BACKGROUND
in plants, sugars  are important molecules that constitute not only metabolites but also nutrients, osmotic and signal molecules.
in numerous species, sucrose is the most prevalent sugar produced in photosynthetic organs  and transported via the phloem over long distances to heterotrophic organs , which depend on a constant supply of carbohydrates  <cit> .
in sink organs, sucrose is either directly imported or cleaved by cell wall-bound invertases into monosaccharides , that can be taken up by the sink cells  <cit> .
in some species, sugar alcohols , such as mannitol, sorbitol and galactinol can also be transported on top of sucrose for long-distance carbon partitioning  <cit> .
in addition to this long-distance transport, sugars can also be allocated in the different organelles of source and sink cells, and more and more biochemical and molecular studies argue for the transport of hexoses into the chloroplast  <cit>  the vacuoles  <cit> , and the golgi apparatus  <cit> .
therefore, it is now clearly established that not only the loading and the unloading of the conducting complex, but also the allocation of sugars into source and sink cells is controlled by sugar transporters mediating the transport of sucrose  <cit> , reducing monosaccharides  <cit> , or polyols  <cit> .
since the cloning of the first monosaccharide transporter in chlorella  <cit> , the first sucrose transporter in spinacia oleracea  <cit> , and the first polyol transporter in apium graveolens  <cit> , many genes belonging to these families have been isolated from various species.
the complete arabidopsis genome has been described to contain  <dig> sucrose transporter-like sequences  <cit>  and a monosaccharide transporter gene family, including  <dig> members grouped into  <dig> subfamilies  <cit> .
furthermore, the evolutionary analysis of plant monosaccharide transporters revealed that these seven subfamilies are ancient in land plants  <cit> .
despite the progress made in identifying genes encoding sugar transporters, little is known about the transcriptional regulation of these genes.
arabidopsis microarray data  and some plant transporter gene expression patterns have indicated that developmental and environmental factors could regulate the expression of sugar transporters.
furthermore, evidence is provided that the expression of some sugar transporter genes is regulated by sugars as described for sugar transporter genes in yeast  <cit> , for vvht <dig>  a grapevine hexose transporter  <cit>  and for sucrose transporter genes from rice, ossut <dig>  <cit>  and sugar beet, bvsut <dig>  <cit> .
all these data suggest that the expression of sugar transporters might be regulated at the transcriptional level by distinct but usually converging signalling pathways, depending on either developmental and environmental cues or metabolic and hormonal signals.
in spite of the evidence for the role of sugar signalling in the transcriptional control of some transporter genes, the in silico analysis of promoter regions of different genes involved in carbon metabolism, sugar storage, mobilization and transport clearly demonstrates the absence of common sugar specific cis-elements  <cit> .
this analysis is consistent with the fact that in plants, several types of transcription factors  are required for sugar signalling and are involved in sugar-regulation of gene expression  <cit> .
considering that the analysis of sugar transporter orthologs in different species might help to better understand their biological function, we analyzed the recently sequenced vitis vinifera genotype pn <dig>  <cit>  in order to identify sugar transporter gene families in this species.
this work will represent the first exhaustive analysis for sugar transporters in ligneous plant as most of the already known sugar transporters have been characterized from herbaceous species.
in woody plants, only  <dig> sucrose transporters have been already described in vitis  <cit>  ,  <dig> in citrus sinensis ,  <dig> in hevea brasiliensis  and one in juglans regia  <cit> .
seven hexose transporters in vitis  <cit> ,  <dig> in juglans regia  <cit>  and few polyol transporters in prunus cerasus  <cit> , in malus domestica  <cit>  and in olea europea  <cit>  were also reported.
furthermore during the last decade, vitis vinifera has become an interesting model to study fruit maturation.
it is now clearly established that the onset of ripening  is characterized by an important accumulation of glucose and fructose in vacuoles of the mesocarp cells  <cit> .
in grapevine, sucrose is the main carbohydrate used for long distance transport and after reaching the phloem of the berry, it is unloaded into the apoplast, possibly cleaved by apoplastic invertases, and sucrose or hexoses can than be transported into the mesocarp.
in the cytoplasm of the mesocarp cells, sucrose and hexoses must be transported into the vacuole via tonoplastic transporters.
the identification and the characterization of sugar transporter genes in vitis vinifera are therefore important steps in understanding the roles of these proteins in grapevine development as well as in grape ripening process and may further highlight our knowledge on plant sugar transporters.
the present study reports on the identification of sucrose and monosaccharide-like transporter genes in the vitis vinifera genome, on their phylogenetic analysis in comparison with arabidopsis transporters, on their promoter sequences analysis.
the construction of specialized cdna macroarrays used to determine the expression pattern for  <dig> of these genes in grapevine vegetative organs and during berry ripening is also described.
RESULTS
identification of sugar transporters from vitis vinifera
blastp searches of the grapevine genome proteome 8× database, using the amino acid sequences of the  <dig> sucrose transporters and the  <dig> monosaccharide transporters from a. thaliana as query, allowed the identification of  <dig> orfs encoding putative sugar transporters in v. vinifera .
among these orfs, only  <dig> encode previously described sucrose transporters  <cit>   and no additional one could be identified.
the  <dig> other orfs seem to encode putative monosaccharide transporters .
phylogenetic analysis of the  <dig> v. vinifera identified protein sequences using the maximum likelihood  method  reveals that sucrose and monosaccharide transporters form two separate groups.
furthermore in agreement with the phylogeny observed for a. thaliana mst  <cit> ,  <dig> distinct subfamilies  could be clearly identified in the vitis monosaccharide transporter group .
vitis vinifera sucrose transporters
the  <dig> amino acids sequences encoding the already described sucrose transporters named vvsuc11/vvsut <dig>  vvsuc <dig>  vvsuc <dig> and vvsut <dig>  <cit>  share  <dig> to 59% similarity between each other and fall into three sucrose transporter subgroups already described   <cit> .
vvsut <dig> and vvsuc <dig> shows 59% similarity and belong to the dicots specific sut <dig> subfamily including high affinity sucrose transporters exhibiting apparent km value between  <dig>  and  <dig> mm.
however, vvsuc <dig> has been described to be a low-affinity/high-capacity sucrose transporter showing a km value for sucrose ranged between  <dig>  and  <dig>  mm  <cit> .
the structure of vvsut <dig> and vvsuc <dig> genes is quite similar, both being around  <dig> bp long and containing  <dig> exons separated by  <dig> introns .
vvsuc <dig> shows  <dig> % similarity with atsuc <dig> and presents an extended domain at the n terminus and an elongated central cytoplasmic loop; two structural characteristics specific to the sut2/suc <dig> subfamily  <cit> .
furthermore, vvsuc <dig> gene is a very long gene  containing  <dig> exons interrupted by  <dig> introns ; such exon/intron organization is also described for atsuc <dig>  the km value for sucrose  reported for this transporter  <cit>  seems, however, higher than that described for other members of this subfamily, showing either a low affinity  or no sucrose transport function.
finally, although vvsuc <dig> has a high affinity for sucrose  it shows  <dig> % similarity with atsuc <dig> and falls into the sut <dig> subfamily including all low-affinity plant sucrose transporters with km value ranging between  <dig> mm and  <dig> mm.
vitis vinifera putative hexose transporters
among the identified orfs,  <dig> showed high similarity  with the atstp  subfamily members.
among these,  <dig> correspond to the already well known v. vinifera hexose transporters named vvht  such as vvht <dig>  vvht <dig>  vvht <dig> , vvht <dig> and vvht <dig>  <cit> , .
therefore, the  <dig> newly identified orfs were named vvht <dig> to vvht <dig>  vvht <dig> amino acid sequence shows  <dig> % similarity with vvht <dig> and the main differences between the two nucleotide sequences reside in some single nucleotide polymorphism and in the length of a microsatellite sequence in the 3'utr region.
considering that the chromosomal location of vvht <dig> and vvht <dig> is not determined, it is difficult to conclude if these sequences represent two independent genes, two alleles of the same gene or possibly one single gene.
vvht <dig> and vvht <dig> share  <dig> % similarity between each other and around 73% with vvht <dig>  interestingly, the three corresponding genes are located in a tandem repeat region, on chromosome  <dig>  in a similar way, vvht <dig> to vvht <dig> form a cluster on chromosome  <dig> and the  <dig> corresponding amino acid sequences show very high similarity .
vvht <dig> and vvht <dig>  which are located nearby on chromosome  <dig>  contain the two first exons and the last exon of a monosaccharide transporter, respectively.
a detailed amino acid sequence analysis revealed that these two partial orfs are wrongly annotated and might constitute a single monosaccharide transporter, in the third exon of which a stop codon  replaces a tryptophan residue .
it is therefore tempting to suggest that this point mutation at the origin of the false annotation, might be due to a sequencing error, but we can not exclude that it could be real.
finally vvht <dig>  vvht <dig> and vvht <dig> are partial mst, whose sequences do not seem to be fully sequenced, missing either the n-ter or the c-ter region, or both.
therefore, considering that vvht <dig> might be identical to vvht <dig> and that vvht <dig> and vvht <dig> are probably a single protein, we can estimate that the grape genome might contain  <dig> putative hexose transporters.
in this vvht subfamily, the exon-intron organization seems to be conserved as all completely sequenced genes contain  <dig> exons separated by  <dig> introns with the exception of vvht <dig> and vvht <dig>  phylogenetic analysis  reveals that the vvht subfamily seems to be divided into two subclades, at the basis of which is located vvht <dig> and vvht <dig> both present on chromosome  <dig>  vvht <dig> is located at the basis of a subclade having a bootstrap value of  <dig> and containing the  <dig> closely related transporters from chromosome  <dig> and  <dig> .
furthermore, if we exclude vvht <dig> and vvht <dig>   <dig> sister-pairs with a strong bootstrap support  could be identified, vvht3/vvht <dig> ; vvht9/vvht <dig>  and vvht2/vvht <dig> .
finally, phylogenetic analysis of a. thaliana and v. vinifera sugar transporter proteins allowed us to identify six ortholog pairs between both species  such as vvht1/atstp <dig> , vvht2/atstp <dig> , vvht3/atstp <dig> , vvht4/atstp <dig> , vvht5/atstp <dig> , vvht13/atstp <dig> .
five of these pairs are supported by bootstrap value of 100%.
vitis vinifera putative tonoplast monosaccharide transporters
we have also identified three orfs, which show the strongest similarity  to the  <dig> a. thaliana tonoplast monosaccharide transporters .
all three vitis orfs show an extended middle loop between the putative trans-membrane helices six and seven in a similar way as the attmt.
gsvivt <dig> is identical to a v. vinifera sugar transporter already mentioned in the literature and called vvht <dig>  <cit> .
considering that it shows a higher similarity with attmt  than with the vvht proteins , we renamed it vvtmt <dig>  similarly, gsvivt <dig> and gsvivt <dig> were called vvtmt <dig> and vvtmt <dig>  respectively.
the exon-intron organization seems to be conserved in the three genes as they contain all  <dig> exons separated by  <dig> introns .
phylogenetic tree performed with vitis sugar transporter amino acid sequences  reveals that the  <dig> vvtmt form a clade, which is closely related to putative myo-inositol transporters  and vacuolar glucose transporters  .
furthermore, phylogenetic analysis using vitis and arabidopsis sequences  confirms that tmt sequences from both species form a single clade, with a strong bootstrap support , but within which low bootstrap values  indicate unresolved nodes and fail to detect sister-pairs between both species.
vitis vinifera putative polyol/monosaccharide transporters
five orfs show highest similarity  with the  <dig> a. thaliana polyol transporters and have been therefore named vvpmt <dig> to vvpmt <dig>  v. vinifera putative polyol transporter amino acids sequences share 40% to  <dig> % similarity between themselves and the corresponding genes present all the same structure with  <dig> exons separated by a single intron.
phylogenetic analysis performed with the a. thaliana and v. vinifera polyol transporters  reveals that vvpmt <dig> and vvpmt <dig> form with atpmt <dig> a separated clade.
vvpmt <dig> is at the basis of a second clade, which can be divided into two groups, one including vvpmt <dig>  atpmt <dig> and atpmt <dig> and the second atpmt <dig>  atpmt <dig>  atpmt <dig> and vvpmt <dig>  only atpmt <dig> and vvpmt <dig> could be identified as putative orthologs.
vitis vinifera putative erd6-like transporters
twenty-two orfs showing strongest similarity with the  <dig> aterd6-like proteins were identified  and share  <dig>  to  <dig> % similarity with each other.
among them,  <dig> orfs correspond to partial sequences in which either the beginning or the end of the protein are not clearly identified.
however after a more precise sequence analysis, we were able to realize the full annotation for gsvivt <dig> and gsvivt <dig>  fourteen orfs are located on chromosome  <dig>  in a region of tandem gene duplications, three other orfs are carried by chromosome  <dig> and two partial orfs by chromosome  <dig>  the  <dig> erd6-like proteins fall into the same subfamily supported by a strong bootstrap value  and  <dig> loci formed  <dig> sister pairs .
the phylogenetic analysis of the amino acid sequences of erd6-like transporters from a. thaliana and v. vinifera  reveals that these transporters can be classified into  <dig> major groups.
a first group includes  <dig> aterd6-like located on the  <dig> chromosomes of a. thaliana and  <dig> vverd6-like located on  <dig> chromosomes  of v. vinifera.
a second small group including transporters from both species  was also identified.
inversely, the two last groups include protein sequences from only one species.
the arabidopsis group contains  <dig> proteins located on three different chromosomes  including erd <dig>  <cit> , sfp <dig> and sfp <dig>  <cit> .
the vitis group includes  <dig> proteins,  <dig> of which are encoded by genes located on chromosome  <dig>
vitis vinifera putative vacuolar glucose transporters
two vitis orfs, named vvvgt <dig> and vvvgt <dig>  show the highest similarity with the  <dig> atvgt -like transporters.
in arabidopsis atvgt <dig> and atvgt <dig> have been shown to be localized in the tonoplast and glucose transport activity has been demonstrated for atvgt <dig>  <cit> .
on the contrary atvgt <dig> is postulated to be localized in chloroplast membrane as this protein presents a n-terminal extension carrying a potential signal for plastid targeting.
phylogenetic tree  indicates clearly that vvvgt <dig> is the closest to atvgt <dig> and atvgt <dig> and that vvvgt <dig>  which presents a n-terminal extension, is more closely related to atvgt <dig>
vitis vinifera putative inositol transporters
we identified  <dig> orfs showing the strongest similarity with the  <dig> atint  already described in arabidopsis.
to our knowledge, only two atint have been already characterized.
atint <dig> is described as a high-affinity, plasma membrane-localized h+/symporter specific for myo-inositol  <cit> .
atint <dig> is a tonoplast-localized h+/inositol symporter that mediates the efflux of inositol that is generated during the degradation of inositol-containing compounds in the vacuolar lumen  <cit> .
the three vitis orfs were named vvint1- <dig> according to their highest similarity with atint .
other vitis vinifera putative monosaccharide transporters
finally,  <dig> orfs show high similarity with the members of the arabidopsis atpglct/atsgb <dig> subfamily, which includes proteins showing homology with a putative glucose transporter  of the chloroplast inner envelope membrane from spinach  <cit>  and with a golgi-localized hexose transporter homolog .
the orf gsvivt <dig> is identical to a v. vinifera sugar transporter already mentioned in the literature and called vvpglt  <cit> .
phylogenetic tree  reveals that inside this subfamily, the proteins separate into  <dig> groups having strong bootstrap support .
vvpglt and atpglct fall into the same group, which includes also sopglct from spinach .
this observation can argue in favor of a chloroplastic localization of vvpglt even if the precise localization of this transporter is not demonstrated.
in a similar way, the fact that vv <dig> and vv <dig> form a second group with at1g <dig> and sgb <dig> indicates that these two vitis putative transporters could be localized in golgi apparatus.
finally vv <dig> forms a third group with at1g <dig>
search for cis-elements putatively involved in the transcriptional regulation of sugar transporter genes
we have identified a  <dig> kb promoter region for each of the  <dig> fully sequenced genes from the four mostly studied sugar transporter families: vvsuc/sut, vvht, vvtmt and vvpmt .
for only three genes vvht <dig> , vvtmt <dig>  and vvpmt <dig> , the identified sequence is shorter due to the presence of an other orf located less than 2kb upstream of these transporter genes.
a place analysis has been applied to these promoter sequences and the  <dig> identified cis-acting elements have been classified per sugar transporter subfamily, for comparison.
cis-elements common to all promoters
in a first approach,  <dig> common cis-regulatory elements conserved in the promoter regions of the  <dig> analyzed sequences have been identified .
only the shortest promoter vvpmt <dig>  is missing three of these elements, namely ciacadianlelhc, pyrimidineboxosramy1a and wboxatnpr <dig>  moreover, these common consensus sequences are highly repetitive displaying up to  <dig> copies into a  <dig> kb promoter.
this might be due to the their limited size , and to their high variability .
these common cis-acting elements are able to confer expression in distinct plant organs, such as leaves, shoots, roots, seeds, and flowers .
they are also responsive to different plant hormones , as well as to several environmental factors .
at least a quarter of these common consensus sequences  are required for the transcriptional regulation by light, and this mainly in leaves and shoots.
this is in agreement with the roles of the studied transporters in sugar allocation between source- and sink-organs.
finally, the presence of the box ciacadianlelhc, absent only in the vvpmt <dig>  vvtmt <dig> and vvht <dig> promoters, strongly suggests the importance of circadian regulation for sugar transporter gene expression.
promoter sequence analysis was performed via place.
cis-element name, sequence motifs and signalling pathway are presented.
up to copies/promoter indicates the highest number of cis-acting element found in one promoter.
* indicates that the motif is found in all promoters except vvpmt <dig>  ** indicates that the motif is found in all promoters except vvpmt <dig>  vvtmt <dig> and vvpmt <dig>
cis-elements present only in a single promoter
a second complementary approach was targeted to unique consensus sequences present in the promoter of only one sugar transporter gene, thereby implying some expression specificity.
the few unique identified cis-elements  are characterized by longer sequences , and usually lacking any nucleotide variability.
interestingly, among the  <dig> gene specific motifs identified,  <dig> are present only in the vvht <dig> promoter,  <dig> in vvtmt <dig>  one in vvht <dig> and another one in vvsuc <dig>  this means that a limited number of gene specific cis-acting elements is concentrated in the promoter regions of few sugar transporter genes.
for example the hexose transporter gene vvht <dig> is the only one among the  <dig> genes studied, displaying  <dig> unique motifs  in its promoter.
finally, another specific cis-element strongly restricted to vvsuc/vvsut genes is the motif mybcoreatcycb <dig> , required for transcriptional regulation of cyclin b <dig> at two different phases of the cell cycle, g1/s and g2/m transitions  <cit> .
promoter sequence analysis was performed via place.
cis-element names, sequence motifs, signalling pathways and the number of copies for each element are presented.
gene indicates the corresponding gene in which the cis-elements are found.
cis-elements involved in sugar regulated transcription
we have studied the transcriptional regulation of sugar transporter genes through the repertory of the main promoter motifs potentially involved in sugar-regulated transcription, and this in combination with other metabolic and hormonal signalling.
additional file  <dig> summarizes the careful comparison of the following consensus sequences: i) elements for sugar responsiveness as the sure boxes  <cit> , the bipartite sucrose box  <dig>  <cit> , the cgacgosamy <dig>  <cit> , the cmsre  <cit> , the sp <dig> and wboxhviso <dig> sequences enabling the binding of some wrky-type proteins at the example of spf and susiba <dig>  <cit> ; ii) sequences common for hormonal and metabolic  signals perception as the s-box for sugar and aba  <cit> , the mybgahv for gibberellins  induction and sugar repression  <cit> ; the garc complex consisting of the amybox <dig> and  <dig>  <cit> , and pyrimidine boxes for gas, aba and sugar regulation.
there is at least one gene for each subfamily displaying the majority of chosen sugar responsive motifs , thus suggesting a possible transcriptional control dependent on sugars as metabolic signals .
the sucrose box  <dig>  is the most frequently found cis-acting element, present at  <dig> to  <dig> copies in all studied promoters except vvht <dig>  vvtmt <dig> and vvpmt <dig>  on the contrary, the cmsre1ibspoa element, involved in sucrose positive regulation is only found in promoter regions of vvht <dig>  vvht <dig> and vvtmt <dig>  the sucrose transporter gene family, is the only one displaying the sure <dig> motif in the promoter regions of vvsuc <dig> and vvsuc <dig>  the sugar responsiveness cgacgosamy <dig> box is carried only by vvht genes , and not by the other subfamilies.
similarly, the s-box  usually closely associated to the light-responsive g-box, is carried also only by vvht genes, namely vvht <dig>  vvht <dig> and vvht <dig>  inversely, the motif mybgahv involved in sugar and ga signalling pathways, is displayed by vvsuc/sut, vvpmt and vvtmt genes, but is lacking in vvht ones.
finally it appears that vvht <dig> and vvht <dig> promoter sequences are the only one to contain a putative garc complex.
a more detailed comparison into the vvht subfamily reveals that vvht <dig> and vvht <dig> promoters are carrying the same cis-elements .
this indicates that as the coding sequences, the promoter regions for these two putative genes present also a very high similarity .
this argues in favor of the assumption that these are either alleles of the same gene or represent the same gene, as already suggested in this study.
such consideration is valuable for another couple of genes mentioned above, vvht <dig> and vvht <dig>  sharing a strong sequence similarity, displaying the same cis-acting elements in their promoter region, and carried on the same chromosome.
a third gene vvht <dig> is present in tandem repeat with both vvht <dig> and vvht <dig>  by the same chromosome, thus suggesting that they may be products of successive duplications.
v. vinifera sugar transporter genes expression in vegetative organs
in order to study the expression pattern of grapevine sugar transporter genes identified above and belonging to the vvsuc/vvsut, vvht, vvtmt, and vvpmt subfamilies, we have developed sugar transporter macroarray membranes.
specific regions for each sugar transporter  have been identified in the 3'utr of the corresponding nucleotide sequences, amplified by pcr using chardonnay genomic dna and spotted on nylon membrane.
vvht <dig>  vvht <dig>  vvht <dig> and vvht <dig> to vvht <dig>  could not be considered in this expression analysis, as it was not possible to found specific dna region for these transporters, due to their high sequence similarity either with vvht <dig> or between each other.
to determine the gene expression patterns in vegetative organs, macroarrays were hybridized with 33p-labelled first-strand cdna synthesized from total rna isolated from young leaves, mature leaves, petioles, stems, roots and tendrils from  <dig> weeks-old grapevine plants grown under aeroponic conditions.
these culture conditions have been used in order to collect all main vegetative organs at the same development stage and in the same conditions and allowed an easy access to the root system without damage.
among the sucrose transporters only vvsuc <dig> is detected at a high level in petioles, stems and tendrils, its transcripts being less abundant in young leaves, mature leaves and roots .
vvsuc <dig> and vvsuc <dig> are detected in all organs but at a weaker level and vvsut <dig> is the less expressed sucrose transporter being only weakly detected in young leaves and roots.
concerning the vvht family ,  <dig> genes  are expressed in all the tested organs at a relatively high level  with vvht <dig> and vvht <dig> being less expressed in young leaves and tendrils, respectively.
vvht <dig> and vvht <dig> seem to present a more specific expression.
vvht <dig> is expressed at a higher level in roots than in the other organs and vvht <dig> is highly expressed in mature leaves and presents a weaker expression in roots and young leaves.
the three other hexose transporters  are weakly detected, indicating a low expression in the tested organs.
the three vvtmt are also detected at a very low level in all organs .
among the polyol/monosaccharide transporters , only vvpmt <dig> could be significantly detected in the six organs.
it shows a strong expression in mature leaves, petioles and tendrils, and a weaker expression in stems, roots and young leaves.
the other vvpmt are weakly expressed in all the tested organs.
in order to validate the results obtained with macroarray hybridizations and to confirm the expression pattern of the sugar transporter genes, we performed a northern blot analysis for few genes.
the results presented in figure  <dig> clearly show similar expression patterns for most of the tested genes.
vvht <dig> shows the weakest expression in young leaves.
vvht <dig> seems to be specifically expressed in roots.
vvht <dig> presents a weak expression in all organs, except in mature leaves.
vvtmt <dig> shows a global weak expression in all organs.
vvpmt <dig> is highly expressed in mature leaves, petioles and tendrils.
finally, vvsuc <dig> shows high amount of transcripts in petioles, stems and tendrils.
few discrepancies were however observed.
first, the expression level detected for vvht <dig> is the highest in mature leaves when detected by northern blot which is not the case using macroarray.
second, vvht <dig> and vvht <dig> show a higher expression in macroarray than in northern blot analysis.
for vvht <dig> signals obtained with northern blot were too low to be correctly quantified.
furthermore, we could confirm using both methods that vvht <dig> is expressed at a higher level than vvht <dig>  vvht <dig>  vvht <dig>  and vvht <dig> in almost all organs.
taken together, all these results indicate that few transporter genes  are the most expressed in almost all vegetative organs and that vvht <dig> and vvht <dig> are more specifically expressed in roots and mature leaves, respectively.
sugar transporter genes expression during grape berry development
in order to study the expression of sugar transporter genes during berry development, we further hybridized the sugar transporters macroarray membranes with 33p-labelled first-strand cdna synthesized from total rna isolated from berries and seeds.
four developmental stages for berries - fruit set , veraison , ripening , ripe berries  - and two for seeds  were used.
among the sucrose transporter genes, vvsuc <dig> and vvsuc <dig> are both expressed during berry development at a similar level to that detected in vegetative organs .
the weakest expression for these two genes is observed in berries at the stage of fruit set.
on the contrary, vvsuc <dig>  the most expressed sucrose transporter gene in vegetative organs is less expressed than vvsuc <dig> and vvsuc <dig> in berries while vvsut <dig> is weakly or not detected.
three hexose transporters are expressed during berries development .
vvht <dig> is expressed during the  <dig> tested stages and presents a maximum at the veraison and during ripening.
inversely, vvht <dig> and vvht <dig> are expressed at an equivalent level during the  <dig> developmental stages.
vvht <dig>  vvht <dig>  vvht <dig>  vvht <dig> and vvht <dig> are poorly or not detected at any stage.
the expression of two putative tonoplast monosaccharide transporters  which is weak at the fruit set increases significantly at the veraison .
furthermore, the expression level of these two genes is higher in berries at the veraison and during ripening than in vegetative organs.
on the contrary, vvtmt <dig> does not seem to be expressed in berries at any stage of development.
polyols transporters are not highly expressed in berries, only vvpmt <dig> is weakly detected during the  <dig> stages and vvpmt <dig> is expressed mainly at the fruit set stage .
macroarray hybridization performed with first-strand cdna synthesized from total rna isolated from seeds reveals clearly that the expression of vvht <dig> and vvht <dig> is very high in seeds and increases during seed development .
two other transporter genes vvsuc <dig> and vvsuc <dig> were also expressed in seeds but at a lower level, and their expression stays stable during the two tested developmental stages .
discussion
phylogenetic analysis of vitis vinifera sugar transporter genes
the search for sugar transporters in the vitis vinifera translated genome has identified  <dig> sucrose and  <dig> putative monosaccharide transporters including  <dig> vvht ,  <dig> vvtmt ,  <dig> vvpmt ,  <dig> vvint ,  <dig> vvvgt ,  <dig> pglt/sgb <dig> and  <dig> erd6-like transporters.
as expected, phylogenetic analysis performed with these sugar transporter proteins revealed that sucrose and monosaccharide transporters form two distinct groups .
this analysis allowed us to identify only  <dig> vitis sucrose transporters, which confirms that, as all other analyzed plants, vitis possesses a small sucrose transporter gene family, in which one gene  belongs to the sut <dig> subfamily.
interestingly, in vitis as in arabidopsis, the vvht and the erd6-like form the largest multigenic subfamilies.
in vitis, this may be due to the presence of  <dig> repeated regions, encompassing vvht and erd6-like genes.
two duplicated regions located on chromosomes  <dig> and  <dig> contain  <dig> and  <dig> vvht, respectively.
the  <dig> other regions carried by chromosomes  <dig> and  <dig> display respectively  <dig> and  <dig> erd6-like genes.
similarly, in arabidopsis, the large expansion of atstp subfamily has been correlated with  <dig> segmental duplications and one tandem duplication as well as the expansion of the aterd6-like subfamily by  <dig> segmental duplications and  <dig> tandem duplications  <cit> .
furthermore, based on significant differences in size observed in the stp and erd6-like subfamilies, between the non-vascular  and the vascular  lineages, it has been suggested that the expansion of these two subfamilies could be related to the evolution of vascular plants.
this is reflecting the increased importance of the sugar transport and sugar transporters in vascular plants  <cit> .
in agreement with this hypothesis, the aterd6/vverd6-like phylogenetic tree  clearly shows that erd <dig> transporters from both species fall into four different groups, two of them containing either aterd <dig> or vverd <dig> transporters only.
this indicates that in both species, the expansion of the erd6-like subfamilies has occurred quite recently, after the separation of these two species.
sugar-responsive elements in sugar transporter gene promoters suggest their regulation by sugars
the in silico search for cis-acting elements reveals several common and highly repetitive motifs in sugar transporter gene promoters.
these cis-acting elements such as dof  proteins, may play a role not only in the regulation of sugar transporter gene expression in terms of activity level, but also plausibly in terms of response specificity via a combinatory control.
such a control has already been suggested for atsuc <dig>  <cit>  the expression of which in the companion cell is regulated by the close cooperation of binding sites for a dof and a putative hd-zip transcription factors.
several transporter gene promoters display an important concentration of sugar-responsive elements suggesting their possible transcriptional regulation by sugars.
to our knowledge, the transcriptional regulation of vvht <dig> by glucose is the only one to be clearly demonstrated  <cit>  and this is confirmed by the fact that vvht <dig> promoter contains the highest number of sugar responsive motifs.
this highlights the power of the in silico analysis as a first step toward the functional characterization of promoter regions.
finally, the mybcoreatcycb <dig> sequence exclusively found in suc/sut promoters is not surprising in regard to the sucrose-dependent induction of cyclin d <dig> gene expression  <cit> , thus suggesting a possible concomitant regulation of some sucrose transporter genes in the cell cycle.
sucrose transporter genes expression in vitis vinifera
the expression patterns detected for the sucrose transporter genes, using macroarrays, are in good agreement with those described in the literature with two main exceptions: the absence of high expression of vvsuc <dig> in seeds and of vvsuc <dig> in roots, as reported by  <cit> .
on the contrary, we confirmed that, in berries, vvsuc <dig> and vvsuc <dig> transcripts are present at all developmental stages and accumulate slightly at the onset of ripening  <cit> .
vvsuc <dig> is closely related to atsuc <dig>  which has been localized in the tonoplast of a. thaliana mesophyll cells  <cit> .
furthermore, vvsuc <dig> falls into the sut2/suc <dig> group which contains very low affinity sucrose transporters for which different putative physiological functions have been proposed including their putative involvement in sucrose import into several sink tissues  <cit> .
therefore, it is tempting to suggest that vvsuc <dig> is probably involved either in phloem unloading or in sucrose import into berry tissues and that vvsuc <dig> might be responsible for sucrose accumulation in berry vacuoles.
this hypothesis will have to be verified with the precise localization of these two transporters.
we noticed that vvsuc <dig> is the most expressed sucrose transporter gene in vegetative organs and that its expression is relatively low in berries.
considering its high expression in petioles, stems and tendrils and the fact that it is closely related to members of the sut <dig> subfamily, vvsuc <dig> is probably responsible for phloem loading and sugar retrieval during long-distance transport.
finally, the weak expression level observed for the less characterized vitis transporter gene vvsut <dig> makes it difficult to assign a specific role for this transporter.
hexose transporter genes expression in vitis vinifera
the present phylogenetic analysis indicates that vvht <dig> shows highest similarity with atstp <dig>  both are high affinity glucose transporters showing km value of  <dig> μm  <cit>  and  <dig> μm  <cit> , respectively.
during the last decade, different authors have reported various expression patterns for vvht <dig> such as a strong expression in berries and young leaves  <cit> , a preferential expression in sink organs  <cit> , an expression in conducting bundle of leaves, petioles and berries  <cit>  or an expression increasing with leaf development  <cit> .
during berry development, vvht <dig> expression was described to show two peaks   <cit>  or to decline rapidly during ripening  <cit> .
this second expression pattern was supported by the detection of vvht <dig> protein only in young green berries  <cit> .
our results  clearly confirm that vvht <dig> belongs to the hexose transporters that are poorly expressed in berries, but is one of the mostly expressed vvht in vegetative organs including leaves, petioles, stems, roots and tendrils.
furthermore, its expression increases during leaf development.
vvht <dig> shows the highest similarity with atstp <dig> which has not been yet characterized.
different reports describing the expression of vvht <dig> have shown that vvht <dig> is weakly expressed in leaves whatever the stage of development  <cit>  and that the transcript level is high in young berries and declines around veraison  <cit> .
our data  confirm not only vvht <dig> expression in leaves and during berry development, but indicate also its weak expression in almost all vegetative organs except for roots in which it seems to be strongly expressed.
vvht <dig> has been described to be one of the mostly expressed vvht in leaves, with increasing expression during leaf development.
in young berries, its expression is high, decreases around veraison and increases again around the phase of sugar storage  <cit> .
the expression pattern determined in our experiment  correlates with that described previously.
however, we found that this transporter, which is expressed in all vegetative organs is also highly expressed in seeds , in which its expression seems to increase during development.
interestingly, vvht <dig> shows the highest similarity with atstp <dig> for which a strong seed expression is also suggested by microarray hybridization data .
although the localisation and the functionality of these transporters are unknown, they might have a determinant function in sugar storage in seeds and/or in embryo development.
vvht <dig> is poorly expressed in all the tested organs and hardly detectable in berries , in accordance with previous report describing a very weak expression in berry and leaf development  <cit> .
vvht <dig> has been characterized as a glucose transporter showing a high affinity for glucose , higher than that reported for atstp <dig>  the closest related arabidopsis transporter.
a physiological role, either to support wounded tissue or in the retrieval of monosaccharides released during cell damage and cell wall degradation, has been proposed for atstp <dig>  based on its induction after wounding  <cit> .
a more precise characterization of vvht <dig> is therefore required to verify if the expression of this gene is also regulated after wounding or in response to other stresses.
vvht <dig> was found to be less expressed than vvht <dig> and vvht <dig> in developing leaves and during berry development  <cit> .
our experiments confirmed that vvht <dig> transcripts are hardly detected in berries, however they are predominant in seeds, at least for the two tested developmental stages .
vvht <dig> shows the highest similarity with atstp <dig> and both have similar high affinity for glucose   <cit> .
furthermore, the expression of these two transporters is described to be induced in response to pathogen attack  <cit> .
this indicates that these genes could be involved in pathogen starvation and/or in a sugar signalling pathway in plant defense.
the presence of four unique cis-acting elements  and of a cluster of three abre motifs  <cit>  in the promoter region of vvht <dig> gene is in agreement with the regulation of its expression by aba and biotic stress but also suggests its involvement in abiotic stress responses.
three putative hexose transporters named vvht <dig>  vvht <dig> and vvht <dig>  that have never been described earlier, have been identified.
our phylogenetic analysis  revealed that vvht <dig> and vvht <dig> are each located at the basis of one of the two duplicated regions involved in the expansion of the vvht subfamily.
furthermore, this analysis allowed us to identify atstp <dig> and vvht <dig> as orthologs, but no orthologs for vvht <dig> and vvht <dig> could be found.
the expression pattern of vvht <dig>  the weak expression of vvht <dig> and vvht <dig> in all vegetative organs and in berries are not sufficient to suggest putative physiological functions for these three transporters.
however recent data indicates that vvht <dig> is induced by necrotrophic fungi and could be involved as vvht <dig> in biotic stress response .
putative tonoplastic monosaccharide transporter genes expression in vitis vinifera
three putative tonoplastic monosaccharide transporters were identified and named vvtmt1- <dig>  expression data indicate that, even if the expression of vvtmt <dig> and vvtmt <dig> is present in all the tested vegetative organs, it is highest in developing berries.
the expression of vvtmt <dig> is described to increase during berry development  <cit>  with a maximum near the start of veraison  <cit> .
our data not only confirm this expression pattern but suggest also that at least two transporters, vvtmt <dig> and vvtmt <dig>  might probably play a significant role during ripening.
although their cellular localisation and their transport activity have not been determined so far, they could be involved in hexose accumulation into vacuoles of berry cells.
furthermore, considering that the expression of attmt <dig> and attmt <dig> has been reported to be induced by drought, salt, and cold treatments  <cit> , it would be interesting to verify, if the expression of the vvtmt genes is also regulated under stress conditions, particularly in vegetative organs, where they are weakly expressed in normal conditions.
polyols transporter genes expression in vitis vinifera
since  <dig>  many polyol transporters have been identified and characterized in sorbitol or mannitol-translocating plants, where they are described to be responsible for the loading of polyols into the phloem and their transfer to sink organs  <cit> .
more recently, polyol transporters have been identified and characterized in non-polyol-translocating species such as a. thaliana  <cit>  which contains  <dig> polyol transporters, the physiological role of which is still unknown.
in vitis, the expression of only one est encoding a putative pmt has been already briefly mentioned in the literature and was shown to be weakly expressed during berry development  <cit> .
our in silico analysis, indicates that the vitis genome contains  <dig> putative polyol transporter genes.
among them only one named vvpmt <dig> was highly expressed in vegetative organs and only at the fruit set.
however as grapevine has not been described as a species transporting polyols in the phloem, the role of this transporter is far from being clear.
CONCLUSIONS
the present work represents the first exhaustive analysis of sugar transporter genes in a woody plant.
the identification of grapevine sugar transporter genes and their comparative analysis with the arabidopsis ones has indicated a strong conservation of these genes between herbaceous and woody plants as well as some expansions of particular functional subfamilies such as hexoses and erd6-like transporters.
in this paper, we developed macroarrays to profile the expression of  <dig> of these transporters simultaneously in different organs.
our results not only confirmed some expression data already described in the literature but also demonstrated that four sugar transporter genes are expressed in almost all vegetative organs , few transporters are more specifically expressed in roots , mature leaves  and/or in seeds  and three others are regulated during berry development  .
the present results might help to elucidate the biological function of sugar transporters in v. vinifera development particularly during berry ripening and would also have a significant impact on our knowledge on plant sugar transporters in general.
the in silico analysis of promoter sequences revealed the presence of cis-regulatory elements involved in sugar signalling, and represents a first step towards the understanding of the regulation of sugar transporter gene expression via metabolic, hormonal and environmental signals.
more and more evidences suggest that sugar transporter genes are regulated under various conditions.
thus, the macroarray analysis described in this paper could constitute a powerful approach to investigate the sugar transporter response to environmental factors in grapevine.

 in plants, sugars are important molecules that constitute not only metabolites but also nutrients, osmotic and signal molecules. in numerous species, sucrose is the most prevalent sugar produced in photosynthetic organs and transported via the phloem over long distances to heterotrophic organs, which depend on a constant supply of carbohydrates. in sink organs, sucrose is either directly imported or cleaved by cell wall-bound invertases into monosaccharides, that can be taken up by the sink cells. in some species, sugar alcohols, such as mannitol, sorbitol and galactinol can also be transported on top of sucrose for long-distance carbon partitioning. in addition to this long-distance transport, sugars can also be allocated in the different organelles of source and sink cells, and more and more biochemical and molecular studies argue for the transport of hexoses into the chloroplast the vacuoles, and the golgi apparatus. therefore, it is now clearly established that not only the loading and the unloading of the conducting complex, but also the allocation of sugars into source and sink cells is controlled by sugar transporters mediating the transport of sucrose, reducing monosaccharides, or polyols. since the cloning of the first monosaccharide transporter in chlorella, the first sucrose transporter in spinacia oleracea, and the first polyol transporter in apium graveolens, many genes belonging to these families have been isolated from various species. the complete arabidopsis genome has been described to contain sucrose transporter-like sequences and a monosaccharide transporter gene family, including members grouped into subfamilies. furthermore, the evolutionary analysis of plant monosaccharide transporters revealed that these seven subfamilies are ancient in land plants. despite the progress made in identifying genes encoding sugar transporters, little is known about the transcriptional regulation of these genes. arabidopsis microarray data and some plant transporter gene expression patterns have indicated that developmental and environmental factors could regulate the expression of sugar transporters. furthermore, evidence is provided that the expression of some sugar transporter genes is regulated by sugars as described for sugar transporter genes in yeast, for vvht a grapevine hexose transporter and for sucrose transporter genes from rice, ossut and sugar beet, bvsut. all these data suggest that the expression of sugar transporters might be regulated at the transcriptional level by distinct but usually converging signalling pathways, depending on either developmental and environmental cues or metabolic and hormonal signals. in spite of the evidence for the role of sugar signalling in the transcriptional control of some transporter genes, the in silico analysis of promoter regions of different genes involved in carbon metabolism, sugar storage, mobilization and transport clearly demonstrates the absence of common sugar specific cis-elements. this analysis is consistent with the fact that in plants, several types of transcription factors are required for sugar signalling and are involved in sugar-regulation of gene expression. considering that the analysis of sugar transporter orthologs in different species might help to better understand their biological function, we analyzed the recently sequenced vitis vinifera genotype pn in order to identify sugar transporter gene families in this species. this work will represent the first exhaustive analysis for sugar transporters in ligneous plant as most of the already known sugar transporters have been characterized from herbaceous species. in woody plants, only sucrose transporters have been already described in vitis, in citrus sinensis, in hevea brasiliensis and one in juglans regia. seven hexose transporters in vitis, in juglans regia and few polyol transporters in prunus cerasus, in malus domestica and in olea europea were also reported. furthermore during the last decade, vitis vinifera has become an interesting model to study fruit maturation. it is now clearly established that the onset of ripening is characterized by an important accumulation of glucose and fructose in vacuoles of the mesocarp cells. in grapevine, sucrose is the main carbohydrate used for long distance transport and after reaching the phloem of the berry, it is unloaded into the apoplast, possibly cleaved by apoplastic invertases, and sucrose or hexoses can than be transported into the mesocarp. in the cytoplasm of the mesocarp cells, sucrose and hexoses must be transported into the vacuole via tonoplastic transporters. the identification and the characterization of sugar transporter genes in vitis vinifera are therefore important steps in understanding the roles of these proteins in grapevine development as well as in grape ripening process and may further highlight our knowledge on plant sugar transporters. the present study reports on the identification of sucrose and monosaccharide-like transporter genes in the vitis vinifera genome, on their phylogenetic analysis in comparison with arabidopsis transporters, on their promoter sequences analysis. the construction of specialized cdna macroarrays used to determine the expression pattern for of these genes in grapevine vegetative organs and during berry ripening is also described.  identification of sugar transporters from vitis vinifera blastp searches of the grapevine genome proteome 8× database, using the amino acid sequences of the sucrose transporters and the monosaccharide transporters from a. thaliana as query, allowed the identification of orfs encoding putative sugar transporters in v. vinifera. among these orfs, only encode previously described sucrose transporters and no additional one could be identified. the other orfs seem to encode putative monosaccharide transporters. phylogenetic analysis of the v. vinifera identified protein sequences using the maximum likelihood method reveals that sucrose and monosaccharide transporters form two separate groups. furthermore in agreement with the phylogeny observed for a. thaliana mst, distinct subfamilies could be clearly identified in the vitis monosaccharide transporter group. vitis vinifera sucrose transporters the amino acids sequences encoding the already described sucrose transporters named vvsuc11/vvsut vvsuc vvsuc and vvsut share to 59% similarity between each other and fall into three sucrose transporter subgroups already described. vvsut and vvsuc shows 59% similarity and belong to the dicots specific sut subfamily including high affinity sucrose transporters exhibiting apparent km value between and mm. however, vvsuc has been described to be a low-affinity/high-capacity sucrose transporter showing a km value for sucrose ranged between and mm. the structure of vvsut and vvsuc genes is quite similar, both being around bp long and containing exons separated by introns. vvsuc shows % similarity with atsuc and presents an extended domain at the n terminus and an elongated central cytoplasmic loop; two structural characteristics specific to the sut2/suc subfamily. furthermore, vvsuc gene is a very long gene containing exons interrupted by introns; such exon/intron organization is also described for atsuc the km value for sucrose reported for this transporter seems, however, higher than that described for other members of this subfamily, showing either a low affinity or no sucrose transport function. finally, although vvsuc has a high affinity for sucrose it shows % similarity with atsuc and falls into the sut subfamily including all low-affinity plant sucrose transporters with km value ranging between mm and mm. vitis vinifera putative hexose transporters among the identified orfs, showed high similarity with the atstp subfamily members. among these, correspond to the already well known v. vinifera hexose transporters named vvht such as vvht vvht vvht, vvht and vvht,. therefore, the newly identified orfs were named vvht to vvht vvht amino acid sequence shows % similarity with vvht and the main differences between the two nucleotide sequences reside in some single nucleotide polymorphism and in the length of a microsatellite sequence in the 3'utr region. considering that the chromosomal location of vvht and vvht is not determined, it is difficult to conclude if these sequences represent two independent genes, two alleles of the same gene or possibly one single gene. vvht and vvht share % similarity between each other and around 73% with vvht interestingly, the three corresponding genes are located in a tandem repeat region, on chromosome in a similar way, vvht to vvht form a cluster on chromosome and the corresponding amino acid sequences show very high similarity. vvht and vvht which are located nearby on chromosome contain the two first exons and the last exon of a monosaccharide transporter, respectively. a detailed amino acid sequence analysis revealed that these two partial orfs are wrongly annotated and might constitute a single monosaccharide transporter, in the third exon of which a stop codon replaces a tryptophan residue. it is therefore tempting to suggest that this point mutation at the origin of the false annotation, might be due to a sequencing error, but we can not exclude that it could be real. finally vvht vvht and vvht are partial mst, whose sequences do not seem to be fully sequenced, missing either the n-ter or the c-ter region, or both. therefore, considering that vvht might be identical to vvht and that vvht and vvht are probably a single protein, we can estimate that the grape genome might contain putative hexose transporters. in this vvht subfamily, the exon-intron organization seems to be conserved as all completely sequenced genes contain exons separated by introns with the exception of vvht and vvht phylogenetic analysis reveals that the vvht subfamily seems to be divided into two subclades, at the basis of which is located vvht and vvht both present on chromosome vvht is located at the basis of a subclade having a bootstrap value of and containing the closely related transporters from chromosome and. furthermore, if we exclude vvht and vvht sister-pairs with a strong bootstrap support could be identified, vvht3/vvht; vvht9/vvht and vvht2/vvht. finally, phylogenetic analysis of a. thaliana and v. vinifera sugar transporter proteins allowed us to identify six ortholog pairs between both species such as vvht1/atstp, vvht2/atstp, vvht3/atstp, vvht4/atstp, vvht5/atstp, vvht13/atstp. five of these pairs are supported by bootstrap value of 100%. vitis vinifera putative tonoplast monosaccharide transporters we have also identified three orfs, which show the strongest similarity to the a. thaliana tonoplast monosaccharide transporters. all three vitis orfs show an extended middle loop between the putative trans-membrane helices six and seven in a similar way as the attmt. gsvivt is identical to a v. vinifera sugar transporter already mentioned in the literature and called vvht. considering that it shows a higher similarity with attmt than with the vvht proteins, we renamed it vvtmt similarly, gsvivt and gsvivt were called vvtmt and vvtmt respectively. the exon-intron organization seems to be conserved in the three genes as they contain all exons separated by introns. phylogenetic tree performed with vitis sugar transporter amino acid sequences reveals that the vvtmt form a clade, which is closely related to putative myo-inositol transporters and vacuolar glucose transporters. furthermore, phylogenetic analysis using vitis and arabidopsis sequences confirms that tmt sequences from both species form a single clade, with a strong bootstrap support, but within which low bootstrap values indicate unresolved nodes and fail to detect sister-pairs between both species. vitis vinifera putative polyol/monosaccharide transporters five orfs show highest similarity with the a. thaliana polyol transporters and have been therefore named vvpmt to vvpmt v. vinifera putative polyol transporter amino acids sequences share 40% to % similarity between themselves and the corresponding genes present all the same structure with exons separated by a single intron. phylogenetic analysis performed with the a. thaliana and v. vinifera polyol transporters reveals that vvpmt and vvpmt form with atpmt a separated clade. vvpmt is at the basis of a second clade, which can be divided into two groups, one including vvpmt atpmt and atpmt and the second atpmt atpmt atpmt and vvpmt only atpmt and vvpmt could be identified as putative orthologs. vitis vinifera putative erd6-like transporters twenty-two orfs showing strongest similarity with the aterd6-like proteins were identified and share to % similarity with each other. among them, orfs correspond to partial sequences in which either the beginning or the end of the protein are not clearly identified. however after a more precise sequence analysis, we were able to realize the full annotation for gsvivt and gsvivt fourteen orfs are located on chromosome in a region of tandem gene duplications, three other orfs are carried by chromosome and two partial orfs by chromosome the erd6-like proteins fall into the same subfamily supported by a strong bootstrap value and loci formed sister pairs. the phylogenetic analysis of the amino acid sequences of erd6-like transporters from a. thaliana and v. vinifera reveals that these transporters can be classified into major groups. a first group includes aterd6-like located on the chromosomes of a. thaliana and vverd6-like located on chromosomes of v. vinifera. a second small group including transporters from both species was also identified. inversely, the two last groups include protein sequences from only one species. the arabidopsis group contains proteins located on three different chromosomes including erd, sfp and sfp. the vitis group includes proteins, of which are encoded by genes located on chromosome vitis vinifera putative vacuolar glucose transporters two vitis orfs, named vvvgt and vvvgt show the highest similarity with the atvgt -like transporters. in arabidopsis atvgt and atvgt have been shown to be localized in the tonoplast and glucose transport activity has been demonstrated for atvgt. on the contrary atvgt is postulated to be localized in chloroplast membrane as this protein presents a n-terminal extension carrying a potential signal for plastid targeting. phylogenetic tree indicates clearly that vvvgt is the closest to atvgt and atvgt and that vvvgt which presents a n-terminal extension, is more closely related to atvgt vitis vinifera putative inositol transporters we identified orfs showing the strongest similarity with the atint already described in arabidopsis. to our knowledge, only two atint have been already characterized. atint is described as a high-affinity, plasma membrane-localized h+/symporter specific for myo-inositol. atint is a tonoplast-localized h+/inositol symporter that mediates the efflux of inositol that is generated during the degradation of inositol-containing compounds in the vacuolar lumen. the three vitis orfs were named vvint1- according to their highest similarity with atint. other vitis vinifera putative monosaccharide transporters finally, orfs show high similarity with the members of the arabidopsis atpglct/atsgb subfamily, which includes proteins showing homology with a putative glucose transporter of the chloroplast inner envelope membrane from spinach and with a golgi-localized hexose transporter homolog. the orf gsvivt is identical to a v. vinifera sugar transporter already mentioned in the literature and called vvpglt. phylogenetic tree reveals that inside this subfamily, the proteins separate into groups having strong bootstrap support. vvpglt and atpglct fall into the same group, which includes also sopglct from spinach. this observation can argue in favor of a chloroplastic localization of vvpglt even if the precise localization of this transporter is not demonstrated. in a similar way, the fact that vv and vv form a second group with at1g and sgb indicates that these two vitis putative transporters could be localized in golgi apparatus. finally vv forms a third group with at1g search for cis-elements putatively involved in the transcriptional regulation of sugar transporter genes we have identified a kb promoter region for each of the fully sequenced genes from the four mostly studied sugar transporter families: vvsuc/sut, vvht, vvtmt and vvpmt. for only three genes vvht, vvtmt and vvpmt, the identified sequence is shorter due to the presence of an other orf located less than 2kb upstream of these transporter genes. a place analysis has been applied to these promoter sequences and the identified cis-acting elements have been classified per sugar transporter subfamily, for comparison. cis-elements common to all promoters in a first approach, common cis-regulatory elements conserved in the promoter regions of the analyzed sequences have been identified. only the shortest promoter vvpmt is missing three of these elements, namely ciacadianlelhc, pyrimidineboxosramy1a and wboxatnpr moreover, these common consensus sequences are highly repetitive displaying up to copies into a kb promoter. this might be due to the their limited size, and to their high variability. these common cis-acting elements are able to confer expression in distinct plant organs, such as leaves, shoots, roots, seeds, and flowers. they are also responsive to different plant hormones, as well as to several environmental factors. at least a quarter of these common consensus sequences are required for the transcriptional regulation by light, and this mainly in leaves and shoots. this is in agreement with the roles of the studied transporters in sugar allocation between source- and sink-organs. finally, the presence of the box ciacadianlelhc, absent only in the vvpmt vvtmt and vvht promoters, strongly suggests the importance of circadian regulation for sugar transporter gene expression. promoter sequence analysis was performed via place. cis-element name, sequence motifs and signalling pathway are presented. up to copies/promoter indicates the highest number of cis-acting element found in one promoter. * indicates that the motif is found in all promoters except vvpmt ** indicates that the motif is found in all promoters except vvpmt vvtmt and vvpmt cis-elements present only in a single promoter a second complementary approach was targeted to unique consensus sequences present in the promoter of only one sugar transporter gene, thereby implying some expression specificity. the few unique identified cis-elements are characterized by longer sequences, and usually lacking any nucleotide variability. interestingly, among the gene specific motifs identified, are present only in the vvht promoter, in vvtmt one in vvht and another one in vvsuc this means that a limited number of gene specific cis-acting elements is concentrated in the promoter regions of few sugar transporter genes. for example the hexose transporter gene vvht is the only one among the genes studied, displaying unique motifs in its promoter. finally, another specific cis-element strongly restricted to vvsuc/vvsut genes is the motif mybcoreatcycb, required for transcriptional regulation of cyclin b at two different phases of the cell cycle, g1/s and g2/m transitions. promoter sequence analysis was performed via place. cis-element names, sequence motifs, signalling pathways and the number of copies for each element are presented. gene indicates the corresponding gene in which the cis-elements are found. cis-elements involved in sugar regulated transcription we have studied the transcriptional regulation of sugar transporter genes through the repertory of the main promoter motifs potentially involved in sugar-regulated transcription, and this in combination with other metabolic and hormonal signalling. additional file summarizes the careful comparison of the following consensus sequences: i) elements for sugar responsiveness as the sure boxes, the bipartite sucrose box, the cgacgosamy, the cmsre, the sp and wboxhviso sequences enabling the binding of some wrky-type proteins at the example of spf and susiba; ii) sequences common for hormonal and metabolic signals perception as the s-box for sugar and aba, the mybgahv for gibberellins induction and sugar repression; the garc complex consisting of the amybox and, and pyrimidine boxes for gas, aba and sugar regulation. there is at least one gene for each subfamily displaying the majority of chosen sugar responsive motifs, thus suggesting a possible transcriptional control dependent on sugars as metabolic signals. the sucrose box is the most frequently found cis-acting element, present at to copies in all studied promoters except vvht vvtmt and vvpmt on the contrary, the cmsre1ibspoa element, involved in sucrose positive regulation is only found in promoter regions of vvht vvht and vvtmt the sucrose transporter gene family, is the only one displaying the sure motif in the promoter regions of vvsuc and vvsuc the sugar responsiveness cgacgosamy box is carried only by vvht genes, and not by the other subfamilies. similarly, the s-box usually closely associated to the light-responsive g-box, is carried also only by vvht genes, namely vvht vvht and vvht inversely, the motif mybgahv involved in sugar and ga signalling pathways, is displayed by vvsuc/sut, vvpmt and vvtmt genes, but is lacking in vvht ones. finally it appears that vvht and vvht promoter sequences are the only one to contain a putative garc complex. a more detailed comparison into the vvht subfamily reveals that vvht and vvht promoters are carrying the same cis-elements. this indicates that as the coding sequences, the promoter regions for these two putative genes present also a very high similarity. this argues in favor of the assumption that these are either alleles of the same gene or represent the same gene, as already suggested in this study. such consideration is valuable for another couple of genes mentioned above, vvht and vvht sharing a strong sequence similarity, displaying the same cis-acting elements in their promoter region, and carried on the same chromosome. a third gene vvht is present in tandem repeat with both vvht and vvht by the same chromosome, thus suggesting that they may be products of successive duplications. v. vinifera sugar transporter genes expression in vegetative organs in order to study the expression pattern of grapevine sugar transporter genes identified above and belonging to the vvsuc/vvsut, vvht, vvtmt, and vvpmt subfamilies, we have developed sugar transporter macroarray membranes. specific regions for each sugar transporter have been identified in the 3'utr of the corresponding nucleotide sequences, amplified by pcr using chardonnay genomic dna and spotted on nylon membrane. vvht vvht vvht and vvht to vvht could not be considered in this expression analysis, as it was not possible to found specific dna region for these transporters, due to their high sequence similarity either with vvht or between each other. to determine the gene expression patterns in vegetative organs, macroarrays were hybridized with 33p-labelled first-strand cdna synthesized from total rna isolated from young leaves, mature leaves, petioles, stems, roots and tendrils from weeks-old grapevine plants grown under aeroponic conditions. these culture conditions have been used in order to collect all main vegetative organs at the same development stage and in the same conditions and allowed an easy access to the root system without damage. among the sucrose transporters only vvsuc is detected at a high level in petioles, stems and tendrils, its transcripts being less abundant in young leaves, mature leaves and roots. vvsuc and vvsuc are detected in all organs but at a weaker level and vvsut is the less expressed sucrose transporter being only weakly detected in young leaves and roots. concerning the vvht family, genes are expressed in all the tested organs at a relatively high level with vvht and vvht being less expressed in young leaves and tendrils, respectively. vvht and vvht seem to present a more specific expression. vvht is expressed at a higher level in roots than in the other organs and vvht is highly expressed in mature leaves and presents a weaker expression in roots and young leaves. the three other hexose transporters are weakly detected, indicating a low expression in the tested organs. the three vvtmt are also detected at a very low level in all organs. among the polyol/monosaccharide transporters, only vvpmt could be significantly detected in the six organs. it shows a strong expression in mature leaves, petioles and tendrils, and a weaker expression in stems, roots and young leaves. the other vvpmt are weakly expressed in all the tested organs. in order to validate the results obtained with macroarray hybridizations and to confirm the expression pattern of the sugar transporter genes, we performed a northern blot analysis for few genes. the results presented in figure clearly show similar expression patterns for most of the tested genes. vvht shows the weakest expression in young leaves. vvht seems to be specifically expressed in roots. vvht presents a weak expression in all organs, except in mature leaves. vvtmt shows a global weak expression in all organs. vvpmt is highly expressed in mature leaves, petioles and tendrils. finally, vvsuc shows high amount of transcripts in petioles, stems and tendrils. few discrepancies were however observed. first, the expression level detected for vvht is the highest in mature leaves when detected by northern blot which is not the case using macroarray. second, vvht and vvht show a higher expression in macroarray than in northern blot analysis. for vvht signals obtained with northern blot were too low to be correctly quantified. furthermore, we could confirm using both methods that vvht is expressed at a higher level than vvht vvht vvht and vvht in almost all organs. taken together, all these results indicate that few transporter genes are the most expressed in almost all vegetative organs and that vvht and vvht are more specifically expressed in roots and mature leaves, respectively. sugar transporter genes expression during grape berry development in order to study the expression of sugar transporter genes during berry development, we further hybridized the sugar transporters macroarray membranes with 33p-labelled first-strand cdna synthesized from total rna isolated from berries and seeds. four developmental stages for berries - fruit set, veraison, ripening, ripe berries - and two for seeds were used. among the sucrose transporter genes, vvsuc and vvsuc are both expressed during berry development at a similar level to that detected in vegetative organs. the weakest expression for these two genes is observed in berries at the stage of fruit set. on the contrary, vvsuc the most expressed sucrose transporter gene in vegetative organs is less expressed than vvsuc and vvsuc in berries while vvsut is weakly or not detected. three hexose transporters are expressed during berries development. vvht is expressed during the tested stages and presents a maximum at the veraison and during ripening. inversely, vvht and vvht are expressed at an equivalent level during the developmental stages. vvht vvht vvht vvht and vvht are poorly or not detected at any stage. the expression of two putative tonoplast monosaccharide transporters which is weak at the fruit set increases significantly at the veraison. furthermore, the expression level of these two genes is higher in berries at the veraison and during ripening than in vegetative organs. on the contrary, vvtmt does not seem to be expressed in berries at any stage of development. polyols transporters are not highly expressed in berries, only vvpmt is weakly detected during the stages and vvpmt is expressed mainly at the fruit set stage. macroarray hybridization performed with first-strand cdna synthesized from total rna isolated from seeds reveals clearly that the expression of vvht and vvht is very high in seeds and increases during seed development. two other transporter genes vvsuc and vvsuc were also expressed in seeds but at a lower level, and their expression stays stable during the two tested developmental stages. discussion phylogenetic analysis of vitis vinifera sugar transporter genes the search for sugar transporters in the vitis vinifera translated genome has identified sucrose and putative monosaccharide transporters including vvht, vvtmt, vvpmt, vvint, vvvgt, pglt/sgb and erd6-like transporters. as expected, phylogenetic analysis performed with these sugar transporter proteins revealed that sucrose and monosaccharide transporters form two distinct groups. this analysis allowed us to identify only vitis sucrose transporters, which confirms that, as all other analyzed plants, vitis possesses a small sucrose transporter gene family, in which one gene belongs to the sut subfamily. interestingly, in vitis as in arabidopsis, the vvht and the erd6-like form the largest multigenic subfamilies. in vitis, this may be due to the presence of repeated regions, encompassing vvht and erd6-like genes. two duplicated regions located on chromosomes and contain and vvht, respectively. the other regions carried by chromosomes and display respectively and erd6-like genes. similarly, in arabidopsis, the large expansion of atstp subfamily has been correlated with segmental duplications and one tandem duplication as well as the expansion of the aterd6-like subfamily by segmental duplications and tandem duplications. furthermore, based on significant differences in size observed in the stp and erd6-like subfamilies, between the non-vascular and the vascular lineages, it has been suggested that the expansion of these two subfamilies could be related to the evolution of vascular plants. this is reflecting the increased importance of the sugar transport and sugar transporters in vascular plants. in agreement with this hypothesis, the aterd6/vverd6-like phylogenetic tree clearly shows that erd transporters from both species fall into four different groups, two of them containing either aterd or vverd transporters only. this indicates that in both species, the expansion of the erd6-like subfamilies has occurred quite recently, after the separation of these two species. sugar-responsive elements in sugar transporter gene promoters suggest their regulation by sugars the in silico search for cis-acting elements reveals several common and highly repetitive motifs in sugar transporter gene promoters. these cis-acting elements such as dof proteins, may play a role not only in the regulation of sugar transporter gene expression in terms of activity level, but also plausibly in terms of response specificity via a combinatory control. such a control has already been suggested for atsuc the expression of which in the companion cell is regulated by the close cooperation of binding sites for a dof and a putative hd-zip transcription factors. several transporter gene promoters display an important concentration of sugar-responsive elements suggesting their possible transcriptional regulation by sugars. to our knowledge, the transcriptional regulation of vvht by glucose is the only one to be clearly demonstrated and this is confirmed by the fact that vvht promoter contains the highest number of sugar responsive motifs. this highlights the power of the in silico analysis as a first step toward the functional characterization of promoter regions. finally, the mybcoreatcycb sequence exclusively found in suc/sut promoters is not surprising in regard to the sucrose-dependent induction of cyclin d gene expression, thus suggesting a possible concomitant regulation of some sucrose transporter genes in the cell cycle. sucrose transporter genes expression in vitis vinifera the expression patterns detected for the sucrose transporter genes, using macroarrays, are in good agreement with those described in the literature with two main exceptions: the absence of high expression of vvsuc in seeds and of vvsuc in roots, as reported by. on the contrary, we confirmed that, in berries, vvsuc and vvsuc transcripts are present at all developmental stages and accumulate slightly at the onset of ripening. vvsuc is closely related to atsuc which has been localized in the tonoplast of a. thaliana mesophyll cells. furthermore, vvsuc falls into the sut2/suc group which contains very low affinity sucrose transporters for which different putative physiological functions have been proposed including their putative involvement in sucrose import into several sink tissues. therefore, it is tempting to suggest that vvsuc is probably involved either in phloem unloading or in sucrose import into berry tissues and that vvsuc might be responsible for sucrose accumulation in berry vacuoles. this hypothesis will have to be verified with the precise localization of these two transporters. we noticed that vvsuc is the most expressed sucrose transporter gene in vegetative organs and that its expression is relatively low in berries. considering its high expression in petioles, stems and tendrils and the fact that it is closely related to members of the sut subfamily, vvsuc is probably responsible for phloem loading and sugar retrieval during long-distance transport. finally, the weak expression level observed for the less characterized vitis transporter gene vvsut makes it difficult to assign a specific role for this transporter. hexose transporter genes expression in vitis vinifera the present phylogenetic analysis indicates that vvht shows highest similarity with atstp both are high affinity glucose transporters showing km value of μm and μm, respectively. during the last decade, different authors have reported various expression patterns for vvht such as a strong expression in berries and young leaves, a preferential expression in sink organs, an expression in conducting bundle of leaves, petioles and berries or an expression increasing with leaf development. during berry development, vvht expression was described to show two peaks or to decline rapidly during ripening. this second expression pattern was supported by the detection of vvht protein only in young green berries. our results clearly confirm that vvht belongs to the hexose transporters that are poorly expressed in berries, but is one of the mostly expressed vvht in vegetative organs including leaves, petioles, stems, roots and tendrils. furthermore, its expression increases during leaf development. vvht shows the highest similarity with atstp which has not been yet characterized. different reports describing the expression of vvht have shown that vvht is weakly expressed in leaves whatever the stage of development and that the transcript level is high in young berries and declines around veraison. our data confirm not only vvht expression in leaves and during berry development, but indicate also its weak expression in almost all vegetative organs except for roots in which it seems to be strongly expressed. vvht has been described to be one of the mostly expressed vvht in leaves, with increasing expression during leaf development. in young berries, its expression is high, decreases around veraison and increases again around the phase of sugar storage. the expression pattern determined in our experiment correlates with that described previously. however, we found that this transporter, which is expressed in all vegetative organs is also highly expressed in seeds, in which its expression seems to increase during development. interestingly, vvht shows the highest similarity with atstp for which a strong seed expression is also suggested by microarray hybridization data. although the localisation and the functionality of these transporters are unknown, they might have a determinant function in sugar storage in seeds and/or in embryo development. vvht is poorly expressed in all the tested organs and hardly detectable in berries, in accordance with previous report describing a very weak expression in berry and leaf development. vvht has been characterized as a glucose transporter showing a high affinity for glucose, higher than that reported for atstp the closest related arabidopsis transporter. a physiological role, either to support wounded tissue or in the retrieval of monosaccharides released during cell damage and cell wall degradation, has been proposed for atstp based on its induction after wounding. a more precise characterization of vvht is therefore required to verify if the expression of this gene is also regulated after wounding or in response to other stresses. vvht was found to be less expressed than vvht and vvht in developing leaves and during berry development. our experiments confirmed that vvht transcripts are hardly detected in berries, however they are predominant in seeds, at least for the two tested developmental stages. vvht shows the highest similarity with atstp and both have similar high affinity for glucose. furthermore, the expression of these two transporters is described to be induced in response to pathogen attack. this indicates that these genes could be involved in pathogen starvation and/or in a sugar signalling pathway in plant defense. the presence of four unique cis-acting elements and of a cluster of three abre motifs in the promoter region of vvht gene is in agreement with the regulation of its expression by aba and biotic stress but also suggests its involvement in abiotic stress responses. three putative hexose transporters named vvht vvht and vvht that have never been described earlier, have been identified. our phylogenetic analysis revealed that vvht and vvht are each located at the basis of one of the two duplicated regions involved in the expansion of the vvht subfamily. furthermore, this analysis allowed us to identify atstp and vvht as orthologs, but no orthologs for vvht and vvht could be found. the expression pattern of vvht the weak expression of vvht and vvht in all vegetative organs and in berries are not sufficient to suggest putative physiological functions for these three transporters. however recent data indicates that vvht is induced by necrotrophic fungi and could be involved as vvht in biotic stress response. putative tonoplastic monosaccharide transporter genes expression in vitis vinifera three putative tonoplastic monosaccharide transporters were identified and named vvtmt1- expression data indicate that, even if the expression of vvtmt and vvtmt is present in all the tested vegetative organs, it is highest in developing berries. the expression of vvtmt is described to increase during berry development with a maximum near the start of veraison. our data not only confirm this expression pattern but suggest also that at least two transporters, vvtmt and vvtmt might probably play a significant role during ripening. although their cellular localisation and their transport activity have not been determined so far, they could be involved in hexose accumulation into vacuoles of berry cells. furthermore, considering that the expression of attmt and attmt has been reported to be induced by drought, salt, and cold treatments, it would be interesting to verify, if the expression of the vvtmt genes is also regulated under stress conditions, particularly in vegetative organs, where they are weakly expressed in normal conditions. polyols transporter genes expression in vitis vinifera since many polyol transporters have been identified and characterized in sorbitol or mannitol-translocating plants, where they are described to be responsible for the loading of polyols into the phloem and their transfer to sink organs. more recently, polyol transporters have been identified and characterized in non-polyol-translocating species such as a. thaliana which contains polyol transporters, the physiological role of which is still unknown. in vitis, the expression of only one est encoding a putative pmt has been already briefly mentioned in the literature and was shown to be weakly expressed during berry development. our in silico analysis, indicates that the vitis genome contains putative polyol transporter genes. among them only one named vvpmt was highly expressed in vegetative organs and only at the fruit set. however as grapevine has not been described as a species transporting polyols in the phloem, the role of this transporter is far from being clear.  the present work represents the first exhaustive analysis of sugar transporter genes in a woody plant. the identification of grapevine sugar transporter genes and their comparative analysis with the arabidopsis ones has indicated a strong conservation of these genes between herbaceous and woody plants as well as some expansions of particular functional subfamilies such as hexoses and erd6-like transporters. in this paper, we developed macroarrays to profile the expression of of these transporters simultaneously in different organs. our results not only confirmed some expression data already described in the literature but also demonstrated that four sugar transporter genes are expressed in almost all vegetative organs, few transporters are more specifically expressed in roots, mature leaves and/or in seeds and three others are regulated during berry development. the present results might help to elucidate the biological function of sugar transporters in v. vinifera development particularly during berry ripening and would also have a significant impact on our knowledge on plant sugar transporters in general. the in silico analysis of promoter sequences revealed the presence of cis-regulatory elements involved in sugar signalling, and represents a first step towards the understanding of the regulation of sugar transporter gene expression via metabolic, hormonal and environmental signals. more and more evidences suggest that sugar transporter genes are regulated under various conditions. thus, the macroarray analysis described in this paper could constitute a powerful approach to investigate the sugar transporter response to environmental factors in grapevine. 
they are distributed through the plant via sugar transporters, which are involved not only in sugar long-distance transport via the loading and the unloading of the conducting complex, but also in sugar allocation into source and sink cells. this study provides an exhaustive survey of sugar transporter genes in vitis vinifera and revealed that sugar transporter gene families in this woody plant are strongly comparable to those of herbaceous species. a search for cis-regulatory elements in the promoter sequences of the most characterized transporter gene families, has revealed that some of them might probably be regulated by sugars. the present results might also have a significant impact on our knowledge on plant sugar transporters. finally, three putative hexose transporter genes show a preferential organ specificity being highly expressed in seeds, in roots or in mature leaves. phylogenetic analysis of protein sequences has indicated that orthologs exist between vitis and arabidospis. this macroarray analysis has revealed that two hexose, one polyol and one sucrose transporter genes, are highly expressed in most vegetative organs. to profile several genes simultaneously, we created a macroarray bearing cdna fragments specific to sugar transporter genes. dedicated macroarrays have provided a vitis sugar transporter genes expression profiling, which will likely contribute to understand their physiological functions in plant and berry development. the availability of the recently released grapevine genome sequence offers the opportunity to identify sucrose and monosaccharide transporter gene families in a woody species and to compare them with those of the herbaceous arabidopsis thaliana using a phylogenetic analysis. the expression of one hexose transporter and two tonoplastic monosaccharide transporter genes are regulated during berry development.
 in higher plants, sugars are not only nutrients but also important signal molecules. they are distributed through the plant via sugar transporters, which are involved not only in sugar long-distance transport via the loading and the unloading of the conducting complex, but also in sugar allocation into source and sink cells. the availability of the recently released grapevine genome sequence offers the opportunity to identify sucrose and monosaccharide transporter gene families in a woody species and to compare them with those of the herbaceous arabidopsis thaliana using a phylogenetic analysis.  in grapevine, one of the most economically important fruit crop in the world, it appeared that sucrose and monosaccharide transporter genes are present in and loci, respectively and that the monosaccharide transporter family can be divided into subfamilies. phylogenetic analysis of protein sequences has indicated that orthologs exist between vitis and arabidospis. a search for cis-regulatory elements in the promoter sequences of the most characterized transporter gene families, has revealed that some of them might probably be regulated by sugars. to profile several genes simultaneously, we created a macroarray bearing cdna fragments specific to sugar transporter genes. this macroarray analysis has revealed that two hexose, one polyol and one sucrose transporter genes, are highly expressed in most vegetative organs. the expression of one hexose transporter and two tonoplastic monosaccharide transporter genes are regulated during berry development. finally, three putative hexose transporter genes show a preferential organ specificity being highly expressed in seeds, in roots or in mature leaves.  this study provides an exhaustive survey of sugar transporter genes in vitis vinifera and revealed that sugar transporter gene families in this woody plant are strongly comparable to those of herbaceous species. dedicated macroarrays have provided a vitis sugar transporter genes expression profiling, which will likely contribute to understand their physiological functions in plant and berry development. the present results might also have a significant impact on our knowledge on plant sugar transporters. 