structures (four and five exons, respectively), SlSUT2 contained 14 exons. Also, SlSUT2 featured an


transporter cDNAs, contrary to their database annotation.

additional cytoplasmic loop typical for SUT2-type proteins, which is not found in SUT1 or SUT4 subfamily members. Our results are consistent with the earlier identification of three tomato sucrose transporters (SlSUT1, SlSUT2 from Barker et al. 2000 and SlSUT4 from Weise et al. 2000). Importantly, we did not identify additional members of the sucrose transporter family. In a phylogenetic analysis the tomato sucrose transporters clustered together with members from the SUT1 (SlSUT1), SUT2 (SlSUT2) and SUT4 (SlSUT4) clades from other organisms (Kühn and Grof 2010). Members of the SUT1 clade from dicots were shown to be localized to the PM in several species and also SlSUT1 was reported to localize to the PM (Kühn et al. 1997). Immunogold-labelling localized SlSUT2 to the PM of sieve elements (Barker et al. 2000). Transient expression

depending on the position of the GFP reporter gene (Schneider et al. 2012). SUT family members are well established as sucrose/H+-symporters (Sauer 2007; Shiratake 2007; Kühn and Grof 2010). Proteins from Arabidopsis and tomato were localized to sieve elements, where they facilitated the loading of sucrose into the phloem. SlSUT1, a high-affinity transporter is thought to be the main importer of apoplastic sucrose into the phloem and knock-down of the SlSUT1 gene resulted in accumulation of photosynthesis products in leaves (Hackel et al. 2006). Members of the SUT2 clade, including SlSUT2 feature additional cytosolic domains and share sequence similarities with yeast sugar sensors (Barker et al. 2000). In tomato, SlSUT2 is probably involved in pollen tube growth (Hackel et al. 2006). Additional roles for SUT/SUCtype transporters were reported for those found exclusively in Arabidopsis. For example, SUC2 was found to play a role in the retrieval of sucrose along the phloem pathway (Srivastava et al. 2008; Srivastava et al. 2009; Gould et al. 2012). Another Arabidopsis-specific transporter (AtSUC5) was recently reported as a biotin transporter, raising the question of substrate specificity of the SUT/SUC family (Pommerrenig et al. 2013). The manipulation of SUTs to improve fruit sugar content seems to be promising. Although ripe tomatoes contain very little sucrose thanks to the action invertases the developing tomato fruit is a sink for sucrose (Roitsch and González 2004). During fruit development sucrose is imported into the fruit and subsequently cleaved to glucose and fructose. These in turn contribute to fruit sweetness or can be used as energy for fruit development. Increasing the sucrose import of developing tomato fruits might therefore increase the sugar content of the ripe fruit. However, tomato sugar content depends on many factors, including source strength,


in protoplasts showed that SlSUT4 fusion proteins localize to the vacuolar membrane or additionally to the ER,

fruit import, invertase activity and intra-fruit transport, so increasing sucrose import to the fruit alone might not be sufficient.

STPs The SUGAR TRANSPORTER PROTEIN family (STP), also called MONOSACCHARIDE TRANSPORTER family (MST) in rice or HEXOSE TRANSPORTER family (HT) in grape is the largest subfamily of STs in tomato with 18 members (Fig. 4). Nine of these were supported by EST evidence. The average AA sequence length was about 500 AA, encompassing 11 or 12 predicted TMDs. The gene models of most of the subfamily members featured three or

Some proteins were predicted as cytosolic, although more than ten TMDs were present. All previously characterized STPs are hexose/H+ symporters and SlSTP2 and 3 localized to the PM when expressed in yeast (Doidy et al. 2012). Their preferred substrate seems to be glucose, but many STPs show a broader substrate specificity, including galactose and mannose. Fructose transport activity was shown for some STPs, but seems to be less common in this family. While most information is available for the Arabidopsis STPs, some tomato STPs were characterized (Büttner 2010). Originally tomato STPs were designated LeHT1 to 3 which does not conform to current nomenclature, so we suggest to refer to them as SlSTP1 to 3. SlSTP1 and SlSTP2 are functional, energy dependent glucose transporter when expressed in yeast (Gear et al. 2000). Four novel tomato STPs (SlSTP6, 8, 10, 14) could be grouped together with SlSTP1, AtSTP1, and VvHT1 in a separate clade. Strikingly, no EST was available for the novel tomato genes. AtSTP1 was the first plant STP shown to transport glucose when expressed in yeast (Sauer et al. 1990). In grape, VvHT1 was proposed to transport hexoses during early fruit development based on gene expression analysis and in situ hybridization experiments (Vignault et al. 2005; Fillion et al. 1999). SlSTP2 was the only tomato STP in a separate subgroup together with one STP each from Arabidopsis, rice and grape, suggesting a conserved origin. In this subgroup VvHT5 seems to have a specialized role in the carbohydrate supply to tissues under biotic stress (Hayes et al. 2010). Similarly, AtSTP13 expression was found to be correlated with programmed cell death following fungal infection (Norholm et al. 2006). AtSTP13 also positively influenced plant growth and N content when overexpressed (Schofield et al. 2009).


four exons. The proteins were predicted to localize to the PM, the vacuolar membrane VM or the chloroplast.

SlSTP3 clustered together with AtSTP7 and VvHT3, all of which showed no sugar transport activity when analyzed in a yeast system (McCurdy et al. 2010; Büttner 2010; Hayes et al. 2007). In this subgroup two novel STPs, supported by EST evidence, were identified in the tomato genome (SlSTP12 and 18). Further research is necessary to find a transport substrate and ultimately the function of STPs from this subgroup. The uncharacterized proteins SlSTP15-17 formed a separate clade, together with VvHT4 and AtSTP3. VvHT4 is a functional hexose transporter when expressed in yeast (Hayes et al. 2007). AtSTP3 is a low affinity transporter of glucose and probably other hexoses (Büttner et al. 2000). Another clade is formed by SlSTP4 together with OsMST1, AtSTP5 and VvHT2. While for VvHT2 no data is available, both OsMST1 and AtSTP5 showed no

tomato STP protein in a small subgroup together with AtSTP4, 9 and 11. Of these, AtSTP9 and AtSTP11 were characterized as functional hexose transporters expressed in Arabidopsis pollen (Schneidereit et al. 2003, Schneidereit et al. 2005). Other pollen-specific STPs from Arabidopsis (AtSTP2, AtSTP6) clustered together with SlSTP5, 7, 11 and 13 (Scholz-Starke et al. 2003, Truernit et al. 1999). It is thus possible, as shown for Arabidopsis, that also tomato has a relatively high number of STPs which are specifically expressed in pollen.

SFPs/ERD6-like In our phylogenetic analysis 10 AA sequences were identified as members of the SUGAR FACILITATOR PROTEIN family (SFP), also called EARLY RESPONSE TO DEHYDRATION 6-like family (ERD6-like) (Fig. 5). For six members EST evidence was found. The length of full-length SFP AA sequences ranged from 396 to 518 AA. Full-length SFP proteins featured between 10 and 12 predicted TMDs and all of them were predicted to localize to the PM. The proteins encoded by the loci SlSFP4, 6, 9 and 10 formed a clade together with the partially characterized AtERD6-like and BvERD6-like proteins. Another clade was formed by SlSFP1, 2 and 5 together with the 11 grape SFPs (VvSFP1 to 11), none of which is characterized. The founding member of the family, AtERD6 was shown to be induced under dehydration and cold stress but any evidence for transport activity is lacking (Kiyosue et al. 1998). Also for two partially characterized proteins from Arabidopsis, AtSFP1 and AtSFP2, no transport evidence is available (Quirino et al. 2001). More recently studies of AtESL1, another member of the ERD6-like subfamily, indicated that ERD6-like members facilitate diffusion of glucose and a range of other hexoses (Yamada et al. 2010). Additionally it was shown that a tri-


glucose transport activity when assayed in yeast (Büttner 2010; Toyofuku et al. 2000). SlSTP9 was the only

leucine motif in the N-terminal region of AtESL1 is necessary for localization to the VM. This motif is also found in SlSFP1, 2 and 8 to 10 (data not shown), indicating a possible VM localization, which contradicts the in silico prediction. Unfortunately, no tomato SFP protein (and also no grape protein) clustered together with the characterized SFPs from Arabidopsis, indicating different functions of SFPs in those species.

PMTs Eight loci putatively encoding POLYOL/MONOSACCHARIDE TRANSPORTER proteins (PMT or PLT) with an average length of ca. 500 AA were identified (Fig. 6). For four family members EST evidence from tomato was

SlPMT4, which had three exons. Full-length SlPMT proteins were predicted to localize to the PM (SlPMT1 to 5) or the VM (SlPMT6 to 8). The general substrate of PMTs seem to be sugar alcohols like sorbitol or mannitol, which replaces sucrose as the long-distance transport form of photoassimilates in some species, most notably fruit trees in the Rosaceae family (Noiraud et al. 2001b). In tomato no major role for sugar alcohols is known, although a functional sorbitol dehydrogenase gene was found to be expressed ubiquitously (Ohta et al. 2005). In Arabidopsis six PMT-type transporters are found. AtPMT5 was described as a non-specific polyol, hexose and pentose transporter expressed in various tissues (Klepek et al. 2005; Reinders 2004). Also in Arabidopsis, AtPMT1 and 2 were characterized as xylitol and fructose transporters expressed in developing xylem and in pollen (Klepek et +

al. 2010). Two proteins from Apium graveolens (celery) were identified as mannitol/H symporters and localized to the plasma membrane of phloem cells (Juchaux-Cachau et al. 2007; Noiraud et al. 2001a). PcSOT1 and 2 were characterized as sorbitol transporters in sink tissues of Prunus cerasus (sour cherry) (Gao et al. 2003). In the phloem of source leaves of apple expression of three sorbitol transporters (MdSOT3 to 5) was detected (Watari et al. 2004). Finally, in Plantago major (common plantain) two sorbitol transporter are expressed in the phloem (Ramsperger-Gleixner et al. 2004). No tomato PMT was found to cluster together with the characterized sorbitol transporters. The AA sequence of SlPMT4 seemed most similar to characterized sorbitol transporters, but this was not supported well according to bootstrap analysis. Together with VvPMT5, SlPMT4 formed a distinctive sub-clade. The SlPMTs 5 to 8 formed a distinct subgroup among the PMTs. Within


found. All PMT genes shared a common structure which consists of two exons separated by an intron, except

this group SlPMT5, 6 and 7 are likely the result of repeated gene duplication events, as these loci are next to each other on chromosome 2.

Smaller families Four less characterized ST families with a smaller number of members are recognized in plant species and were also found in the tomato genome (Fig. 7). They are discussed together, although they do not form a distinct clade among the STs. These families are the H+/Na+ MYO-INOSITOL TRANSPORTERS also called INOSITOL TRANSPORTER family (INT or IRT), the PLASTIDIC GLUCOSE TRANSLOCATOR family (pGlcT), the

family (VGT). Due to their predicted localization and function members of the pGlcT-, the TMT- and the VGTfamilies are promising targets in the search for novel transport proteins controlling the movement of sugars between cellular compartments in fruits, e.g. the accumulation of glucose in the vacuoles of mesocarp cells.

INTs In the tomato genome four loci putatively encoding INT family members were identified. Of these SlINT1 and 4 were supported by EST evidence. The confirmed gene models of these two SlINTs showed six exons, while SlINT3 was predicted to have four exons. Members of the INT subfamily had between 497 and 578 AA and featured 10 to 12 predicted TMDs. All proteins were predicted to localize to the PM. +

INT-type transport proteins are thought to be H -symporters of inositols. They were first characterized in the halophyte Mesembryanthemum crystallinum (Chauhan et al. 2000). Later three INT genes from Arabidopsis were characterized. AtINT1 encodes a myo-inositol transporter localized to the VM (Schneider et al. 2008), while AtINT2 and 4 were shown to be localized to the PM (Schneider et al. 2007; Schneider et al. 2006). So far, no physiological role for INT proteins was reported.

pGlcTs Four loci encoding pGlcT-type transporters were identified in the tomato genome, all of which were supported by EST evidence. The length of the encoded proteins was 484 to 545 AA forming 10 or 12 predicted TMDs.


TONOPLAST MONOSACCHARIDE TRANSPORTER family (TMT) and the VACUOLAR GLUCOSE TRANSPORTER

Confirmation of the predicted gene models by EST sequencing revealed 12 to 14 exons. The proteins were predicted to localize to mitochondria (SlpGlcT1), the PM (SlpGlcT2) or the chloroplast (SlpGlcT3 and 4). Each tomato pGlcT protein formed its own respective clade in our phylogenetic analysis and clustered together with one or two pGlct proteins from Arabidopsis, grape and rice. In plants, plastid-localized glucose transporters were first identified in spinach with the implication of a role in the export of starch breakdown products from chloroplasts during the night (Weber et al. 2000). Similar results were more recently obtained by using Arabidopsis knockout mutants of AtpGlcT (Cho et al. 2011). Starch is found in the early stages of tomato fruit development but is almost completely absent in ripe fruits (Yin et al.

export the breakdown products of starch from plastids thus makes pGlcT proteins candidates for further research into fruit sugar accumulation. In olive trees one pGlcT-type protein was reported to be expressed during fruit development (Butowt et al. 2003). Tomato SlpGlcT1 was found to cluster together with AtpGlcT and OepGlcT in a phylogenetic analysis. SlpGlcT2 however was more comparable to AtSGB1 (SUPPRESSOR OF G-PROTEIN BETA1). AtSGPB1 was identified in a mutant screening as a suppressor of the agb1 (arabidopsis gprotein β) phenotype, which leads to defects in early development (Wang et al. 2006). Furthermore AtSGB1 was shown to localize to Golgi vesicles. SlpGlcT4 was found to be most similar to OsGMST1. Knockdown of OsGMST1 led to reduced tolerance against high NaCl conditions and slightly reduced glucose and fructose content. In rice OsGMST1 was shown to localize to the Golgi apparatus (Cao et al. 2011).

TMTs Three members of the SlTMT family were detected, but only SlTMT1 and 2 had ESTs available. TMTs are large proteins of more than 700 AA in most species, including tomato. The gene models consist of five (SlTMT1 and 2) or six (SlTMT3) exons. All three proteins are predicted to localize to the PM despite their sequence similarity with Arabidopsis or rice VM-transporters. So far, TMTs from Arabidopsis and rice have been characterized. In Arabidopsis AtTMT1 and 2 were localized to the VM and characterized as glucose or fructose/H+ antiporters, importing sugars into the vacuole (Wormit et al. 2006; Schulz et al. 2011; Wingenter et al. 2010). Comparable results were obtained for the rice OsTMT1


2010). It can be speculated that the degradation of starch contributes to fruit sugar content. The ability to

and 2 proteins (Cho et al. 2010). While the Arabidopsis TMTs respond to environmental cues, like cold stress on the transcript level, the rice TMTs did not.

VGTs The smallest family of STs analysed in this study were the VGTs with two members identified in tomato. Both members had EST clones available. The sequenced ESTs were found to encode 546 (SlVGT1) and 504 (SlVGT2) AA long proteins, respectively. They had 9 and 12 predicted TMDs, respectively and were predicted to localize to the chloroplast and the PM, despite their name. The gene models showed 14 exons.

protein from Arabidopsis was characterized so far (Aluri and Büttner 2007). In yeast and in Arabidopsis protoplasts AtVGT1 localized to the VM and was shown to import glucose and to a lesser extent also fructose into the vacuole in an ATP-dependent manner. Although Atvgt1 mutants were shown to have a germination defect and a late flowering phenotype, no specific physiological function was shown.

Expression analysis Although Micro-Tom ESTs from 35 STs could be identified, only 29 of those ESTs encoded a full-length ST proteins. To get insight into putative functions of ST genes we analyzed gene expression of these 29 STs in different Micro-Tom tissues by semi-quantitative RT-PCR (Fig. 8). Since we were most interested in the functions of STs during fruit development we included tissues from developing fruits at nine different time points in our analysis. Several STs showed tissue- and development-specific dynamic expression patterns which is a first hint at their putative functions. In our analysis we found SlSUT1 and SlSUT4 to be expressed ubiquitously, with a slight increase in the course of fruit development. In contrast to previous data we could not detect SlSUT2 expression in any tissue. Barker et al. 2000 used RNA-gel blotting to detect a weak signal of SlSUT2 in leaves, stems and roots. Their method seems to be more sensitive but probably also less specific than the PCR-based detection used here. SlSUT2 was reported to be expressed predominantly in anthers, which were not sampled separately in this study (Hackel et al. 2006). As in other species SlSUT1 and SlSUT4 are thought to import sucrose into specialized phloem cells


VGT proteins form a small subfamily with two or three members in all species analysed here. Only one VGT

in source leaves (Sauer 2007). The fact that they are expressed also in typical sink tissue like roots or the developing fruit is a strong indication that they also play a role in sink tissues, for example in phloem unloading or import into sink parenchyma cells. Thus, SlSUTs should be considered targets for the manipulation of tomato sweetness. SlSTP1 and SlSTP3 were reported to be expressed predominantly in sink tissue with highest expression in young fruits (Gear et al. 2000). In our analysis SlSTP1 transcript could be detected in all analyzed tissues, while SlSTP3 was expressed at detectable levels only in flowers and developing fruits 14 DAP (days after pollination). SlSTP2 expression was detected in young leaves, in shoots, in developing fruits 14 DAP and to a lesser extent

could be detected in any tissue despite EST evidence. SlSTP12 was expressed in all analyzed tissues, with strongest expression in leaves and fruits 14 DAP. Also SlSTP15, which is expressed only in developing fruits showed strongest expression at 14 DAP. Additionally, SlSTP2, 3 and 18 showed a distinct expression at 14 DAP. This expression profile indicates that the expression of SlSTP genes in developing fruits at around 14 DAP plays a critical role for sugar accumulation in ripe fruits. Expression of SlSTP2 and SlSTP3 was analyzed previously in the pericarp layer (Dibley et al. 2005). The developmental expression profile in the pericarp differed from that found here in the whole fruit, which might be explained by the different sampling and maybe by the use of different tomato cultivars. Based on expression patterns, SlSTP2, 3, and 18 might play a role in fruit development around 14 DAP, whereas SlSTP12 and 15 are important during the later stages of fruit development. Expression around 14 DAP might be in connection with the proposed symplastic-to-apoplastic switch of sugar supply to the developing tomato fruit (Ruan and Patrick 1995). In the earlier stages of fruit development sugars are thought to be distributed within the fruit mainly symplastically through plasmodesmata. At some point in development the sucrose which reaches the fruit is exported to the apoplast and cleaved to glucose and fructose by cell wall-bound invertases. These monosaccharides then need to be distributed apoplastically and reimported into fruit parenchyma cells, which might the reason for the increased expression of several SlSTP genes at 14 DAP. Two members of the SFP family, SlSFP2 and SlSFP5, were expressed during the later stages of fruit development. SlSFP4 expression was weak and restricted to the leaves. Expression of SlSFP6 was restricted to vegetative tissues and the early stages of fruit development. SlSFP10 was expressed in the roots and in flowers.


also in mature leaves. Expression of SlSTP7 was restricted to the roots. No expression of SlSTP11 and SlSTP16

Based on this data SlSFP2 and SlSFP5 might contribute to fruit development. However, since the substrate specificity of SFP-type transporters is not very well characterized it is not possible to propose a more specific function. Of the PMT family, only SlPMT4 showed strong dynamic expression during the late stages of fruit development. SlPMT3 transcript was not detectable in any tissue. SlPMT5 showed only weak expression in mature leaves and roots. Expression of SlpGlcT1 was detected in all tissues, except fruits 3 DAP. SlpGlcT2 and 3 were expressed at several stages during fruit development with SlpGlcT3 showing strong expression at 7 DAP. Expression of SlpGlcT4 was not detected in any tissue. The two SlTMT genes and SlINT4 were expressed in most analyzed tissues. SlTMT2 showed strong, consistent expression in vegetative tissues, while during fruit

expressed in all analyzed tissues SlVGT2 was expressed only in leaves and during the later stages of fruit development, with strongest expression during the Orange stage of fruit development. Because of its increasing expression levels in the course of fruit development SlVGT2 is worth further investigation.

Conclusion

In this study 52 putative sugar transporter genes were detected in the tomato genome. An expression analysis of selected genes revealed tissue and developmental stage-specific gene expression of some members. In the next step specific roles for these genes should be found. This will likely result in a more complete picture about sugar mobility during development of fleshy fruits (Ludewig and Flugge 2013). For example, it is unclear what the individual contribution of each SUT transporter to the different steps in phloem transport is. It is also worth investigating the function of the monosaccharide transporters found to be expressed in a dynamic, fruitspecific manner. After cleavage of sucrose to glucose and fructose in the developing fruit the hexoses have to be transported within the fruit to the storage parenchyma for storage, presumably into the vacuoles. These steps require monosaccharide transporters, which have not been identified yet.


development expression was weaker, except 14 DAP and during the Breaker stage. While SlVGT1 was

Materials and Methods

Identification of Solanum lycopersicum sugar transporter genes Tomato loci putatively encoding sugar transporter (ST) proteins were retrieved from the tomato reference genome (ITAG release 2.3 SL2.40)(Sato et al. 2012). The TBLASTX tool on http://www.solgenomics.net was

used to identify loci encoding putative STs. Amino acid sequences of known members from each recognized ST

sequences were then used for phylogenetic analysis and in silico predictions of gene features. The identified CDSs were used to find EST clones containing ST cDNAs from the TOMATOMICS database (Aoki et al. 2010) or from the Sol Genomics Network. After consolidation of the data, the most similar EST clone for each putative ST locus was obtained and sequenced to verify the current gene model. All EST sequences were submitted to the DNA Data Bank of Japan (http://www.ddbj.nig.ac.jp/) (accession numbers: [DDBJ: AB845639] to [DDBJ: AB845668]).

Multiple sequence alignments and phylogenetic analysis Final classification of ST genes into subfamilies and subgroups was done according to phylogenetic analysis. Multiple sequence alignments, using AA sequences from predicted proteins (ITAG release 2.3 SL2.40) or from sequenced ESTs were made using the CLUSTAL alignment function in the CLC Main Workbench software (CLC Bio, Aarhus, Denmark). Phylogenetic trees were build using the Neighbor-joining algorithm in the same software and visualized using Treeview (Page 2002) and Dendroscope (Huson and Scornavacca 2012).

In silico prediction of subcellular localization and transmembrane helical domains Prediction of subcellular localization of putative STs was performed using the WoLFPSORT algorithm (http://wolfpsort.seq.cbrc.jp) (Horton et al. 2007). Prediction of transmembrane helical domains was performed using TMHMM Server v.2.0 (http://www.cbs.dtu.dk/services/TMHMM/)(Krogh et al. 2001).


subfamily from tomato or from Arabidopsis were used as queries. The retrieved gene models and amino acid

Plant material and growth conditions Solanum lycopersicum plants for gene expression analysis were of the dwarf cultivar ‘Micro-Tom’. Plants were grown on soil in a growth chamber (Biotron LPH-350S, NK Systems, Osaka, Japan) with a light regime of 16 h of light /8 h darkness at 25°C and 60% relative humidity. Plants were watered twice a week with tap water. Fertilizer (Otsuka Chemicals, Osaka, Japan) was applied once per week.

Plant tissues from young leaves, mature leaves, roots, shoots, flowers and from developing fruits 3, 7, 14, 21 and 28 days after fertilization and during the Breaker, Orange and Red stages of fruit development were harvest into liquid nitrogen. Vegetative tissues were harvested from ca. six week old plants. Samples of young leaves included developing, not fully expanded leaves, samples of mature leaves included fully expanded, nonsenescent leaves. RNA from developing fruits 14 and 21 days after fertilization was isolated using the RNA Suisui-R kit (Rizo, Tsukuba, Japan). RNA from all other tissues was isolated using TRIzol reagent (Life Technologies, Carlsbad, USA) following the manufacturer’s protocol. Quality and quantity of the RNA was assessed using a spectrophotometer. RNA was stored at -80°C. cDNA was prepared using the PrimeScript RT reagent Kit with gDNA Eraser (Clontech, Mountain View, USA) according to the manufacturer’s specification. For each 20 µl reaction 500 ng of total RNA was used.

RT-PCR expression analysis Semi-quantitative RT-PCR was performed using 0.1 µl of cDNA as a template and EmeraldAmp GT PCR Mastermix (Clontech, Mountain View, USA). For each primer pair the PCR program was empirically adjusted. All primers were tested for specificity by trying obtain a PCR product using vector DNA containing ESTs from other subfamily members as a template (data not shown). As a control the constitutively expressed SlUBQ (Solyc01g056940) gene was used. PCR products were analyzed using 1% agarose gels stained for nucleic acids with Ethidium Bromide. Primer sequences and PCR conditions are described in Supplemental Table 1.


RNA isolation and cDNA synthesis

Funding: This work was supported by: Program for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry from Bio-oriented Technology Research Advancement Institution (BRAIN) and by Grant-in-Aids for Scientific Research from The Japan Society for the Promotion of Science (JSPS).

Disclosure: No conflicts of interest declared.

Author Contributions KS conceived and designed experiments. SR, MA, HM and TY performed experiments. KA and DS provided

Acknowledgments We thank Dr. Shogo Matsumoto and Dr. Shungo Otagaki for helpful discussions and the National Bioresource Project (NBRP)-Tomato in Japan and the Sol Genomic Network (SGN) for providing cDNA clones. Also we thanks Dr. Takashi Akihiro from the University of Shimane for comments on our manuscript.


cDNA clones. SR and KS wrote the manuscript.

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+

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Tables

Table 1: Comprehensive nomenclature and feature list of 52 sugar transporters identified in tomato.

SUT

STP

SFP

Gene Name

Locus

Best Hit EST No.

AA1

TMD2

LOC3

SlSUT1 SlSUT2 SlSUT4 SlSTP1 SlSTP2 SlSTP3 SlSTP4 SlSTP5 SlSTP6 SlSTP7 SlSTP8 SlSTP9 SlSTP10 SlSTP11 SlSTP12 SlSTP13 SlSTP14 SlSTP15 SlSTP16 SlSTP17 SlSTP18 SlSFP1 SlSFP2 SlSFP3 SlSFP4 SlSFP5 SlSFP6 SlSFP7 SlSFP8 SlSFP9 SlSFP10

Solyc11g017010.1 Solyc05g007190.2 Solyc04g076960.2 Solyc02g079220.2 Solyc09g075820.2 Solyc07g006970.2 Solyc04g074070.1 Solyc01g010530.1 Solyc08g080300.1 Solyc03g005140.1 Solyc00g009030.1 Solyc01g008240.2 Solyc03g006650.1 Solyc06g054270.2 Solyc05g018230.2 Solyc03g005150.2 Solyc03g078600.1 Solyc03g093400.2 Solyc03g093410.2 Solyc03g094170.1 Solyc12g008320.1 Solyc01g098490.2 Solyc02g005180.2 Solyc01g080680.2 Solyc04g080460.2 Solyc01g098500.2 Solyc12g089180.1 Solyc09g074230.2 Solyc01g098560.2 Solyc02g062750.2 Solyc02g085170.2

LEFL1045AF06 LEFL2035O24 LEFL1070DB04 SGN-E398079 LEFL1033AD12 LEFL2015N15 Not Found Not Found Not Found LEFL3132D24 Not Found # SGN-E360972 Not Found LEFL1093CG01 LEFL1033AG05 Not Found Not Found LEFL1086CC04 LEFL1049CD03* Not Found LEFL3049D18 Not Found LEFL3012N18* SGN-E211041# LEFL1070CE02 LEFL1019BF02 LEFL1001AD10 SGN-E282759 Not Found Not Found SGN-E547709

512 605 501 524 524 514 510 490 529 488 397 506 519 490 517 488 438 500 515 242 516 461 396 469 487 462 491 481 466 518 484

11 11 12 12 12 11 11 11 12 11 7 11 12 11 12 11 8 12 12 4 10 12 10 11 12 10 12 12 12 12 12

PM PM VM/ER PM PM PM VM PM VM PM PM CS CS PM PM PM PM PM PM CL PM PM PM PM PM PM PM PM PM PM PM

Remarks LOC (Kühn et al. 1997) LOC (Barker et al. 2000) LOC (Schneider et al. 2012) EST not full length (Δ1-377); LeHT1 (Gear et al. 2000) LOC: PM in yeast, LeHT2 (Gear et al. 2000) LOC: PM in yeast, LeHT3 (Gear et al. 2000)

EST not full length (Δ1-36) short C-terminus

short N-terminus

short N- and C-terminus

EST not full length (Δ1-12), LeST3 (Garcia-Rodriguez et al. 2005)

(continued on next page)


(Table 1 continued) Gene Name

Locus

Best Hit EST No.

AA1

TMD2

LOC3

Remarks

SlPMT1 Solyc02g078600.2 Not Found 514 10 PM SlPMT2 Solyc07g024030.2 Not Found 478 12 PM SlPMT3 Solyc12g010690.1 LEFL2033E02 520 12 PM SlPMT4 Solyc01g109460.2 LEFL1006BG12 542 10 PM SlPMT5 Solyc02g062890.1 LEFL3155L03 510 11 PM SlPMT6 Solyc02g062860.2 Not Found 498 12 VM SlPMT7 Solyc02g062870.2 Not Found 498 12 VM SlPMT8 Solyc02g081710.1 SGN-E359943 488 12 VM Micro-Tom EST differs from reference genome SlINT1 Solyc06g073420.2 SGN-E324058 497 10 PM failed to grow EST clone SlINT2 Solyc08g048290.2 SGN-E312790# 527 11 PM SlINT3 Solyc12g099070.1 Not Found 581 12 PM SlINT4 Solyc11g012450.1 LEFL1003DF04 578 12 PM SlpGlcT1 Solyc02g086160.2 LEFL2053A18 545 12 MM SlpGlcT2 Solyc06g066600.2 LEFL2053F03* 492 10 PM SlpGlcT3 Solyc07g020790.2 LEFL2036E03 484 10 CL SlpGlcT4 Solyc07g049310.2 LEFL2028B16 541 10 CL SlTMT1 Solyc03g032040.2 LEFL1012BC07 726 10 PM SlTMT2 Solyc04g082700.2 LEFL1032BA11 739 11 PM SlTMT3 Solyc02g082410.2 Not Found 708 11 PM SlVGT1 Solyc03g078000.2 LEFL1094BD10 546 9 CL SlVGT2 Solyc03g096950.2 LEFL2044B22 504 12 PM 1 The amino acid sequence length was either confirmed by EST sequencing or predicted using SL2.40 gene models. 2 The number of transmembrane domains was predicted by TMHMM Server v2.0. 3 The subcellular localizations were predicted by WoLFPSORT or determined experimentally as indicated under “Remarks”. PM plasma membrane, VM vacuolar membrane, CL chloroplast, MM mitochondrion NU nucleus, ER endoplasmatic reticulum, CS cytosol * The sequenced ESTs contained a 2 bp deletion (STP16) or unspliced intronic sequence (SFP2, pGlcT2) (assumed to be a cloning artifacts) leading to a frameshift. Further analyses were performed using the corrected gene models. # The ordered EST clone contained another, non-sugar transporter cDNA.

PMT

INT

pGlcT TMT VGT


Figure legends

Figure 1: Phylogenetic analysis of tomato sugar transporters. Shown is a phylogenetic tree generated by the neighbor-joining method derived from a CLUSTAL alignment of AA sequences from tomato. Numbers at internal nodes show the results of bootstrapping analysis (n = 1000). Colored boxes indicate different sugar transporter subfamilies.

Figure 2: Exon-Intron structure of 52 sugar transporter genes identified in tomato. Shown is a graphic representation of the gene models of all 52 sugar transporters identified in this study. UTRs are shown as hatched boxes, exons are shown as black boxes and introns are shown as black lines. Gene models are based on

Figure 3: Phylogenetic analysis of the SUCROSE TRANSPORTER family. Shown is a phylogenetic tree generated by the neighbor-joining method derived from a CLUSTAL alignment of AA sequences from SUT/SUC subfamily members from tomato (this study, bold type label, red lines) and selected family members from Arabidopsis thaliana AtSUC1 (At1g71880), AtSUC2 (At1g22710), AtSUC3 (At2g02860), AtSUC4 (At1g09960), AtSUC5 (At1g71890), AtSUC6 (At5g43610), AtSUC7 (At1g66570), AtSUC8 (At2g14670), AtSUC9 (At5g06170); Oryza sativa OsSUT1 (Os03g0170900), OsSUT2 (Os12g0641400), OsSUT3 (Os10g0404500), OsSUT4 (Os12g44380), OsSUT5 (Os02g0576600); Vitis vinifera VvSUC11/VvSUT1 (HQ323256), VvSUC12 (HQ323257), VvSUC27 (HQ323258), VvSUT2 (HQ323259); Solanum tuberosum StSUT1 (CAA48915), StSUT4 (AAG25923.2); Zea mays ZmSUT1 (BAA83501); Nicotiana tabacum NtSUT4 (BAI60050); Pisum sativum PsSUF1 (DQ221698), PsSUF4 (DQ221697); Phaseolus vulgaris PvSUF1 (DQ221700); Spinacia oleraceae SoSUT1 (Q03411); Hordeum vulgare HvSUT2 (Q9M423); Lotus japonicas LjSUT4 (CAD61275); Populus tremula x alba PtaSUT4 (HM749900). Numbers at internal nodes show the results of bootstrapping analysis (n = 1000).

Figure 4: Phylogenetic analysis of the SUGAR TRANSPORTER PROTEIN family. Shown is a phylogenetic tree generated by the neighbor-joining method derived from a CLUSTAL alignment of AA sequences from STP subfamily members from tomato (this study, bold type label, red lines) and characterized family members from Arabidopsis thaliana AtSTP1 (At1g11260), AtSTP2 (At1g07340),AtSTP3 (At5g61520), AtSTP4 (At3g19930), AtSTP5 (At1g34580), AtSTP6 (At3g05960), AtSTP7 (At4g02050), AtSTP9 (At1g50310), AtSTP11 (At5g23270), AtSTP13 (At5g26340), AtSTP14 (At1g77210); Oryza sativa OsMST1 (BAB19862.1), OsMST2 (Os03g39710), OsMST3 (Os07g01560), OsMST4 (AAQ24871.1), OsMST5 (Os08g08070), OsMST6 (AAQ24872.1), OsMST8 (Os01g38670); Vitis vinifera VvHT1 (HQ323260), VvHT2 (HQ323261), VvHT3/VvHT7 (HQ323262), VvHT4 (HQ323263), VvHT5 (HQ323264); Juglans regia JrHT1 (DQ026508), JrHT2 (DQ026509,); Olea europaea OeMST2 (DQ087177) and Ananas comosus AcMST1 (EF460876). Numbers at internal nodes show the results of bootstrapping analysis (n = 1000).

Figure 5: Phylogenetic analysis of the SUGAR FACILITATOR PROTEIN/EARLY RESPONSE TO DEHYDRATION 6 protein family. Shown is a phylogenetic tree generated by the neighbor-joining method derived from a CLUSTAL alignment of AA sequences from ERD6/SFP subfamily members from tomato (this study, bold type label, red lines); Arabidopsis thaliana

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sequenced ESTs, or in the case of lacking EST evidence, in silico predictions (ITAG release 2.3 SL2.40) are used.

AtERDL6 (At1g75220), AtERD6 (At1g08930), AtSFP1 (At5g27350), AtSFP2 (At5g27360), AtERD6-like1 (At1g08890), AtESL1 (At1g08920), AtESL2 (At1g08900) and 12 additional uncharacterized Arabidopsis proteins indicated by their AGI-code and Beta vulgaris (beet) BvERD6l (U43629). Grape proteins sequences were taken from (Afoufa-Bastien et al. 2010): VvSFP1 HQ323290, VvSFP2 HQ323291, VvSFP3 HQ323292, VvSFP4 HQ323293, VvSFP5 HQ323294, VvSFP6 HQ323295, VvSFP7 HQ323296, VvSFP8 HQ323297, VvSFP9 HQ323298, VvSFP10 HQ323299, VvSFP11 HQ323300, VvSFP12 HQ323301, VvSFP13 HQ323302, VvSFP14 HQ323303, VvSFP15 HQ323304, VvSFP16 HQ323305, VvSFP17 HQ323306, VvSFP18 HQ323307, VvSFP19 HQ323308, VvSFP20 HQ323309, VvSFP21 HQ323310, VvSFP22 HQ323311. Numbers at internal nodes show the results of bootstrapping analysis (n = 1000).

Figure 6: Phylogenetic analysis of the POLY/MONOSACCHARIDE TRANSPORTER family. Shown is a phylogenetic tree generated by the neighbor-joining method derived from a CLUSTAL alignment of AA sequences from PMT/PLT subfamily members from tomato (this study, bold type label, red lines); Arabidopsis thaliana (At4g36670); Vitis vinifera VvPMT1 (HQ323285), VvPMT2 (HQ323286), VvPMT3 (HQ323287), VvPMT4 (HQ323288), VvPMT5 (HQ323289); Apium graveolens ApMaT1 (AF215837), ApMaT2 (AF480069); Plantago major PmPLT1 (CAD58709), PmPLT2 (CAD58710); Malus domestica MdSOT1 (AY237400), MdSOT2 (AY237401), MdSOT3 (BAD42343), MdSOT4 (BAD42344), MdSOT5 (BAD4234); Prunus cerasus PcSOT1 (AF482011), PcSOT2 (AY100638). Numbers at internal nodes show the results of bootstrapping analysis (n = 1000).

Figure 7: Phylogenetic analysis of five tomato sugar transporter protein families. Shown is a phylogenetic tree generated by the neighbor-joining method derived from a CLUSTAL alignment of AA sequences from five ST subfamilies from tomato (this study, bold type label); Arabidopsis thaliana AtINT1 (At2g43330), AtINT2 (At1g30220), AtINT3 (At2g35740), AtINT4 (At4g16480), AtAtTMT1 (At1g20840), AtTMT2 (At4g35300), AtTMT3 (At3g51490), AtSGB1 (At1g79820), AtpGlcT (At5g16150), AtpGlcT-like1 (At1g67300) AtpGlcT-like2 (At1g05030), AtVGT1 (At3g03090), AtVGT2 (At5g17010), AtVGT3 (At5g59250); Oryza sativa OsITR1 (Os04g41460), OsITR2 (Os07g05640), OsITR3 (Os04g43210), OsTMT1 (Os10g39440), OsTMT2 (Os02g13560), OsTMT3 Os03g03680, OsTMT4 (Os11g40540), OsTMT5 (Os02g58530), OsTMT6 (Os11g28610), OspGlcT (Os01g04190), OsGMST1 (Os09g23110); OspGlcT-like1 (Os02g17500), OspGlcT-like2 (Os09g27900), OsVGT1 (Os03g60820), OsVGT2 (Os10g42830), Vitis vinifera VvINT1 (HQ323314), VvINT2 (HQ323315), VvINT3 (HQ323316), VvTMT1 (HQ323282), VvTMT2 (HQ323283), VvTMT3 (HQ323284), VvpGlT (HQ323320), VvpGlcT2(HQ323319), VvpGlcT3 (HQ323318), VvpGlcT4(HQ323317), VvVGT1 (HQ323312), VvVGT2 (HQ323313), Olea europaea OepGlcT (AY036055), Spinacia oleracea SopGlcT (AF215851), Mesembryanthemum crystallinum McITR1 (AF280431), McITR2 (AF280432). Numbers at internal nodes show the results of bootstrapping analysis (n = 1000). Colored background indicate different subfamilies of STs.

Figure 8: Expression analysis of selected tomato sugar transporters. Shown is a semi-quantitative RT-PCR analysis of tomato sugar transporters. RNA was extracted from the indicated tissues, transcribed to cDNA and used as a template for PCR. + indicates reactions using the respective EST-containing vector as a template. Gene-specific primers (amplicons ca. 200 bp) were used to analyze expression levels by PCR. UBQ indicates a tomato ubiquitin gene used as a constitutively expressed control gene. DAP = days after pollination. Results are representative of two technical replicates for each tissue.

30


AtPMT1 (At2g16120), AtPMT2 (At2g16130), AtPMT3 (At2g18480), AtPMT4 (At2g20780), AtPMT5 (At3g18830), AtPMT6

Supplemental Data

Supplemental Table 1: Sequences of oligonucleotides and PCR program settings used for gene expression analysis. Shown are the sequences of the forward (FWD) and the reverse (REV) primer used to analyze the expression of each tomato sugar transporter. Below each primer pair the PCR program used for each target gene is given.


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Figure 1 205x337mm (150 x 150 DPI)


Figure 3 169x174mm (150 x 150 DPI)


Figure 4 197x291mm (150 x 150 DPI)


Figure 5 564x846mm (600 x 600 DPI)


Figure 6 143x143mm (150 x 150 DPI)


Figure 7 323x684mm (150 x 150 DPI)


Figure 8 404x721mm (150 x 150 DPI)

Genome-wide identification and expression analysis of aquaporins in tomato.

Genome-wide identification, phylogeny, and expression analysis of the SWEET gene family in tomato.

Comprehensive Genomic Identification and Expression Analysis of the Phosphate Transporter (PHT) Gene Family in Apple.

Genome-Wide Identification and Expression Analysis of Two-Component System Genes in Tomato.

Genome-wide identification, phylogeny and expression analysis of GRAS gene family in tomato.

Genome-wide identification and expression analysis of E2 ubiquitin-conjugating enzymes in tomato.

Genome-Wide Identification and Expression Analysis of Calcium-dependent Protein Kinase in Tomato.

Identification and Functional Characterization of a Tonoplast Dicarboxylate Transporter in Tomato (Solanum lycopersicum).

Genome-Wide Identification, Classification, and Expression Analysis of Amino Acid Transporter Gene Family in Glycine Max.

Genome-Wide Function, Evolutionary Characterization and Expression Analysis of Sugar Transporter Family Genes in Pear (Pyrus bretschneideri Rehd).

Modeling sugar metabolism in tomato fruit.

GxySBA ABC transporter of Agrobacterium tumefaciens and its role in sugar utilization and vir gene expression.

Evolution of the Apicomplexan Sugar Transporter Gene Family Repertoire.

Genome-Wide Identification, Evolution, and Expression Analysis of the ATP-Binding Cassette Transporter Gene Family in Brassica rapa.

Genomewide analysis of phytochrome proteins in the phylum Basidiomycota.

Genome-wide identification, phylogenetic analysis, expression profiling, and protein-protein interaction properties of TOPLESS gene family members in tomato.

Structural insight into the PTS sugar transporter EIIC.

The tomato 66.3-kD polyphenoloxidase gene: molecular identification and developmental expression.

The SOD Gene Family in Tomato: Identification, Phylogenetic Relationships, and Expression Patterns.

Analysis of tomato polygalacturonase expression in transgenic tobacco.

Expression, purification and structural analysis of functional GABA transporter 1 using the baculovirus expression system.

Identification of a broad-specificity nucleoside transporter with affinity for the sugar moiety in Giardia intestinalis trophozoites.

Genome-wide identification and expression profiling of the copper transporter gene family in Populus trichocarpa.

Identification of a tomato gene for the ethylene-forming enzyme by expression in yeast.