Fatma Lecourieux

Christian Kappel

David Lecourieux

Alejandra Serrano

Elizabeth Torres

Patricio Arce-Johnson

Serge Delrot
email: serge.delrot@bordeaux.inra.fr

An update on sugar transport and signalling in grapevine

Keywords: Fruit biology, grapevine, signalling, stress, sugar, transport

In addition to their role as a source of reduced carbon, sugars may directly or indirectly control a wide range of activities in plant cells, through transcriptional and post-translational regulation. This control has been studied in detail using Arabidopsis thaliana, where genetic analysis offers many possibilities. Much less is known about perennial woody species. For several years, various aspects of sugar sensing and signalling have been investigated in the grape (Vitis vinifera L.) berry, an organ that accumulates high concentrations of hexoses in the vacuoles of flesh cells. Here we review various aspects of this topic: the molecular basis of sugar transport and its regulation by sugars in grapevine; the functional analysis of several sugar-induced genes; the effects of some biotic and abiotic stresses on the sugar content of the berry; and finally the effects of exogenous sugar supply on the ripening process in field conditions. A picture of complex feedback and multiprocess regulation emerges from these data.

Introduction

Sugar transport and allocation of assimilates between sources and sinks are major parameters controlling crop productivity [START_REF] Gifford | Crop productivity and assimilate partitioning[END_REF]. In addition to their role as a supply of energy and carbon backbone for growth and development in plants, sugars directly or indirectly control a wide range of processes, including photosynthesis, sugar transport itself, nitrogen uptake, defence reactions, secondary metabolism, and hormonal balance [START_REF] Smeekens | Sugar signals and molecular networks controlling plant growth[END_REF]. This control is mainly based on the regulation of gene expression [START_REF] Koch | Carbohydrate-modulated gene expression in plants[END_REF], but it may also affect mRNA stability, and protein translation and stability [START_REF] Wiese | A conserved upstream open reading frame mediates sucrose-induced repression of translation[END_REF]. Monosaccharides (glucose, fructose, mannose, or galactinol) and disaccharides (sucrose, trehalose, and sucrose analogues) may affect gene expression. The genes affected encode sugar transporters, components of the photosystems such as chlorophyll a/bbinding protein and plastocyanin, enzymes of carbohydrate metabolism (small subunit of RubisCO, α-amylase, invertase, sucrose synthase, and ADPG-prophosphorylase), enzymes of secondary metabolism (phenylalanine ammonia lyase and other enzymes of the flavonoid biosynthesis pathway), and transcription factors [START_REF] Koch | Carbohydrate-modulated gene expression in plants[END_REF][START_REF] Saigne | Effect of methyl jasmonate in combination with carbohydrates on gene expression of PR proteins, stilbene and anthocyanin accumulation in grapevine cell cultures[END_REF][START_REF] Agasse | Sugar transport and sugar sensing in grape[END_REF][START_REF] Roitsch | Source-sink regulation by sugar and stress[END_REF][START_REF] Smeekens | Sugar-induced signal transduction in plants[END_REF][START_REF] Smeekens | Sugar signals and molecular networks controlling plant growth[END_REF][START_REF] Nicolas | The grape berry-specific basic helix-loop-helix transcription factor VvCEB1 affects cell size[END_REF][START_REF] Tognetti | Sucrose signaling in plants. A world yet to be explored[END_REF]. The perception of sugars or sugar fluxes by membrane or cytosolic sensors is called sugar sensing, and the pathways that mediate sugarcontrolled responses constitute sugar signalling. Components of sugar sensing and signalling provide the molecular basis through which plant cells adapt their activity as a function of their sugar status. In plants, these processes have been described for different sugars and are intimately interconnected with hormonal signalling pathways [abscisic acid (ABA), ethylene, and cytokinin] [START_REF] Gibson | Sugar and phytohormone response pathways: navigating a signalling network[END_REF][START_REF] Rolland | Sugar sensing and signaling in plants[END_REF][START_REF] Rolland | Sugar sensing and signalling in plants: conserved and novel mechanisms[END_REF][START_REF] Grigston | D-Glucose sensing by a plasma membrane regulator of G signaling protein, AtRGS1[END_REF][START_REF] Ramon | Sugar sensing and signalling[END_REF][START_REF] Hanson | Sugar perception and signaling-an update[END_REF]. Several additional sugar-derived signalling systems were recently identified and described as important players in plant development because they integrate sugar availability and developmental programmes [START_REF] Smeekens | Sugar signals and molecular networks controlling plant growth[END_REF]. These systems include trehalose-6 phosphate (T6P) [START_REF] Ponnu | Trehalose-6-phosphate: connecting plant metabolism and development[END_REF][START_REF] Dai | Metabolic profiling reveals coordinated switches in primary carbohydrate metabolism in grape berry (Vitis vinifera L.), a non-climacteric fleshy fruit[END_REF], sucrose non-fermenting-related kinase-1 (SnRK1), and the target of rapamycin (TOR) kinase complex [START_REF] John | Plant TOR signaling componenets[END_REF]. All these processes are interconnected and were recently shown to play key roles in plant responses to abiotic stresses by maintaining the homeostasis between sugar availability and plant metabolism [START_REF] Smeekens | Sugar signals and molecular networks controlling plant growth[END_REF][START_REF] Robaglia | Sensing nutrient and energy status by SnRK1 and TOR kinases[END_REF].

Grapevine (Vitis vinifera L.) is a woody perennial plant providing fruit species used as fresh fruit, dried raisins, and for wine making and distillation of liquors. In 2011, vineyards occupied ~7585 thousands hectares throughout the world, and total grape production reached 69.2 Mt (OIV report 2012; http://www.oiv.int). Ripe grape berries contain a very high concentration of glucose and fructose (~1 M each) in the vacuole of flesh cells [START_REF] Fontes | Grape berry vacuole: a complex and heterogeneous membrane system specialized in the accumulation of solutes[END_REF]. The sink/source ratio is an important parameter for the sensory properties of the berries, and is manipulated through viticultural practices which affect the number of leaves and clusters. Grapevine is therefore a woody species particularly relevant for the study of sugar transport and sugar signalling, and the present review will summarize the data obtained on these topics. Initial work on sugar signalling in grape mainly concerned the regulation of anthocyanin biosynthesis, and of sugar transport (for reviews, see [START_REF] Agasse | Sugar transport and sugar sensing in grape[END_REF][START_REF] Davies | Source/sink relationships and molecular biology of sugar accumulation in grape berries[END_REF], but recent data indicate that other major processes such as cell growth are also controlled by sugars in the grape berry. A picture of complex feedback and multiprocess regulation emerges from these data.

Sugar transport and sugar transporters in grapevine

Physiological background

In grapevine, as in many other dicotyledons, sucrose is the form of long-distance transport in the phloem. The ripening grape berry is a strong sink for dry matter transported from current photosynthesis and wood reserves [START_REF] Coombe | The grape berry as a sink[END_REF]. The sugars accumulated in the vacuoles of mesocarp cells account for 65-91% of the fresh weight in a mature berry. They start to accumulate in a linear way when the berries reach the veraison stage, which corresponds to the trigger of ripening. At this stage, the berries also change colour and begin to soften, which indicates a coordinated reprogramming of cell activities. Grapevine is a non-climacteric fruit, and the molecular signals controlling ripening are still debated (see [START_REF] Kühn | Berry ripening: recently heard through the grapevine[END_REF]. However, sugar and ABA accumulation in the berries follow a similar pattern.

Since the sugars accumulated in the vacuoles of flesh cells are glucose and fructose, the accumulation of these hexoses in the berries involves the activity of sucrose-metabolizing enzymes, sucrose transporters, and monosaccharide transporters (MSTs; [START_REF] Agasse | Sugar transport and sugar sensing in grape[END_REF]. The events occurring at veraison were studied in detail in a Vitis vinifera×Vitis labrusca hybrid. [START_REF] Zhang | A shift of phloem unloading from symplasmic to apoplasmic pathway is involved in developmental onset of ripening in grape berry[END_REF] conducted electron microscope analysis, measured the activity of sucrose-degrading enzymes (invertases and sucrose synthases), and studied the movement of symplastic tracers and of companion cellexpressed green fluorescent protein (GFP)-tagged viral protein. They demonstrated that a shift from a symplasmic to an apoplasmic unloading pathway occurs at or just prior to the onset of ripening. In addition, they observed an increase in the expression and activity of cell wall invertases that was concomitant with a rise in apoplastic sugar concentration and osmotic pressure. Apoplastic phloem unloading coupled with solute accumulation in the berry apoplast may be responsible for the decline in xylem water influx into ripening grape berries [START_REF] Keller | Ripening grape berries remain hydraulically connected to the shoot[END_REF].

Sugar concentrations in the skin are generally lower than those in the flesh tissue [START_REF] Coombe | Distribution of solutes within the developing grape berry in relation to its morphology[END_REF], and hexose concentration gradients have been detected in the flesh (low hexose concentration near the brush, high at the stylar end). Active uptake of d-glucose in the flesh is only detected after the beginning of ripening, while it can be measured at both pre-and post-veraison stage in isolated skin pieces [START_REF] Coombe | Sugar accumulation by grape berry pericarp cells. I. Sugar uptake by skin segments[END_REF]. However, a high proportion of the sugar uptake by skin is diffusive, whatever the sampling time, in contrast to the mesocarp tissue where the uptake is principally active.

Sucrose transporters

Analysis of the grape genome sequence suggests that sucrose transporter genes constitute a small multigenic family of four members in this species [START_REF] Agasse | Sugar transport and sugar sensing in grape[END_REF][START_REF] Afoufa-Bastien | The Vitis vinifera sugar transporter gene family: phylogenetic overview and macroarray expression profiling[END_REF]. Three sucrose transporter cDNAs have been cloned from Shiraz and Cabernet Sauvignon berries (VvSUC11; AF021808, also identified as VvSUT1 AF182445; VvSUC12 AF021809; and VvSUC27 AF021810) and characterized as proton-dependent sucrose transporters by heterologous expression in Saccharomyces cerevisiae. VvSUC11 and VvSUC12 are intermediate affinity sucrose transporters (K m 0.9 mM and 1.4 mM, respectively) [START_REF] Ageorges | Characterization of an active sucrose transporter gene expressed during the ripening of grape berry (Vitis vinifera L.)[END_REF][START_REF] Manning | Functional characterization of two ripening-related sucrose transporters from grape berries[END_REF], and VvSUC27 is a low affinity sucrose transporter (K m of 8-10 mM; [START_REF] Zhang | Functional characterization of a LAHC sucrose transporter isolated from grape berries in yeast[END_REF]. VvSUT2, whose sequence is close to that of VvSUC27, is weakly expressed in the berries [START_REF] Afoufa-Bastien | The Vitis vinifera sugar transporter gene family: phylogenetic overview and macroarray expression profiling[END_REF].

The sucrose transporters expressed in the berries must fulfil two opposite functions: maintain sucrose in the conducting bundles (through uptake and/or retrieval) until it reaches the site of unloading, and, at this site, they must allow its leakage or mediate efflux. Heterologous expression in yeast indicates that these three sucrose transporters act as protonsucrose symporters, and are thus expected to mediate sucrose uptake (in the phloem, or in the mesocarp cells). Whether one or several of these transporters may act in the reverse mode [START_REF] Carpaneto | Phloem-localized, proton-coupled sucrose carrier ZmSUT1 mediates sucrose efflux under the control of the sucrose gradient and the proton motive force[END_REF] and may play a role in sucrose efflux from the phloem has not yet been tested. Such reverse mode function may not be necessary if the transmembrane motive force that maintains constant uptake and retrieval of sucrose along the conducting complex, against a very high concentration gradient, decreases at the site of unloading. VvSUC11 and VvSUC12 transcripts concomitantly increase with postvéraison sugar accumulation, in contrast to VvSUC27 transcripts whose amounts significantly decrease at this stage [START_REF] Davies | Three putative sucrose transporters are differentially expressed in grapevine tissues[END_REF]. The different expression profile and the different K m of VvSUC27 clearly indicate a function that differs from that of the other SUC transporters. Unfortunately, no immunolocalization data are available to determine precisely the cellular and subcellular localization of these transporters.

AtSUC4 (Arabidopsis thaliana) and HvSUT2 (barley), which belong to group 4 of the sucrose transporters family [START_REF] Sauer | Molecular physiology of higher plant sucrose transporters[END_REF], have low affinity for sucrose (K m =5-6 mM), are expressed in sink organs, and are localized in the vacuolar membrane. In Lotus japonicus, LjSUT4 functions in the proton-coupled uptake of sucrose and possibly other glucosides into the cytoplasm from the vacuole [START_REF] Reinders | Functional analysis of LjSUT4, a vacuolar sucrose transporter from Lotus japonicus[END_REF]. VvSUC11 is the closest homologue to AtSUC4 (68%), but it has a higher affinity for sucrose (K m =0.88 mM) than AtSUC4. No data on its subcellular localization are available. Although SUT2/SUC3 was initially described as a sucrose sensor located in the plasma membrane of sieve tubes in tomato [START_REF] Barker | SUT2, a putative sucrose sensor in sieve elements[END_REF][START_REF] Reinders | Protein-protein interactions between sucrose transporters of different affinities colocalized in the same enucleate sieve element[END_REF], this conclusion was later questioned [START_REF] Sauer | Molecular physiology of higher plant sucrose transporters[END_REF], and its putative sucrose-sensing function no longer seems to be valid. The closest homologue to AtSUC3 in the grape genome is VvSUC12, but it has a lower K m (1.4 mM) than AtSUC3 (11.7 mM).

Hexose transporters

In grape, 59 putative hexose transporter homologues have been identified based on protein motif recognition [START_REF] Samson | FLAGdb++: a database for the functional analysis of the Arabidopsis genome[END_REF][START_REF] Jaillon | The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla[END_REF][START_REF] Agasse | Sugar transport and sugar sensing in grape[END_REF][START_REF] Afoufa-Bastien | The Vitis vinifera sugar transporter gene family: phylogenetic overview and macroarray expression profiling[END_REF]. Six full-length cDNAs encoding MSTs and named VvHT1-VvHT6 (V. vinifera hexose transporter; VvHT1 AJ001061; VvHT2 AY663846; VvHT3 AY538259 and AY854146; VvHT4 AY538260; VvHT5 AY538261; VvHT6 AY861386, DQ017393) have been cloned from various cultivars such as Pinot noir, Ugni blanc, Chardonnay, Cabernet Sauvignon, and Syrah [START_REF] Fillion | Cloning and expression of a hexose transporter gene expressed during the ripening of grape berry[END_REF][START_REF] Vignault | VvHT1 encodes a monosaccharide transporter expressed in the conducting complex of the grape berry phloem[END_REF][START_REF] Hayes | Isolation, functional characterization, and expression analysis of grapevine (Vitis vinifera L.) hexose transporters: differential roles in sink and source tissues[END_REF]. In spite of the high number of putative monosaccharide genes in the grape genome, no other VvHT was identified in grape berries, suggesting that the most important transporters for sugar accumulation in this organ have already been cloned. VvHT1, VvHT2, and particularly VvHT3 are highly expressed compared with the other VvHT genes, at all stages of berry development. The diversity of hexose transporter genes expressed during berry development is consistent with the shift from a symplastic to an apoplastic phloem unloading pathway that occurs prior to veraison. With the exception of VvHT6, all VvHTs isolated so far from grape berries present high homologies with AtSTPs; that is, functional hexose transporters. VvHT1, the first transporter of this family identified in grape [START_REF] Fillion | Cloning and expression of a hexose transporter gene expressed during the ripening of grape berry[END_REF], and functionally characterized [START_REF] Vignault | VvHT1 encodes a monosaccharide transporter expressed in the conducting complex of the grape berry phloem[END_REF], is homologous to AtSTP1.

Plasma membrane hexose transporters

The plasma membrane localization of VvHT1, VvHT4, and VvHT5 has been demonstrated by immunofluorescence, immunolabelling, and GFP fusion proteins [START_REF] Vignault | VvHT1 encodes a monosaccharide transporter expressed in the conducting complex of the grape berry phloem[END_REF][START_REF] Hayes | Isolation, functional characterization, and expression analysis of grapevine (Vitis vinifera L.) hexose transporters: differential roles in sink and source tissues[END_REF]. Functional studies based on expression in a hxt-null mutant yeast EBY VW 4000 showed that VvHT1, VvHT4, and VvHT5 encode highaffinity, H + -dependent glucose transporters. VvHT1 has a higher affinity for glucose (K m of 70 μM) than VvHT4 and VvHT5 (K m ~150 μM and 100 μM, respectively) and displays broad substrate specificity, being able to transport galactose, xylose, and glucose analogues such as 3-O-methyl-d-glucose (3-OMeG). Uptake of radiolabelled d-[U-14 C]fructose in grape cells also displays Michaelis-Menten kinetics, with a V max similar to that measured for glucose uptake, suggesting that both glucose and fructose are transported by the same protein, although the K m for fructose is much higher than the K m for glucose [START_REF] Conde | Pathways of glucose regulation of monosaccharide transport in grape cells[END_REF]. Both VvHT1 transcripts and protein levels [START_REF] Conde | Pathways of glucose regulation of monosaccharide transport in grape cells[END_REF] are much higher at prevéraison stages, indicating that it is not directly responsible for the post-véraison sugar accumulation [START_REF] Vignault | VvHT1 encodes a monosaccharide transporter expressed in the conducting complex of the grape berry phloem[END_REF][START_REF] Conde | Pathways of glucose regulation of monosaccharide transport in grape cells[END_REF]. VvHT1 transcripts are abundant in the phloem region of the conducting bundles of the leaf, petiole, and berry [START_REF] Vignault | VvHT1 encodes a monosaccharide transporter expressed in the conducting complex of the grape berry phloem[END_REF]. This localization, and its high affinity for hexoses, suggest that it could be involved in the retrieval of minor amounts of hexoses leaking from the conducting complex.

While VvHT4 transport activity may be restricted to glucose, VvHT5 is able to transport both glucose and fructose [START_REF] Hayes | Isolation, functional characterization, and expression analysis of grapevine (Vitis vinifera L.) hexose transporters: differential roles in sink and source tissues[END_REF]. VvHT5 transcripts are relatively more abundant at late ripening, but their amount remains low. VvHT3, whose transcript levels are reduced at véraison but high at both green and ripening stages [START_REF] Hayes | Isolation, functional characterization, and expression analysis of grapevine (Vitis vinifera L.) hexose transporters: differential roles in sink and source tissues[END_REF], is homologous to AtSTP7, which has not been functionally characterized. VvHT3 is not able to transport any of the tested radiolabelled sugars in the deficient yeast model [START_REF] Vignault | VvHT1 encodes a monosaccharide transporter expressed in the conducting complex of the grape berry phloem[END_REF][START_REF] Hayes | Isolation, functional characterization, and expression analysis of grapevine (Vitis vinifera L.) hexose transporters: differential roles in sink and source tissues[END_REF], and its transport function is also unknown. A gene (re)named VvHT8 has a high similarity to VvHT1 (99.4%) and was identified as a target of grape selection that has led to high sugar contents in domesticated grapes [START_REF] Xin | The effects of artificial selection on sugar metabolism and transporter genes in grape[END_REF].

Tonoplast hexose transporters

VvHT2 and VvHT6 seem to be localized in the tonoplast (C. Vignault et al., unpublished), and VvHT6 has high sequence similarity to the previously described tonoplast transporter of A. thaliana AtTMT2 [START_REF] Afoufa-Bastien | The Vitis vinifera sugar transporter gene family: phylogenetic overview and macroarray expression profiling[END_REF]. The TMT (tonoplast monosaccharide transporter) subfamily of MFS (major facilitator superfamily) transporters contains tonoplast hexose-proton antiporters. This family is represented by three members in V. vinifera [START_REF] Afoufa-Bastien | The Vitis vinifera sugar transporter gene family: phylogenetic overview and macroarray expression profiling[END_REF]. VvTMT1 has been cloned, localized, and characterized as a functional MST localized in the tonoplast of grape cells [START_REF] Zeng | Cloning, localization and expression analysis of a new tonoplast monosaccharide transporter from Vitis vinifera L[END_REF]. Its expression decreases strongly during berry development. Unlike VvTMT1, VvHT6/TMT2 transcripts are highly accumulated at véraison [START_REF] Terrier | Isogene specific oligo arrays reveal multifaceted changes in gene expression during grape berry (Vitis vinifera L.) development[END_REF][START_REF] Deluc | Transcriptomic and metabolite analyses of Cabernet Sauvignon grape berry development[END_REF], suggesting that this transporter may be responsible for vacuolar accumulation of hexose at the inception of ripening. VvHT2, whose expression is mainly associated with véraison [START_REF] Terrier | Isogene specific oligo arrays reveal multifaceted changes in gene expression during grape berry (Vitis vinifera L.) development[END_REF][START_REF] Hayes | Isolation, functional characterization, and expression analysis of grapevine (Vitis vinifera L.) hexose transporters: differential roles in sink and source tissues[END_REF]C. Vignault et al., unpublished), is homologous to AtSTP5, but its transport activity has not yet been characterized.

Others

Chen et al. (2010) identified a new class of sugar transporters, named SWEETs, found in Caenorhabditis elegans, plants, and mammals, and mediating glucose efflux. AtSWEET1, cloned from A. thaliana, was co-expressed in human HEK293T cells, which have low endogenous glucose uptake activity, as assessed with FLIPglu600mD13V, a high-sensitivity fluorescence resonance energy transfer (FRET) glucose sensor. The glucose-dependent response of the sensor demonstrated that AtSWEET1 can mediate both uptake across the plasma membrane and efflux into the endoplasmic reticulum. SWEET1 seems to function as a bidirectional uniporter/facilitator, which is largely pH independent. It has a low affinity for glucose, and does not efficiently complement mannose, fructose, and galactose uptake deficiencies of yeast mutants [START_REF] Chen | Sugar transporters for intercellular exchange and nutrition of pathogens[END_REF]. Sixteen SWEET homologues may be identified in the grapevine genome (Fig. 1A) but none has yet been studied in detail. Among these homologues, only three are expressed significantly during berry development (Fig. 1B). The SWEET gene that is the most expressed in the berries is close to AtSWEET15 [START_REF] Chen | Sucrose efflux mediated by SWEET proteins as a key step for phloem transport[END_REF].

Mechanisms involving endocytosis have been described in a species accumulating concentrations of mannitol in the fruit (olive tree; [START_REF] Conde | An Hg-sensitive channel mediates the diffusional component of glucose transport in olive cells[END_REF], but they have not been explored in grapevine.

Control of sugar transporters by sugars

The promoter region (2500 bp upstream of the site for initiation of transcription) of VvHT1 contains several sugarresponsive elements [START_REF] Fillion | Cloning and expression of a hexose transporter gene expressed during the ripening of grape berry[END_REF], and this led us to investigate the possibility that the expression of this sugar transporter gene may be regulated by the sugar concentration in the cell. Different lengths of the promoter were transcriptionally fused to the β-glucuronidase (GUS) reporter gene and used to transform tobacco. The VvHT1 promoter directed expression in sink organs. Glucose (58 mM), sucrose (58 mM), and the non-transported sucrose isomer palatinose doubled the GUS activity conferred by the VvHT1 promoter in tobacco cells, whereas fructose did not affect it [START_REF] Atanassova | Sugar-regulated expression of a putative hexose transport gene in grape[END_REF]. These effects were the strongest with the 2.4 kb promoter, which contains all putative sugarresponsive elements (activating and repressing), but they were Fig. 1. Phylogenic tree and berry expression of SWEET transporter genes in grape. (A) Evolutionary tree of grapevine and Arabidopsis SWEET transporters inferred by using the maximum likelihood method implemented in the MEGA5 software [START_REF] Tamura | MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods[END_REF]. Grapevine SWEET were identified by their PFAM [START_REF] Punta | The Pfam protein families database[END_REF] also significant with the 0.3 kb promoter, which contains only activating sugar boxes. The induction of VvHT1 expression by both sucrose and palatinose was confirmed in the homologous grape berry cell culture. These data provided the first example of a sugar transporter induced by both glucose and sucrose in higher plants. The fact that palatinose, a sucrose homologue that is only very poorly absorbed by plant cells, may up-regulate VvHT1 expression may suggest the existence of a plasma membrane sucrose sensor and of signalling pathways that start from the plasma membrane. Although induction of VvHT1 expression by sucrose does not require transport, the glucosyl moiety of the sucrose molecule is necessary for sugar sensing [START_REF] Atanassova | Sugar-regulated expression of a putative hexose transport gene in grape[END_REF].

The proximal 160 bp region of the VvHT1 promoter upstream of the 'TATA box' was used to develop a onehybrid approach (Cakir et al., 2003). This region, which contains two positive sugar-responsive motifs, is a perfect 'sucrose box 3' encompassing an imperfect SURE1 sequence. The screening allowed the identification of a grape ASR (abscisic acid, stress, ripening induced gene), named VvMSA, interacting with these DNA elements (Cakir et al., 2003). Gel shift assays confirmed specific binding of VvMSA to the two sugar-responsive elements present in the proximal region of the promoter. VvMSA and VvHT1 share similar patterns of expression during the ripening of grape. Both genes are inducible by sucrose in grape berry cell culture. The positive regulation of VvHT1 promoter activity by VvMSA was also shown in plants by co-expression experiments. In contrast to the data obtained in grape, the Asr1 transcripts levels were negatively correlated with glucose content and mRNA levels of the hexose transporter Ht2 in tubers of transgenic potato plants [START_REF] Frankel | ci21A/Asr1 expression influences glucose accumulation in potato tubers[END_REF].

In grape cell suspension, sugar induction of VvMSA was strongly enhanced by ABA, suggesting that VvMSA is involved in a common transduction pathway of sugar and ABA signalling (Cakir et al., 2003). Several ASR orthologous and paralogous genes are transcriptionally regulated by ABA and sugars (Dominguez et al., 2013, and references therein). [START_REF] Saumonneau | Interaction of grape ASR proteins with a DREB transcription factor in the nucleus[END_REF] described some polymorphism for grape ASRs and characterized them as chromosomal non-histone proteins. By a yeast two-hybrid approach and functional analysis, they identified a protein partner of VvMSA which was characterized as an APETALA2 domain transcription factor named VvDREB. Later, three ASR genes were isolated from two grape varieties, Cabernet Sauvignon and Ugni blanc [START_REF] Saumonneau | November 2020 and response to glucose and abscisic acid[END_REF]. One was specific for Cabernet Sauvignon, one for Ugni blanc, and one was common to both varieties. Functional analysis of the promoters of these genes demonstrated the organ-preferential expression conferred on the GUS reporter gene, and the specific phloem tissue localization. These ASR may play a role in the fine tuning of hormonal and metabolic signalling which allows the integration of environmental cues [START_REF] Saumonneau | November 2020 and response to glucose and abscisic acid[END_REF].

ASRs are not only involved in the control of hexose uptake in heterotrophic organs. Recently, [START_REF] Dominguez | ASR1 mediates glucose-hormone cross talk by affecting sugar trafficking in tobacco plants[END_REF] suggested that in tobacco (Nicotiana tabacum) ASRs were involved in the signalling cascade of interactions among glucose, ABA, and gibberellins. The fine regulation mediated by ASR through hexokinase1 and Snf1-related kinase on glucose metabolism resulted in dwarfism, reduced carbon dioxide assimilation, and accelerated leaf senescence. [START_REF] Conde | Pathways of glucose regulation of monosaccharide transport in grape cells[END_REF] used grape heterotrophic suspensioncultured cells as a model system to study glucose transport and its regulation. Glucose uptake in these cells was mediated by a high-affinity, broad-specificity H1-dependent transport system (K m =0.05 mM) superimposed on first-order kinetics. The glucose concentration in the medium tightly regulated the transcription of VvHT1 as well as the amount of VvHT1 protein and monosaccharide transport activity. The other MST genes expressed in grape berry (VvHT2, VvHT3, VvHT4, VvHT5, and VvHT7) were poorly expressed and responded weakly to glucose. Various sugars were tested for their ability to repress VvHT1 expression: 2-DOG (2-deoxyd-glucose), which can be phosphorylated, 3-OMeG, which cannot be phosphorylated, and mannoheptulose (MHL), a potent inhibitor of hexokinase. VvHT1 transcription was strongly repressed by glucose and 2-DOG, but not by 3-OMeG or glucose plus MHL, indicating a hexokinasedependent repression of transcription. Although OMeG, and glucose plus MHL did not affect the amount of VvHT1 transcripts, they decreased the glucose uptake capacity of the cells. This was due to the reduction of VvHT1 protein in the plasma membrane. High glucose down-regulated VvHT1 transcription and glucose uptake, whereas low glucose increased these parameters. These data provide an example showing control of plant sugar transporters by their own substrate at both transcriptional and post-transcriptional levels. In this experimental system, glucose regulated its own uptake through hexokinase-dependent repression of transcription, and through hexokinase-independent post-transcriptional regulation.

Sugar regulation of genes involved in the anthocyanin biosynthesis pathway

Flavonoids play an important role in defence against pathogens, protection from UV radiation, and coloration of flowers and fruits. The anthocyanins, which belong to the flavonoid group, are mainly accumulated in the skin cells, giving colour to the berries of the red and black varieties, and also influencing the quality of the berry. The three most abundant anthocyanins in grape are 3-monoglucoside, 3-acetylglucoside, and 3-p-coumarylglucoside [START_REF] Boss | Analysis of the expression of anthocyanin pathway genes in developing Vitis vinifera L. cv Shiraz grape berries and the implications for pathway regulation[END_REF].

The anthocyanin content of the grape berry increases concomitantly with sugar accumulation at the post-veraison stages [START_REF] Coombe | Research on development and ripening of the grape berry[END_REF][START_REF] Vitrac | Sugar sensing and Ca 2+ -calmodulin requirement in Vitis vinifera cells producing anthocyanins[END_REF][START_REF] Deluc | Water deficit alters differentially metabolic pathways affecting important flavor and quality traits in grape berries of Cabernet Sauvignon and Chardonnay[END_REF]. The addition of different concentrations of sucrose (100-150 mM) at day 7 (the end of the exponential growth phase) to suspension cultures of Gamay Fréaux increases the anthocyanin production up to 12-fold compared with cells without sucrose addition [START_REF] Larronde | Regulation of polyphenol production in Vitis vinifera cell suspension cultures by sugars[END_REF]. Addition of glucose or fructose (150 mM) also increased the anthocyanin content, although fructose is less effective than sucrose or glucose. In contrast, mannitol and sorbitol (150 mM) do not stimulate anthocyanin accumulation in grape cells, suggesting that the effect of sucrose or glucose is not osmotic. The amount of sucrose added to the culture medium did not affect grape cell proliferation, indicating that the effect on anthocyanin content is not indirectly due to cell division [START_REF] Larronde | Regulation of polyphenol production in Vitis vinifera cell suspension cultures by sugars[END_REF]. Similar results were observed in cell suspensions of different grape cultivars (Cabernet Sauvignon, Barbera berries, and Gamay Red) [START_REF] Vitrac | Sugar sensing and Ca 2+ -calmodulin requirement in Vitis vinifera cells producing anthocyanins[END_REF][START_REF] Gollop | Regulation of the leucoanthocyanidin dioxygenase gene expression[END_REF][START_REF] Belhadj | Effect of methyljasmonate in combination with carbohydrates on gene expression of PR proteins, stilbene and anthocyanin accumulation in grapevine cell cultures[END_REF][START_REF] Ferri | Increasing sucrose concentrations promote phenylpropanoid biosynthesis in grapevine cell cultures[END_REF].

Several genes involved in the anthocyanin biosynthesis pathway have been identified, cloned, and characterized (see [START_REF] Kühn | Berry ripening: recently heard through the grapevine[END_REF]. Several transcription factors regulating this pathway are known so far. Among these, the MYB transcription factor family that controls many important processes in plants [START_REF] Matus | Quality-related clades and conserved gene structure organization across Vitis and Arabidopsis genomes[END_REF][START_REF] Dubos | MYB transcription factors in Arabidopsis[END_REF] is the most studied. The expression of several structural genes and transcription factors involved in the anthocyanin biosynthesis pathway is regulated by sugars in petunia [START_REF] Shoseyov | Sugars enhance the expression of gibberellin-induced genes in developing petunia flowers[END_REF] and Arabidopsis [START_REF] Tsukaya | Sugar-dependent expression of the CHS-A gene for chalcone synthase from Petunia in transgenic Arabidopsis[END_REF][START_REF] Solfanelli | Sucrosespecific induction of the anthocyanin biosynthetic pathway in Arabidopsis[END_REF].

Concerning grape, the expression of both LDOX (leucoanthocyanidin dioxygenase) and DFR (dihydroflavonol-4-reductase) was up-regulated by treatment of cv. Gamay Red cell cultures with 8.7 mM sucrose [START_REF] Gollop | Regulation of the leucoanthocyanidin dioxygenase gene expression[END_REF]. When cell cultures were transformed with a DFR::GUS fusion gene, the transcripts of both GUS and endogenous DFR were more abundant after sucrose treatment [START_REF] Gollop | Expression of the grape dihydroflavonol reductase gene and analysis of its promoter region[END_REF]. The transcripts and protein amounts of F3H (flavanone 3-hydroxylase) protein were also increased in grape berries incubated with different sugars. The F3H protein in berries peaked after 1 h and 2 h treatment with glucose (100 mM) and sucrose (150 mM), respectively [START_REF] Zheng | Sugars induce anthocyanin accumulation and flavanone 3-hydroxylase expression in grape berries[END_REF]. F3H transcripts peaked at 1 h (with 100 mM glucose and 100 mM fructose) or at 2 h (with 150 mM sucrose), and then decreased immediately.

Since anthocyanin biosynthesis is localized in the skin cells of the berries, except in teinturier varieties, the sugar content of the skin cells may not be directly related to the total sugar content of the berries. Also, cell suspensions provide a convenient experimental system, but the data derived from this system should be extrapolated with caution to berry physiology.

Recent field experiments tested the effects of mannose (150 mM), the C-2 epimer of glucose, on anthocyanin biosynthesis (A. Serrano et al., unpublished). For many plants, mannose is toxic, because it is actively phosphorylated by hexokinases into mannose-6-phosphate, which decreases the pool of Pi. However, there are some mannose-insensitive plants which have the ability to convert mannose-6-phosphate to fructose 6-phosphate by mannose-6-phosphate isomerase, which is not toxic to the cell (Heilman et al., 1997). In grape cell cultures, mannose (20-100 mM) induces a concentrationdependent accumulation of anthocyanins and is not toxic for the grape cells [START_REF] Vitrac | Sugar sensing and Ca 2+ -calmodulin requirement in Vitis vinifera cells producing anthocyanins[END_REF]. However, the stimulation of anthocyanin accumulation by mannose (100 mM) is only 70% of what is observed with the same concentration of sucrose. Unlike mannose, 2-deoxyglucose is toxic for the grape cells. In non-toxic concentrations, it does not affect the anthocyanin content. Thus, within the non-metabolizable sugars, mannose is a good candidate to induce the accumulation of anthocyanins in grape. To test mannose as an inductor of anthocyanin biosynthesis, it was added exogenously to cluster grape before veraison in field experiments. In parallel, other sugars that may induce anthocyanin biosynthesis were tested. Mannose had an important effect on the coloration of the berries, inducing early coloration compared with control treatment (A. Serrano et al., in preparation). The addition of other sugars was less effective than that of mannose, and mannitol had no effect.

Sugars and biotic stress: effects of viral infection on sugar content of grape berries

More than 58 different viruses may affect V. vinifera, and many severely impact grapevine physiology and cause significant economic losses [START_REF] Martelli | Directory of infectious diseases of grapevines and viroses and virus-like diseases of the grapevine[END_REF][START_REF] Vega | Compatible GLRaV-3 viral infections affect berry ripening decreasing sugar accumulation and anthocyanin biosynthesis in Vitis vinifera[END_REF]. Among them, Grapevine leaf-roll-associated virus-3 (GLRaV-3) is one of the most widespread viruses worldwide, and one of the most important diseases affecting grapevines [START_REF] Martelli | Directory of infectious diseases of grapevines and viroses and virus-like diseases of the grapevine[END_REF][START_REF] Vega | Compatible GLRaV-3 viral infections affect berry ripening decreasing sugar accumulation and anthocyanin biosynthesis in Vitis vinifera[END_REF]. It may alter primary and secondary metabolism in both the leaves and the berries. Thus, it may induce leaf deformation, deficiency in grape cluster development, and alterations in fruit quality, associated with the reduction of the sugar and anthocyanin contents in red cultivars, which dramatically affect the ripening process [START_REF] Bertamini | Effect of grapevine leafroll on the photosynthesis of field grown grapevine plants (Vitis vinifera L. cv. Lagrein)[END_REF][START_REF] Vega | Compatible GLRaV-3 viral infections affect berry ripening decreasing sugar accumulation and anthocyanin biosynthesis in Vitis vinifera[END_REF]. In the leaves of both Carmenere and Cabernet Sauvignon grape cultivars, GLRaV-3 infection induces genes related to sugar metabolism, such as sugar transporters and glycosyl transferases [START_REF] Espinoza | Gene expression associated with compatible viral diseases in grapevine cultivars[END_REF]. The genes of the flavonoid biosynthetic pathway are up-regulated in symptomatic leaves of a red-fruited wine grape cultivar infected with GLRaV-3 [START_REF] Gutha | Modulation of flavonoid biosynthetic pathway genes and anthocyanins due to virus infection in grapevine (Vitis vinifera L.) leaves[END_REF]. Among them, CHS3 (chalcone synthase 3), F3′5′H (flavonoid 3′,5′-hydroxylase), F3H1, LDOX, and LAR1 (leucoanthocyanidin reductase 1) showed a >10-fold increase and MybA1 a 19-fold increase upon virus infection. As a result, there was an increase in the flavonol, proanthocyanidin, and anthocyanin contents of the leaves of infected plants.

In the berries, global transcript profiling showed that GLRaV-3 affects a wide range of biological processes at both veraison and ripening stages [START_REF] Vega | Compatible GLRaV-3 viral infections affect berry ripening decreasing sugar accumulation and anthocyanin biosynthesis in Vitis vinifera[END_REF]. Glucose and fructose accumulation were reduced during ripening of virus-infected vines, and, in parallel, the expression of genes implicated in sugar metabolism and transport was affected by the virus [START_REF] Vega | Compatible GLRaV-3 viral infections affect berry ripening decreasing sugar accumulation and anthocyanin biosynthesis in Vitis vinifera[END_REF]. Among these genes, VvHT1 and VvMSA were significantly repressed in virus-infected berries at most developmental stages analysed. All these results suggest that the decrease in the sugar levels in the berry of GLRaV-3-infected plants is mediated through effects on sugar transport and metabolism.

GLRaV-3 infection also decreased the anthocyanin content and the transcript levels of structural and regulatory genes of the anthocyanin biosynthesis pathway. Among these, CHS2 mRNA levels were up-regulated in immature infected berries, but repressed at the ripening stage. The expression of the UFGT (UDP glucose: flavonoid-3-O-glucosyltransferase) gene was strongly down-regulated by the virus in infected ripening berries. In berries of virus-infected vines, the transcripts of MYBPA1 (a transcription factor that regulates the anthocyanin biosynthesis pathway) were induced before veraison, but repressed at ripening, and those of MYBA1 were downregulated during ripening [START_REF] Vega | Compatible GLRaV-3 viral infections affect berry ripening decreasing sugar accumulation and anthocyanin biosynthesis in Vitis vinifera[END_REF]. Given that anthocyanin synthesis is regulated at least in part by sugars, it is tempting to conclude that reduction in the sugar level of ripe berries by GLRaV-3 infection may down-regulate the genes related to the anthocyanin biosynthesis pathway, and consequently reduce the anthocyanin content.

Sugars and abiotic stress: effects of heat stress on galactinol synthesis

Recently, [START_REF] Pillet | VvGOLS1 and VvHsfA2 are involved in the heat stress responses in grapevine berries[END_REF] showed that high temperature locally applied to V. vinifera Cabernet Sauvignon clusters induces the accumulation of galactinol in berries. This process was mediated, at least in part, through the action of VvHsfA2 (heat stress factor A2) and VvGolS1 (galactinol synthase 1). In plants, galactinol acts mainly as a galactosyl donor for biosynthesis of RFOs (raffinose family oligosaccharides). RFOs, such as raffinose and stachyose, fulfil important functions including carbon translocation and storage, and are defined as compatible solutes with a protective role during abiotic stress [START_REF] Pillet | VvGOLS1 and VvHsfA2 are involved in the heat stress responses in grapevine berries[END_REF]. Despite a significant galactinol accumulation, no raffinose and stachyose were detected in berries subjected to long-term heat exposure [START_REF] Pillet | VvGOLS1 and VvHsfA2 are involved in the heat stress responses in grapevine berries[END_REF]. Two hypotheses were proposed to explain these results. The first one suggests that the RFOs accumulated during the early stage of heat exposure were rapidly catabolized to provide metabolizable energy and carbon skeletons. The second one proposed that galactinol might not act as a galactosyl donor and RFO synthesis regulator in heat-stressed berries, but rather as an endogenous molecular signal. Galactinol was recently described as a signalling molecule involved in pathogen-induced systemic resistance [START_REF] Kim | Galactinol is a signaling component of the induced systemic resistance caused by Pseudomonas chlororaphis O6 root colonization[END_REF]. Since both biotic and abiotic stimuli share some common features in their stress mechanisms, [START_REF] Valluru | Myo-inositol and beyondemerging networks under stress[END_REF] proposed that galactinol and RFOs function as signals as well as true ROS (reactive oxygen species) scavengers. If galactinol can signal heat stress to trigger adaptive responses in grape berries, its exact function in stressed berries remains to be determined, as do the consequences of its accumulation for fruit development and fruit quality.

Sugar and berry development

A transcriptomic approach was initiated using Cabernet Sauvignon berry cells treated or not with sucrose [START_REF] Lecourieux | A sugar inducible protein kinase, VvSK1, regulates sugar transport and accumulation in grapevine cells[END_REF] in order to obtain an overall idea of all the genes potentially affected by cell sugar status. Total RNA were extracted and hybridized with 70-mer oligoarrays bearing a set of 14 562 unigenes (Qiagen Operon Array-Ready Oligo Set for the Grape Genome Version 1.0). A genome ontology category enrichment analysis indicated that the most affected processes belong to signal transduction pathways, cell wall modifications, protein synthesis and degradation, abiotic and biotic stresses, secondary metabolism, lipid metabolism, and redox modifications. Here, we will describe a few molecular players that were significantly up-regulated by sucrose supply. These genes belong to the families of protein kinases and transcription factors.

Protein kinases are major components of intracellular signal transduction enabling plant cells to respond rapidly to environmental cues. VvSK1 was identified as a sugar-(and ABA-) inducible protein kinase affecting hexose transport and soluble sugar accumulation in grape cells [START_REF] Lecourieux | A sugar inducible protein kinase, VvSK1, regulates sugar transport and accumulation in grapevine cells[END_REF]. This protein is 468 amino acids long and belongs to clade III of the GSK3 family. VvSK1 is only expressed in sink organs (flowers, berries, and roots). During berry development, its transcripts decreased after the green stage and increased again after the veraison stage when both sugars and ABA accumulate [START_REF] Davies | Treatment of grape berries, a nonclimacteric fruit, with a synthetic auxin retards ripening and alters the expression of developmentally regulated genes[END_REF][START_REF] Pan | Abscisic acid activates acid invertases in developing grape berry[END_REF]. Interestingly, both sucrose and ABA were able to increase VvSK1 expression in grape cell suspensions, underlining the tight interaction between sugars and hormone signalling pathways [START_REF] Smeekens | Sugar-induced signal transduction in plants[END_REF][START_REF] Finkelstein | ABA and sugar interactions regulating development: cross-talk or voices in a crowd?[END_REF][START_REF] Léon | Sugar and hormone connections[END_REF][START_REF] Rolland | Sugar sensing and signalling in plants: conserved and novel mechanisms[END_REF]. Additionally, the use of glucose analogues (2-DOG and 3-OMeG) and of a hexokinase inhibitor (MHL) showed that the sugar control of VvSK1 is dependent on a hexokinase pathway. The production of transgenic grape cell suspensions overexpressing VvSK1 allowed a better understanding of its role in the control of sugar uptake in response to sugar supply. Indeed, in these transgenic cells, the rate of glucose uptake was increased 3-to 5-fold, and the amount of glucose and sucrose accumulation was more than doubled, while the amount of starch was not affected. This suggested that VvSK1 is involved in the control of sugar compartmentation, a hypothesis further supported by the capacity of VvSK1 to trigger transcript accumulation of four MSTs (VvHT3, VvHT4, VvHT5, and VvHT6). However, among these transporters, it is more likely that the control of VvHT6 and VvHT3 that accumulates parallel to the accumulation of hexoses in the berries [START_REF] Deluc | Transcriptomic and metabolite analyses of Cabernet Sauvignon grape berry development[END_REF][START_REF] Hayes | Isolation, functional characterization, and expression analysis of grapevine (Vitis vinifera L.) hexose transporters: differential roles in sink and source tissues[END_REF] could mainly contribute to the VvSK1-mediated regulation of sugar import during berry development. Finally, this work underlines the importance of protein kinases and therefore of phosphorylation events in the control of sugar uptake and accumulation by sugar, but also provided a good tool for investigating the metabolic and physiological consequences of the deregulation of such processes.

Transcription factors are key elements that control cell metabolism reprogramming and expression of key genes involved in berry development and ripening. Different genes encoding transcription factors were identified by transcriptomic analysis of both sucrose-treated cells and developing grape berries (D. Lecourieux et al., unpublished data; D. Glissant and S. Delrot, unpublished data). Among these, two showed interesting patterns. The first one belongs to the bZIP family and was linked to berry ripening processes (D. Lecourieux et al., unpublished) and the second one, VvCEB1 (V. vinifera cell elongation bHLH 1), belongs to the basic helix-loop-helix (bHLH) family. VvCEB1 was recently shown to affect cell size in grape. bHLH proteins belong to a vast multigenic family of transcriptional regulators present in both animals and plants, and that intervene in numerous physiological and developmental processes [START_REF] Pires | Origin and diversification of basic-helix-loophelix proteins in plants[END_REF]. In plants, bHLH proteins function as transcriptional regulators modulating secondary metabolism pathways, fruit dehiscence, carpel and epidermal development, phytochrome signalling, and stress responses [START_REF] Ramsay | MYB-bHLH-WD40 protein complex and the evolution of cellular diversity[END_REF][START_REF] Castillon | Phytochrome interacting factors, central players in phytochrome-mediated light signaling networks[END_REF][START_REF] Pires | Origin and diversification of basic-helix-loophelix proteins in plants[END_REF][START_REF] Feller | Evolutionary and comparative genomics of MYB and bHLH plant transcription factors[END_REF].

VvCEB1 belongs to the subfamily XII of the bHLH transcription factors [START_REF] Pires | Origin and diversification of basic-helix-loophelix proteins in plants[END_REF] containing members involved in growth regulation [START_REF] Szécsi | BIGPETALp, a bHLH transcription factor is involved in the control of Arabidopsis petal size[END_REF][START_REF] Kay | A bacterial effector acts as a plant transcription factor and induces a cell size regulator[END_REF]. VvCEB1 expression was reduced by auxin treatment, while its transcripts predominantly accumulated in the expanding tissues of the berry (mesocarp and exocarp) with a maximum at veraison when the amounts of auxins are low. The heterologous analysis of its promoter activity in tomato confirmed that VvCEB1 was specifically expressed in the fruit and that this expression was maintained during the fruit expansion period. The production of grape embryos overexpressing VvCEB1 revealed that its overexpression resulted in the disruption of embryonic development and tissues organization by affecting cell size and polarity. Indeed, these embryos grew faster, and failed to acquire bilateral symmetry and to reach a more important size because of the presence of larger cells, thus suggesting a role for VvCEB1 in the stimulation of cell expansion and in the control of auxin homeostasis. This hypothesis was confirmed by the study of auxin-and cell expansion-related genes in grape embryos overexpressing VvCEB1. Among the VvCEB1-stimulated genes, we found IAA genes such as VvIAA19 that was recently shown to promote growth in Arabidopsis [START_REF] Kohno | Auxin non responsive grape Aux/IAA19 is a positive regulator of plant growth[END_REF], SAUR genes that accumulate in the soybean hypocotyl elongation zone [START_REF] Gee | Tissue-specific and organ specific expression of soybean auxin-responsive transcripts GH3 and SAURs[END_REF] or that simulate cell expansion [START_REF] Spartz | The SAUR19 subfamily of SMALL AUXIN UP RNA genes promote cell expansion[END_REF], and GH3 genes that were associated with longan fruit growth and ripening [START_REF] Kuang | Two GH3 genes from longan are differentially regulated during fruit growth and development[END_REF]. Many genes associated with cell wall metabolism (XET, PECL, AMY, AGP, and EXP) and water transport (AQP) were also strongly up-regulated in VvCEB1overexpressing embryos, in agreement with a role for VvCEB1 in the stimulation of cell expansion processes. Altogether, this study highlighted the ability of sugar to act as a signalling molecule. The examples described above showed how a sugar signal could (i) regulate the fruit sugar content by modulating VvSK1 action; and (ii) regulate the final size of the fruit by controlling VvCEB1-mediated cell expansion.

Concluding remarks

The present status of our knowledge related to sugar transport and sugar sensing in grapevine is summarized in Fig. 2. This review underlines the complexity of sugar transport and sugar control as well as the gaps in our knowledge. The molecular players leading to the accumulation of high concentrations of glucose and frucose in the vacuoles of flesh cells are still loosely characterized. A lot has been learnt on the sugar control of transporters, which may be positive or negative, depending on the sugar concentration, and may involve a hexokinase-dependent or independent pathway, and transcriptional and protein regulation. The simultaneous presence of up-and down-regulation of several pathways (hexokinase dependent and independent), as well as the multilevel regulations (transcripts and proteins) described for sugar transport pinpoints the major role of these compounds as nutrients and signals, and the importance of carbon economy for the plant. Beyond transport, the sugar status directly or indirectly affects other cellular activities such as phenylpropanoid metabolism, cell wall metabolism, auxin homeostasis, and ripening, and ultimately berry growth, ripening, and adaptation to stress. Interactions between the sugar and ABA signalling pathway play a key role, also underlined by the parallel accumulation of sugars and ABA in the berry after veraison. In many cases, results have been obtained with cell suspensions rather than intact berries. Their physiological relevance must be checked systematically in the berry organ, and whole berry studies need to be developed. Although most studies on sugar sensing in grapevine have been conducted with sucrose, glucose, and fructose, recent data indicate that other sugars such as galactinol and trehalose may also play a significant role in the regulation of berry metabolism, ripening, and adaptation to stress. Quicker progress in grapevine research will obviously depend on the development of new functional genomic tools adapted to this slow growing, large sized, and not easily transformed plant.

  Fig.1. Phylogenic tree and berry expression of SWEET transporter genes in grape. (A) Evolutionary tree of grapevine and Arabidopsis SWEET transporters inferred by using the maximum likelihood method implemented in the MEGA5 software[START_REF] Tamura | MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods[END_REF]. Grapevine SWEET were identified by their PFAM[START_REF] Punta | The Pfam protein families database[END_REF] domain PF03083. (B) Boxplot of gene expression profiles over a microarray developmental series made by Fasoli et al. (2012) for the three most berry-specific VvSWEET genes: Vv01s0146g00260, Vv17s0000g00820, and Vv17s0000g00830. Red dots indicate median expression intensities for berry samples. Data were obtained from NCBI GEO (Barrett et al., 2013; accession no. GSE36128) and RMA normalized using the R/oligo package (Carvalho and Irizarry, 2010).

Fig. 2 .

 2 Fig. 2. Summary diagram of sugar transport and sugar-sensing processes described so far in grapevine. bZIP, basic leucine zipper; CEB1, Vitis vinifera cell elongation bHLH protein 1; DREB, Vitis vinifera drought responsive element binding protein; HXK, hexokinase; MSA, Vitis vinifera maturation stress abscisic acid gene (ASR family); VvHT1-VvHT6, Vitis vinifera hexose transporter 1-VvHT6; VvSK1: Vitis vinifera glycogen synthase kinase 3 protein kinase 1; X, unknown intermediate.

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Acknowledgements

The authors thank the ECOS Sud programme (no. C11B01) for its support. Work at ISVV is supported by INRA, the University of Bordeaux, Bordeaux Sciences Agro, the Regional Council of Aquitaine, and the Conseil Interprofessionnel du Vin de Bordeaux.


Christian Artigues
email: christian.artigues@laas.fr

Pierre Lopez
email: pierre.lopez@laas.fr

Oliver Polo Mejía
email: oliver.polo-mejia@laas.fr

Structural and Experimental Comparisons of Formulations for a Multi-Skill Project Scheduling Problem with Partial Preemption

Keywords: RCPSP, Multi-skill, Partial preemption, Mixed-integer programming, Constraint programming

Problem statement

Preemptive scheduling problems assume that all resources are released during preemption periods, and that they can be used to perform other activities. However, in certain cases, constraints require that a subset of resources remains allocated to the activity when it has been interrupted, to ensure safety for example. Suppose one must execute an experimental activity that requires an inert atmosphere for its execution. In practice, one can stop this activity and allow the technicians and some of the equipment to be used in other activities. However, safety and operational constraints force us to preserve the inert atmosphere even when the activity is stopped (before its end). In other words, one cannot release the equipment that ensures the inert atmosphere during the preemption periods. Traditional preemptive schedule models cannot represent this behaviour since they assume that all resources are released during the preemption periods. Until now, the only way to model this activity, while respecting safety requirements, was to declare it as "non-preemptive". However, this decision can increase the project makespan, especially when the activities have restrictive time windows and the availability/capacity of the resources vary over time. We call partial preemption the possibility of only releasing a subset of resources during the preemption periods.

We are concerned here in multi-skill project scheduling problem (MSPSP) (Bellenguez and Néron 2012). We present in this extended abstract a new variant of the MSPSP that uses the concept of partial preemption. The variant of the problem under study is then called Multi-Skill Project Scheduling Problem with Partial Preemption (MSPSP-PP). To the best of our knowledge, it has not been studied yet in the scientific literature.

In the MSPSP-PP, if an activity is interrupted, we release only a subset of resources while seizing the remainder. We can then classify the set I of activities to be scheduled into three types according to the possibility of releasing the resources during the preemption periods: 1) Non-preemptive activities (N P ), if none of the resources can be released; 2) Partially preemptive activities (P P ), if a subset of resources can be released; and 3) Preemptive activities (P ), if all resources can be set free. In our case, the partial preemption is only related to mono-skilled resources, and we made the hypothesis that resources can always be released during preemption periods.

Our objective in the MSPSP-PP is to find a feasible schedule that minimises the total duration of the project (C max ). Finding a solution consists in determining the periods during which each activity is executed and also which resources will execute the activity in every period; all this, while respecting the resources capacity and the activities characteristics. We must schedule these activities on renewable resources with limited capacity; they can be cumulative mono-skilled resources (machines or equipment) or disjunctive multiskilled resources (technicians) mastering a given number of skills. Multi-skilled resources can respond to more than one skill requirement per activity and may execute it partially (except for non-preemptive activities where technicians must perform the whole activity). An activity is defined by its duration (D i ), its precedence relationships (set E), its requirements of resources, its requirements of skills, the minimum number of technicians needed to perform it, and the subset of preemptive resources. Activities might or not have either a deadline or a release date. Figure 1 illustrates an example of an MSPSP-PP instance and a possible solution. The complexity of the MSPSP with partial preemption can be established using the classical RCPSP (Resource-Constrained Project Scheduling Problem) as a starting point. For each instance of the RCPSP, we can match an instance of the MSPSP with partial preemption, where all resources are mono-skilled, and none of the resources can be preempted. Thus, we can define the RCPSP as a particular case of the MSPSP with partial preemption. Since the RCPSP has been proved to be strongly NP-hard [START_REF] Błazewicz | Scheduling subject to resource constraints: classification and complexity[END_REF], we can, therefore, infer that the MSPSP with partial preemption is also strongly NP-hard.

We propose five formulations for the MSPSP-PP using Mixed-Integer/Linear Programming (MILP) and Constraint Programming (CP).

MILP formulations

We present below five time-indexed formulations of the problem over a discretized horizon H. These formulations generalize the ones presented in [START_REF] Polo-Mejía | Mixed-integer and constraint programming formulations for a multi-skill project scheduling problem with partial preemption[END_REF][START_REF] Polo-Mejía | Mixed-integer/linear and constraint programming approaches for activity scheduling in a nuclear research facility[END_REF]. All models are based on on/off binary variables Y i,t stating whether an activity i is in process in time period t, on/off binary variables O j,i,t = 1 if technician j is assigned to activity i during period t, binary variable S j,i = 1 if technician j is assigned to non-preemptive activity i (this variable is used to express that any technician assigned to a non-preemptive activity must remain assigned until its completeness). For any partially preemptive activity i, an on/off binary variable P p i,t = 1 if activity i is preempted in time period t. For the three first models, step binary variable Z i,t = 1 if partially preemptive or non-preemptive activity i starts in time period t or before and step binary variable W i,t = 1 if partially preemptive or non-preemptive activity i ends in time period t or after. We only provide a subset of the constraints of the first model (MSPP1a): precedence constraints (1) and the constraints (2-8) that link variables Y , P p, Z, W , S, O and C max , the project makespan. The other constraints are standard resource constraints and operator availability constraints.

D i * (1 -Y l,t ) ≥ |H| t =t Y i,t ∀(i, l) ∈ E, ∀t ∈ H (1) Z i,t ≥ Y i,t ∀i / ∈ P , ∀t ∈ H, ∀t ≤ t (2) W i,t ≥ Y i,t ∀i / ∈ P , ∀t ∈ H, ∀t ≥ t (3) P p i,t = Z i,t + W i,t -Y i,t -1 ∀i ∈ P P , ∀t ∈ H (4) Z i,t + W i,t -Y i,t = 1 ∀i ∈ N P , ∀t ∈ H (5) O j,i,t ≥ S j,i + Y i,t -1 ∀i ∈ N P , ∀j ∈ J, ∀t ∈ H (6) O j,i,t ≤ S j,i ∀i ∈ N P , ∀j ∈ J, ∀t ∈ H (7) C max ≥ t * Y i,t ∀i ∈ I, ∀t ∈ H (8)
Given the variables W i,t and Z i,t , we can replace the precedence constraints (1) by a disaggregated version below, yielding the second model (MSPP1b), while the third model (MSPP1c) includes both constraints (1) and ( 9).

Z l,t + W i,t ≤ 1 ∀(i, l) ∈ E, ∀t ∈ H (9)
We also propose two mixed continuous-time/discrete-time models (MSPP2a and MSPP2b), replacing binary variables W i,t and Z i,t by continuous time variables G i and F i representing the start and completion times of activity i, respectively. We replace constraints (1-5) by:

F i + 1 ≤ G l ∀(i, l) ∈ E (10) P p i,t ≤ 1 -Y i,t ∀i ∈ P P , ∀t ∈ H (11)
F i -G i + 1 ≤ D i + t∈H P p i,t ∀i ∈ P P (12)
F i -G i + 1 ≤ D i ∀i ∈ N P (13)
F i ≥ t * Y i,t ∀i ∈ I, ∀t ∈ H (14) G i ≤ t * Y i,t + (1 -Y i,t ) * |H| ∀i ∈ I (15)
It remains to express the fact that partial preemption variables P p i,t must be equal to 0 outside the execution interval of i. We either use the following constraints using variables Y (MSPP2a):

P p i,t ≤ t t =1 Y i,t ; P p i,t ≤ |H| t =t Y i,t ∀i ∈ P P , ∀t ∈ H (16)
or the following ones using variables F and G (MSPP2b):

F i ≥ t * P p i,t ; G i ≤ t * P p i,t -(1 -P p i,t ) * |H| ∀i ∈ P P , ∀t ∈ H (17)
In Section 3, we compare the proposed MILP formulations in terms of LP relaxation strength and we provide a computational comparison with the constraint programming formulation described in [START_REF] Polo-Mejía | Mixed-integer/linear and constraint programming approaches for activity scheduling in a nuclear research facility[END_REF].

3 Structural and computational comparisons

Structural comparison of the MILP formulations

Using the transformation G i = |H| -t∈H Z i,t + 1 and F i = t∈H W i,t we show that the constraints in model MSPP2 involving the F i and G i variables are all implied by the constraints of model MSPP1 augmented with the transformation. As the computational experiments show that there are instances where MSPP1 has a strictly better LP relaxation than MSPP2, this yields the following result: Theorem 1. Formulation MSPP1b is tighter than formulations MSPP2a and MSPP2b.

We could not prove a dominance relation between MSPP1a and MSPP1b, which was corroborated by the experiments fox which some bounds provided by the MSPP1a relaxation are better than those provided by the MSPP1b relaxation and vice-versa, which justifies the proposal of MSPP1c.

Computational experiments on the MILP and CP formulations

For computational tests, we use CPLEX 12.7 and CP Optimizer 12.7 for solving the MILP models and the CP models, respectively (using the default configuration and limiting the number of threads used by the solvers at 8). The computation time was limited to 10 minutes. We use the four sets of 30 activities instances of (Polo et al. 2019), each of them having 50 instances and a different proportion of preemption types. The activity durations are between 5 to 10 time units. There are up to 15 skills, 8 cumulative resources, 8 technicians (multi-skilled resources) divided into two teams, 20% of activities with time windows, the density of precedence relationships is low, and an average optimum C max between 70 and 90 time units.

First of all, the MILP formulations are only efficient when the number of preemptive activities is high. For these instances, model MSPP1b provides the larger number of optimal solutions but model MSPP2b is faster and has a better average gap. There was no perceptible advantage for the integrated model MSPP1c. These computational results confirm one more time that a theoretically stronger formulation does not necessarily imply better practical performance. The MILP MSPP2b model outperforms the CP one when the percentage of preemptive activities is high, proving the optimality of a higher number of instances, and giving a lower average gap. CP, on the other hand, gives better results when this percentage is low. One could then say that the two methods are complementary.

Future research should be done in order to develop a hybrid method that better exploit the characteristics of each instance.

Fig. 1 .

 1 Fig. 1. Example of an MSPSP-PP instance.