Clemence Henry

Amelie Rabot

Maryse Laloi

Eric Mortreau

Monique Sigogne

Nathalie Leduc

Rémi Lemoine

Soulaiman Sakr

Alain Vian

Sandrine Travier
email: sandrine.travier@univ-angers.fr

Clémence Henry

Amélie Rabot

Sandrine Pelleschi-Travier

Regulation of RhSUC2, a sucrose transporter, is correlated with the light control of bud burst in Rosa sp

Keywords: axillary bud, photocontrol, photomorphogenesis, sink strength, sugar loading

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

The regulation of sugar transport, allocation and mobilization represents a major event in plant adaptation to environment. In most higher plants, sucrose is the major carbohydrate for long-distance transport through the vascular system [START_REF] Zimmermann | List of sugars and sugar alcohols in sieve-tube exudates[END_REF]. Sucrose partitioning between its site of synthesis (source tissues), the leaf mesophyll and sink tissues depends on equilibrium between organs. Among them, buds are actively growing heterotrophic organs, whose development depends upon their capacity to import and metabolize sugars [START_REF] Cottignies | The hydrolysis of starch as related to the interruption of dormancy in the ash bud[END_REF][START_REF] Rinne | Seasonal changes in bud dormancy in relation to bud morphology, water and starch content, and abscisic acid concentration in adult trees of Betula pubescens[END_REF]Maurel et al. 2004a,b).

Previous work in ligneous plants demonstrated that bud burst is accompanied by mobilization of stem carbohydrates: starch in Quercus robur [START_REF] Alaoui-Sossé | Rythmic growth and carbon allocation in Quercus robur. Starch and sucrose[END_REF], mainly hexoses in Prunus persica (Maurel et al. 2004a) and sucrose in Juglans regia [START_REF] Decourteix | Sucrose (JrSUT1) and hexose (JrHT1 and JrHT2) transporters in walnut xylem parenchyma cells: their potential role in early events of growth resumption[END_REF]. In Rosa sp., bud outgrowth is associated with mobilization of local and distant (surrounding stem portion) sucrose reserves [START_REF] Girault | Sugars are under light control during bud burst in Rosa sp[END_REF]). These findings are in accordance with the stimulation of both sugar (glucose and sucrose) uptake [START_REF] Marquat | Dormancy in vegetative buds of peach: relation between carbohydrate absorption potentials and carbohydrate concentration in the bud during dormancy and its release[END_REF]Maurel et al. 2004a) and the activity of plasma membrane H+-ATPase [START_REF] Aue | Change in plasma membrane ATPase activity during dormancy release of vegetative peach-tree buds[END_REF] in bursting buds of Prunus persica. Despite all this research, little is known about the involvement or the regulation of soluble sugar transport in bud outgrowth.

Plant architecture is strongly determined by the capacity of buds to grow after release of apical dominance. In several species including Rosa sp., bud outgrowth is a complex process involving cell division, new organogenesis and elongation [START_REF] Zieslin | Components of axillary bud inhibition in rose plants. I. The effect of different plant parts (correlative inhibition)[END_REF][START_REF] Khayat | Environmental factors involved in the regulation of sprounting of basal buds in rose plants[END_REF][START_REF] Marcelis-Van Acker | Development of axillary buds of rose in vitro[END_REF][START_REF] Girault | Light controls shoot meristem organogenic activity and leaf primordia growth during bud burst in Rosa sp[END_REF], and is controlled by a genetic program and environmental cues. In Rosa sp., much evidence showed that light (in quantity and quality) is a powerful factor modulating bud outgrowth [START_REF] Healy | The effect of light quality on 'bottom-breaks' in roses[END_REF][START_REF] Mor | Characterisation of the light reaction in promoting the mobilizing ability of rose shoot tips[END_REF][START_REF] Khayat | Environmental factors involved in the regulation of sprounting of basal buds in rose plants[END_REF][START_REF] Girault | Light controls shoot meristem organogenic activity and leaf primordia growth during bud burst in Rosa sp[END_REF]. Light triggers a high bud sink strength through sugar accumulation (sucrose and glucose), concomitantly with an increase in both transcript levels and enzymatic activity of acid vacuolar invertase in buds [START_REF] Girault | Sugars are under light control during bud burst in Rosa sp[END_REF]. Neither sucrose accumulation nor sugar metabolism stimulation was found to occur in the dark, where bud burst was totally prevented [START_REF] Girault | Sugars are under light control during bud burst in Rosa sp[END_REF]. Thus, light induction of bud burst is likely to require higher sucrose import, through an up-regulation of sugar transporters.

Sugar transport at the cellular level follows either the symplastic pathway (through the plasmodesmata) or the apoplastic pathway, which is mediated by plasma membrane-localized sugar transporters. These proteins, known as sugar transporters or sugar carriers, mainly catalyse active energy-dependent transport [START_REF] Delrot | Plasma membrane transporters: a machinery for uptake of organic solutes and stress resistance[END_REF][START_REF] Kühn | The sucrose transporter StSUT1 localizes to sieve elements in potato tuber phloem and influences tuber physiology and development[END_REF]. They are trans-membrane proteins, with 12 characteristic loops of the major facilitator superfamily (MFS) [START_REF] Lemoine | Sucrose transporters in plants: update on function and structure[END_REF]. Many sucrose transporter (named SUC or SUT) genes have been so far isolated from herbaceous and woody plants such as Betula pendula [START_REF] Wright | Changes in carbon allocation and expression of carbon transporter genes in Betula pendula Roth. colonized by the ectomycorrhizal fungus Paxillus involutus (Batsch) Fr. Plant[END_REF], Citrus sp. [START_REF] Li | Sugars regulate sucrose transporter gene expression in citrus[END_REF], Hevea brasiliensis [START_REF] Dusotoit-Coucaud | Molecular characterisation of the sucrose transporter HbSUT1B in relation to rubber production in latex cells[END_REF][START_REF] Tang | The sucrose transporter HbSUT3 plays an active role in sucrose loading to laticifer and rubber productivity in exploited trees of Hevea brasiliensis (para rubber tree)[END_REF], Juglans regia [START_REF] Decourteix | JrSUT1, a putative xylem sucrose transporter, could mediate sucrose influx into xylem parenchyma cells and be up-regulated by freeze-thaw cycles over the autumn-winter period in walnut tree (Juglans regia L.)[END_REF], Malus domestica [START_REF] Fan | Apple sucrose transporter SUT1 and sorbitol transporter SOT6 interact with cytochrome b5 to regulate their affinity for substrate sugars[END_REF] and Vitis vinifera [START_REF] Davies | Three putative sucrose transporters are differentially expressed in grapevine tissues[END_REF][START_REF] Ageorges | Identification and functional expression in yeast of a grape berry sucrose carrier[END_REF].

Genome sequencing demonstrated that sucrose transporters are encoded by a small multigenic family: five genes in Oryza sativa [START_REF] Braun | Genetic control of carbon partitioning in grasses: roles of sucrose transporters and tie-dyed loci in phloem loading[END_REF] or nine genes in Arabidopsis thaliana [START_REF] Baud | The AtSUC5 sucrose transporter specifically expressed in the endosperm is involved in early seed development in Arabidopsis[END_REF]. In this latter, seven genes encode functional sucrose transporters, whereas two genes (AtSUC6 and AtSUC7) code aberrant and nonfunctional proteins [START_REF] Sauer | AtSUC8 and AtSUC9 encode functional sucrose transporters, but the closely related AtSUC6 and AtSUC7 genes encode aberrant proteins in different Arabidopsis ecotypes[END_REF]). Based on their sequence homology and biochemical activity, SUTs were assigned into either three [START_REF] Aoki | The sucrose transporter gene family in rice[END_REF], four [START_REF] Sauer | Molecular physiology of higher plant sucrose transporters[END_REF] or five groups [START_REF] Braun | Genetic control of carbon partitioning in grasses: roles of sucrose transporters and tie-dyed loci in phloem loading[END_REF][START_REF] Kühn | Sucrose transporters of higher plants[END_REF]. Most of these sucrose transporters are localized in the plasma membrane, while some of them are found in the tonoplast [START_REF] Sauer | Molecular physiology of higher plant sucrose transporters[END_REF].All plasma membrane sucrose transporters have been identified as energy-dependent proton sucrose/H+ transporters, except the sucrose facilitators (SUFs) isolated from Pisum sativum and Phaseolus vulgaris. SUFs catalyse bidirectional sucrose transport in a pH-and energy-independent manner [START_REF] Zhou | A suite of sucrose transporters expressed in coats of developing legume seeds includes novel pH-independent facilitators[END_REF].

SUTs are involved in: (1) loading sucrose into the phloem from the apoplast of the source cell; (2) maintaining a high sucrose concentration in the phloem; and (3) unloading to sink cells (Williams, Lemoine & Sauer 2000 for review). Their role in diverse sink tissues has been well documented in the literature. For example, in potato tuber, specific antisense repression of SUT1 was accompanied by a reduction of phloem unloading [START_REF] Kühn | The sucrose transporter StSUT1 localizes to sieve elements in potato tuber phloem and influences tuber physiology and development[END_REF]; fertility of tomato fruits was highly reduced in LeSUT2 antisense plants [START_REF] Hackel | Sucrose transporter LeSUT1 and LeSUT2 inhibition affects tomato fruit development in different ways[END_REF]; in grape, VvSUC27 is highly expressed after flowering [START_REF] Davies | Three putative sucrose transporters are differentially expressed in grapevine tissues[END_REF][START_REF] Zhang | Functional characterization of a LAHC sucrose transporter isolated from grape berries in yeast[END_REF]. Although the bursting bud is considered an active sink organ and requires a high sugar import to satisfy its carbon demand, very little is known on the involvement of this kind of plasma membrane transporter in this morphogenetic process.

Sucrose transporter expression is highly regulated at both transcriptional and post-transcriptional level [START_REF] Williams | Sugar transporters in h i g h e rp l a n t s-ad iversity of roles and complex regulation[END_REF]Kühn & Grof 2010 for review). Wounding [START_REF] Meyer | Wounding enhances expression of AtSUC3, a sucrose transporter from Arabidopsis sieve elements and sink tissues[END_REF], salt stress [START_REF] Noiraud | The sucrose transporter of celery. Identification and expression during salt stress[END_REF], temperature [START_REF] Decourteix | JrSUT1, a putative xylem sucrose transporter, could mediate sucrose influx into xylem parenchyma cells and be up-regulated by freeze-thaw cycles over the autumn-winter period in walnut tree (Juglans regia L.)[END_REF]) and light [START_REF] Shakya | Characterization of source-and sink-specific sucrose/H + symporters from carrot[END_REF][START_REF] Matsukura | Sugar uptake and transport in rice embryo. Expression of companion cell-specific sucrose transporter (OsSUT1) induced by sugar and light[END_REF][START_REF] Chincinska | Sucrose transporter StSUT4 from potato affects flowering, tuberization, and shade avoidance response[END_REF][START_REF] Kühn | Sucrose transporters of higher plants[END_REF] evoked mRNA accumulation. Furthermore, these genes were also regulated by endogenous factors such as sugar concentration [START_REF] Weber | A role for sugar transporters during seed development: molecular characterization of a hexose and a sucrose carrier in fava bean seeds[END_REF][START_REF] Chiou | Sucrose is a signal molecule in assimilate partitioning[END_REF][START_REF] Li | Sugars regulate sucrose transporter gene expression in citrus[END_REF][START_REF] Tang | The sucrose transporter HbSUT3 plays an active role in sucrose loading to laticifer and rubber productivity in exploited trees of Hevea brasiliensis (para rubber tree)[END_REF] or the phytohormones, gibberellins [START_REF] Chincinska | Sucrose transporter StSUT4 from potato affects flowering, tuberization, and shade avoidance response[END_REF][START_REF] Chen | Hormone and sugar effects on rice sucrose transporter OsSUT1 expression in germinating embryos[END_REF]), ethylene (Dusotoit-Coucaud et al. 2009) and cytokinins [START_REF] Ehness | Co-ordinated induction of mRNAs for extracellular invertase and a glucose transporter in Chenopodium rubrum by cytokinins[END_REF]. These changes may result from direct action of physical or biochemical signals, or indirectly through modulation of sugar concentrations in cells.

In Rosa sp., previous data from our laboratory involved light as an essential environmental factor targeting the bud itself to trigger its development in a beheaded plant system [START_REF] Girault | Light controls shoot meristem organogenic activity and leaf primordia growth during bud burst in Rosa sp[END_REF]. This light-mediated bud burst is concomitant with the stimulation of sugar metabolism through an up-regulation of a vacuolar acid invertase [START_REF] Girault | Sugars are under light control during bud burst in Rosa sp[END_REF] and local accumulation of sugars (sucrose and glucose). In addition, the essential need of light for bud burst is specific to Rosa sp., and was not found in any tested model plants such as Arabidopsis, poplar or tomato [START_REF] Girault | Light controls shoot meristem organogenic activity and leaf primordia growth during bud burst in Rosa sp[END_REF]. Within this context, the determination of the mechanism of sucrose unloading in rose buds is a first step towards the understanding of the mechanism by which light modulates bud break and ultimately plant architecture.

This work aimed to provide insight into the role of sucrose transporters in the photocontrol of bud burst. Here, we present sucrose transporter isolation, their expression in buds exposed to light/dark conditions and their tissuespecific expression pattern. We determined the sucrose uptake kinetics of one of them, RhSUC2, using a baker's yeast system. Its active role in bud sucrose import to promote bud burst under light in Rosa is discussed.

MATERIALS AND METHODS

Plant material and growth conditions

Plants (Rosa hybrida 'Radrazz') were propagated by cuttings from a single plant genotype as previously described [START_REF] Girault | Light controls shoot meristem organogenic activity and leaf primordia growth during bud burst in Rosa sp[END_REF]. Experiments were performed on plants displaying an emerging flower bud, hereafter referred to as a 'visible flower bud' (VFB). At this stage, the plant bore at least seven leaflet leaves, and all axillary buds remained dormant because of the apical dominance exerted by the shoot apical meristem. In all experiments, we studied axillary bud of the third proximal leaflet leaves (named 'bud of interest'). For this study, two experimental models were used:

Morphological analyses

After 6 d of light/dark treatment, the length of the distal bud was measured using a digital caliper (0-150 mm) and the total number of leaf-like organs (including scales, preformed leaves at different development stages and leaf primordia) was determined after manual dissection of the bud, under a stereo-dissecting microscope.

Sugar content analyses

Sugar content analyses were performed using 40 mg of frozen tissues. Tissues were ground to a fine powder and homogenized with 400 mL of extraction buffer [45 mm HEPES (pH 7.0), 9 mm MgCl2, 0.9 mm ethylenediaminetetraacetic acid (EDTA), 2.6 mm dithiothreitol (DTT), 10% ethylene glycol, 0.02% Triton X-100] and centrifuged 3 min at 12 000 g (4 °C). The supernatant was collected and used for sugar (starch, sucrose, d-glucose and d-fructose) content determination using a Konelab 20i sequential automat (Thermo Electron, Vantaa, Finland) and the ENZYTEC system (SCIL Diagnostics, Martinsried, Germany).Analyses were performed on three independent biological samples.

C sucrose accumulation analyses

Bud sugar accumulation analyses were carried out on buds harvested from beheaded plants grown as previously described. After harvesting, buds were washed for 30 min in 300 mL of control solution (100 mm mannitol, 20 mm MES, 0.25 mm CaCl2, 0.25 mm MgCl2, pH 5.5) with or without 0.5 mm CCCP (Sigma, Évry, France). Buds were then soaked and transferred to solid control media supplemented with 1mm [U-14C] sucrose (0.5 mCi mL -1 , 21.8 kBq mmol -1 , Perkin Elmer, Les Ulis, France) and 0 or 0.5 mm CCCP. The sectioned part of bud was the only region directly in contact with the labelled media. The incubation was performed for 2 h, then buds were washed three times for 5 min with 500 mL of control solution. Tissues were treated with 300 mL of digesting solution (Triton 0.1X, 13% HClO4, 12% H2O2) for 12 h at 55 °C. Four millilitres of liquid scintillation cocktail (Ecolite(+)TM liquid scintillation fluid; MP Biomedicals, Paris, France) was added and the radioactivity measured in a liquid scintillation counter (Packard, Tricarb 1900TR, Les Ulis, France). Sugar uptake was calculated from the radioactivity measurements. Each experiment was done three times with three to five pooled buds. Buds grown under darkness were kept under darkness during sugar uptake assays.

RNA extraction and cDNA synthesis

Buds were collected from the two experimental models (beheaded plant and in vitro single node explant), frozen in liquid nitrogen and stored at -80 °C until use. For plants grown in darkness, tissue collection was performed under green light. Frozen tissues (60-100 mg) were ground using mortar, pestle and liquid nitrogen in the presence of 10% PVP40 (w/w). Total RNA was then isolated using NucleoSpin RNA Plant Kit (Macherey-Nagel, Paris, France), its quantity evaluated with a NanoDrop spectrophotometer (Fisher Scientific, Illkirch, France) and its quality checked by gel electrophoresis. RNA (1 mg) was reverse transcribed with oligo(dT)20 primer using SuperScript III Reverse transcriptase kit (Invitrogen, Paris, France). Genomic DNA contamination was verified by PCR using specific primer designed against an intronic region of RhGAPDH gene. cDNA was purified with QIAquick PCR Purification Kit (QIAGEN, Les Ulis, France) and diluted 400 times with water and stored at -20 °C until needed.

Sequence identification and cloning

A screen of rose genomic resources (URGI, http://urgi. versailles.inra.fr/ and Genome Database for Rosaceae (GDR), http://www.rosaceae.org) allowed us to identify a single Expressed Sequenced Tag (RhSUC3, CL_RO_ 3183.1-19612540) that potentially encodes a sucrose transporter. Other putative sucrose transporters were PCR amplified from different tissues using the degenerate primers RhSUCa

[forward-C(AGCT)TT(CT)TT(CT)A TGGC(GC)GT(GCT)GG/reverse -TC(AT)(CT)(GT) (AGC)CCCATCCA(AG)TC(AGT)GT] and RhSUCb [forward -CT(AG)AC(CT)CC(AGCT)TA(CT)GT(ACT) CA(AG)CT/reverse - GG(AG)AACCA(AGCT)GC (AGT)ATCCA(AG)T].
The PCR products were subsequently cloned into pGEM-T-Easy vector and sequenced (Genome Express, Grenoble, France).

The RhSUC2 full-length cDNA sequence was isolated using RACE-PCR (Marathon cDNA Amplification Kit; Clontech, Paris, France) with cDNA made from buds harvested from beheaded light-grown plants. The RhSUC2 3′ and 5′-end amplifications were performed with BD Advantage 2 PCR Enzyme System (BD Biosciences, Paris, France) with AP1 and RhSUC2-F1 (CCATCTTCGTGGTCGGG TTCTGGAT) or RhSUC2-R1 (GGATACGAGAGCGA GAATGGTGAGGGC) primers. PCR products were then cloned into the pGEM-T-Easy vector and sequenced.

Gene expression analyses

Organ-specific gene expression was assessed on purified cDNA produced from dormant and bursting axillary buds, flower buds, young and mature leaves, stems and roots. Amplifications were performed in standard conditions using 0.5 U of GoTaq Flexi DNA polymerase (Promega, Charbonnières les bains, France) in a final volume of 25 mL. Reactions were set up with following primer pairs: SUC1 (forward-CGGCTGGATCGCTAAACGTT/ reverse-TGAGGTTGGCGCAGTAAATGTC), SUC2 (forward-CTACACCCACCTCCACAAGATG/reverse-A CCAAGGATCTCGCTGAAGA), SUC3 (forward-AGA CCGACTCGGTGTCGATTC/reverse-GCTCATCATCC ATCCCCATCA) and SUC4 (forward-TTGGCAATATT CTTGGCTATG-CAA/reverse-CGCACGCGGAGGTAA GTGTA), and for calibration SAND1 (forward-TCTC CTCATTTGAACCAACACACA/reverse-AGGGCCACC TACGCCATCAT). PCR products were separated on agarose gel. Experiments were repeated at least three times.

Real-time PCR reactions were performed using the iQ SYBR Green Supermix (Bio-Rad, Marnes-la-Coquette, France) and fluorescence was detected using Chromo4 Real-time PCR Detector (Bio-Rad). Each RT-PCR reaction was done in triplicate on three independent biological samples. Primer efficiency was determined as described by [START_REF] Rasmussen | Quantification on the LightCycler[END_REF], and the abundance of targeted transcript was normalized with the SAND1 transcript level and expressed relative to the control condition (before beheading) using the following formula: R = [(Etarget) DCT target (control treated) /(Ereference) DCT reference (control treated) ] (Pfaffl 2001).

RhSUC2 functional validation

The functionality of RhSUC2 was carried out in baker's yeast (Saccharomyces cerevisiae). We used the SUSY7 strain (suc2::URA3 malO leu2 trpl/YIP128A2-SUSY) for transformation [START_REF] Riesmeier | Isolation and characterization of a sucrose carrier cDNA from spinach by functional expression in yeast[END_REF]. Full-length CDS of RhSUC2 was amplified from the cDNA library using the following primers: forward (GAGAGAA TTCCCAGCAATGGAAGTCGAAACCACCCA) and reverse (GAGACTCGAGCGATCGAAATCAGTGGA ATGCAGCAGCT). PCR product was purified with QIAquick PCR Purification Kit (QIAGEN), digested with EcoRI and XhoI (Promega, Yerres, France) and cloned into digested pDR296 expression vector. This plasmid is derived from pDR195, using TRP1 instead of URA3 as the selection gene [START_REF] Rentsch | NTR1 encodes a high affinity oligopeptide transporter in Arabidopsis[END_REF].

Sucrose uptake assays were performed on transformed yeast (with the empty pDR296 or the RhSUC2-pDR296) grown to early logarithmic phase in YNB medium supplemented with 2% glucose. Cells were washed and resuspended with 50 mm MES buffer (pH 4.5) to reach a final OD600nm value of 0.3. The pH dependency study was performed with cells resuspended in distilled water. Aliquots (100 mL) of cell suspension were incubated in 100 mLo fa solution containing 50 mm MES (pH 4.5), 1 mm [U-14C] sucrose (0.5 mCi mL -1 , 18.43 kBq mmol -1 , Perkin Elmer) at 28 °C for 1, 2, 5 and 10 min for time course assays. Concentration dependence assays were performed by using the same solution containing 0.2-10 mm [U-14C] sucrose (0.5 mCi mL -1 ) at 28 °C for 2 min. For pH dependency study, cells were incubated with 100 mL of a solution containing 100 mm MES (pH 4.0-7.0) and 1 mm [U-14C] sucrose (0.5 mCi mL -1 ) at 28 °C for 2 min. Sugar specificity and inhibitor studies were monitored with 100 mL of a solution containing 50 mm MES (pH 4.5) and 5 mm [U-14C] sucrose (0.5 mCi mL -1 ) added with 50 mm of competing sugars or inhibitors. Cells were incubated for 2 min at 28 °C.The reactions were stopped by adding 8 mL of cold water and immediate filtration on glass microfibre filters (25 mm, Fisher Bioblock, Illkirch, France). This step was repeated once. Radioactivity incorporated into cells collected on filters was evaluated using a liquid scintillation counter.

Statistical analyses

The Student or Mann-Whitney tests were used to compare two conditions. To compare more than two conditions, the Kruskal-Wallis test possibly followed by pairwise comparisons with the Mann-Whitney tests and Bonferroni correction were carried out. The statistical computations were performed using the PAST software (http://folk.uio.no/ ohammer/past/).

RESULTS

Effect of beheading and sugar supply on rose bud break, elongation and organogenesis under light

Control buds (before treatments) are fully dormant with adherent scales (Fig. 1a). After apex removal, a 6 d light exposure was sufficient to induce the outgrowth of 90% of rose buds (Table 1). At this stage, developed leaves (harbouring leaflets) had just emerged from the bud (Fig. 1b). In contrast, when grown 6 d in vitro in the absence of sugars, the majority of buds (81%) were merely swollen and only 19% outgrowth (Fig. 1c). Supplying buds with the nonmetabolizable sugar (mannitol) also caused the buds to enlarge (Fig. 1d), but did not induce any outgrowth (0% at 100 and 250 mm, and 6% at 500 mm). In contrast to mannitol, 100 mm sorbitol did induce bud burst (81%), while higher amounts (250 and 500 mm) did not (Fig. 1e). Interestingly, sucrose gave rise to a massive bud burst, with a maximum of 100% at 250 mm, where buds exhibited a similar morphology to those on beheaded plants (Fig. 1f).

Bud elongation and organogenesis were also analysed (Table 1). Buds grown for 6 d in the absence of sugars showed very limited growth compared to plants before treatment and beheaded plants, while the mannitol-treated ones were nearly indistinguishable from plants before treatment. However, a stimulation of organogenesis was found in buds incubated with a high concentration (250 mm) of mannitol. Buds supplied with 100 mm sorbitol showed a partial stimulation of elongation (2.3-fold when compared to buds grown without sugar), while higher concentrations (250 mm) caused a response similar to that induced by mannitol. Sucrose showed varying responses: 100 and 250 mm stimulated both elongation (5.5-and 5.1-fold), and organogenesis (one and three additional primordia) when compared to buds grown without sugar, respectively. In addition, 500 mm sucrose caused a weaker stimulation of these two processes (2.2-fold for elongation and no additional primordia was synthesized, when compared to buds grown without sugar).

Effect of sugar supply on rose bud sugar content under light

Glucose, fructose, sucrose and starch contents were analysed after 72 h (3 d) in buds supplied in vitro with 250 mm sugars (Fig. 2). Dormant buds (control) contained high quantities of sucrose (7.36 Ϯ 2.46 mg g -1 FW) and low quantities of glucose (1.15 Ϯ 0.23 mg g -1 FW), fructose (0.78 Ϯ 0.11 mg g -1 FW) and starch (0.45 Ϯ 0.21 mg g -1 FW). Buds grown without sugars exhibited a reduction of all soluble sugar contents (2.2-to 2.3-fold relative to dormant buds), whereas starch content was not significantly reduced. Supplying buds with 250 mm mannitol induced a reduction of their sucrose content (3.4-fold relative to dormant buds), whereas hexose and starch contents were not affected by the treatment. Bud sugar contents remained unchanged relative to control only when buds were supplied with 250 mm sucrose.

Sucrose accumulation into buds of beheaded plants under light and dark conditions

Total radio-labelled sucrose accumulation into buds 96 h after beheading and exposure to light or dark conditions was analysed (Table 2). Dormant buds (control) were able to take up sucrose (4.62 Ϯ 0.76 mBq per bud). Sucrose absorption by buds was significantly induced by light (11.93 Ϯ 0.98 mBq per bud), but only slightly by dark (6.80 Ϯ 2.31 mBq per bud). To check whether sucrose uptake was mediated by an active process, radio-labelled sucrose accumulation into buds was performed in the absence or presence of 0.5 mm of CCCP (a protonophore) under light and dark condition. CCCP highly reduced sucrose accumulation into buds exposed to light (56%) and dark (44%) (data not shown). This result indicates that an electrochemical gradient across the plasma membrane is essential for sucrose accumulation into buds.

Isolation of four putative sucrose transporters (RhSUC1, 2, 3 and 4)

Data from Unité de Recherche en Génomique et Bio-Informatique (URGI) and GDR databases allowed us to identify a partial cDNA sequence (637 bp), corresponding to the 5′-end of a putative sucrose transporter gene from R. hybrida: RhSUC3 (CF349302) [START_REF] Guterman | Rose scent: genomics approach to discovering novel floral fragrance-related genes[END_REF].

We designed degenerated primers directed against conserved domains of the previously isolated sucrose transporters from several species to identify additional putative sucrose transporters in R. hybrida. Three partial cDNA sequences were obtained and referred as to RhSUC1 (HO762757, 367 bp), RhSUC2 (755 bp) and RhSUC4 (HO762758, 397 bp), based on their orthologs in Arabidopsis thaliana. Predicted protein sequences of partial and complete isolated R. hybrida sucrose transporters were compared with other protein sequences (NCBI/Protein) using ClustalW, and the resulting phylogenetic tree was visualized with Treeview (Fig. 3). RhSUC1 partial putative protein shares the highest sequence similarity with HbSUT1A (68%) [START_REF] Dusotoit-Coucaud | Molecular characterisation of the sucrose transporter HbSUT1B in relation to rubber production in latex cells[END_REF]) and RcSCR1 (67%) [START_REF] Eisenbarth | Sucrose carrier RcSCR1 is involved in sucrose retrieval, but not in sucrose unloading in growing hypocotyls of Ricinus communis L[END_REF], and 66% identity with AtSUC1 being its closest sequence in Arabidopsis. RhSUC2 putative protein shares 73, 72, 70 and 66% identity

RhSUC1, 2, 3 and 4 expression in R. hybrida organs

The putative sucrose transporters differ greatly in their expression pattern in the tested organs (Fig. 4). RhSUC1 transcript is mainly accumulated in stems, and to a much lower extent in mature leaves and flower buds. RhSUC2 strongly accumulated in bursting buds and in stems. This transcript also showed a slight accumulation in young leaves. No accumulation of RhSUC2 was found in mature leaves, suggesting its expression is specific to sink tissues. RhSUC3 and RhSUC4 showed a low, but constitutive transcript accumulation in all tested organs.

Transcript pattern of RhSUC2, 3 and 4 into buds of beheaded plants under light and dark conditions

The relative gene expression of R. hybrida sucrose transporters was assessed on beheaded plants grown under light or total darkness from 24 to 96 h (Fig. 5). Untreated plants were used as control. No expression of RhSUC1 was detected in buds under light or dark conditions. RhSUC2 was the only transporter displaying significant difference in transcript accumulation in buds between light and dark conditions from 48 to 96 h after beheading. Its transcript level was induced (3.5-to 4-fold) within 24 h after beheading, under light and darkness. This induction was stable in light over the time course (until 96 h), but not in darkness where RhSUC2 transcript level dropped and returned to its basal level 48 h after beheading.

RhSUC3 and RhSUC4 transcript levels were unaffected by beheading, or by the presence or absence of light over the time course frame (until 96 h).

Functional characterization of RhSUC2 in baker's yeast

As RhSUC2 expression is correlated to the light-induced bud break, its transport activity was tested in a heterologous system, baker's yeast. We expressed RhSUC2 cDNA in the SUSY7 yeast strain, which is deficient in both cell wall invertase and maltose utilization, and expresses a potato sucrose synthase [START_REF] Riesmeier | Isolation and characterization of a sucrose carrier cDNA from spinach by functional expression in yeast[END_REF]. Our data (Fig. 6; Table 3) demonstrate that RhSUC2 encodes a functional sucrose/H+ co-transporter.

Sucrose uptake of RhSUC2 cDNA complemented cells was clearly higher (31.5-fold after 10 min) than those complemented with the empty pDR296 vector. RhSUC2 sucrose uptake was constant and did not level off during the 10 min time course. It was pH dependant with an optimum at pH 4.5 (Fig. 6b). The protonophore CCCP and the -SH group blocker PCMBS that are well characterized as sucrose/H+ transporter inhibitors caused a massive drop in sucrose uptake (75 and 91%, respectively relative to the control Table 3). The affinity of RhSUC2 for sucrose was also investigated at pH 4.5 for sucrose concentrations ranging from 0.1 to 5 mm (Fig. 6c). A Lineweaver-Burk transformation of data revealed, at this pH, that RhSUC2 displayed a Km value of 2.99 mm and a Vmax of 21.4 nmol sucrose mg -1 protein min -1 (Fig. 6d). Interestingly, RhSUC2 sucrose transport activity was repressed (63%) in the presence of 5 mm sucrose, a concentration close to the Km value.

The substrate specificity of RhSUC2 was assessed in the presence of an excess of various unlabelled sugars (25 mm) (Table 3). Among all tested sugars, sucrose and maltose were the only ones to compete with 14 C-sucrose uptake (reduction of 79 and 57% of sucrose uptake activity when compared to control, respectively). In contrast, glucose and fructose stimulated sucrose uptake.

Effect of auxin on RhSUC2, 3 and 4 transcript level in buds

RhSUC2, 3 and 4 transcript level in buds grown in vitro in the presence of MS media alone or supplemented with 0.1, 1o r1 0mm of synthetic auxin (NAA) for 72 h under light was estimated by quantitative RT-PCR. Dormant buds before treatment served as control (Fig. 7).

Compared to dormant buds, RhSUC2 transcript level is higher in buds grown 72 h in vitro on MS media under light. This light-induced bud break and RhSUC2 expression were reduced in MS medium supplemented with 1 and 10 mm of synthetic auxin (NAA). No difference was found between samples of MS and 0.1 mm NAA-supplemented MS. Under the same experimental condition, transcript level of RhSUC3 and RhSUC4 was insensitive to auxin application.

DISCUSSION

Light-mediated bud break requires sugar influx into bud

In Rosa sp., light is critical for bud burst after apical dominance release through beheading (Mor & Halevy 1980;Mor et al. 1980;[START_REF] Girault | Light controls shoot meristem organogenic activity and leaf primordia growth during bud burst in Rosa sp[END_REF]. Light is perceived by the bud itself, and promotes organogenesis and the outgrowth of the foliar primordia [START_REF] Girault | Light controls shoot meristem organogenic activity and leaf primordia growth during bud burst in Rosa sp[END_REF]. These processes are accompanied by a shift in sugar metabolism (NCBI/Protein) were used to construct a phylogenetic tree. Partial or complete Rosa hybrida sucrose transporter predicted protein sequences were compared to sucrose transporters from other plant species using ClustalW. Phylogenetic tree was visualized with Treeview. Phylogenetic groups defined by [START_REF] Sauer | Molecular physiology of higher plant sucrose transporters[END_REF] are indicated. Accession numbers of presented sucrose transporter sequences are: AtSUC1 (Arabidopsis thaliana; NP_177333), AtSUC2 (NP_173685), AtSUC3 (NP_178389), AtSUC4 (NP_172467), AtSUC5 (NP_177334), AtSUC6 (NP_199174), AtSUC7 (NP_176830), AtSUC8 (NP_179074), AtSUC9 (NP_196235), DcSUT1 (Daucus carota, Y16766), DcSUT2 (Y16768), HbSUT1A (Hevea brasiliensis, DQ985466), HbSUT1B (AM492537), HbSUT2A (DQ985467), HbSUT2B (DQ985465), HbSUT2C (AM491808), HbSUT4 (EF067335), HbSUT5 (EF067333), JrSUT1 (Juglans regia, AAU11810), LeSUT1 (Lycopersicon esculentum, CAA57726), LeSUT2 (AAG12987), LeSUT4 (AAG09270), MdSUT1 (Malus ¥ domestica, AAR17700), NtSUT1 (Nicotiana tabacum, X82276), NtSUT3 (AAD34610), OsSUT1 (Oryza sativa, AAF90181), OsSUT2 (Q0ILJ3), OsSUT3 (BAB68368), OsSUT4 (BAC67164), OsSUT5 (BAC67165), PmSUC1 (Plantago major, CAI59556), PmSUC2 (X75764), PmSUC3 (CAD58887), PsSUF1 (Pisum sativum, ABB30163), PsSUF4 (ABB30162), PvSUF1 (Phaseolus vulgaris, ABB30165), PvSUT1 (ABB30164), RcSCR1 (Ricinus communis, CAA83436), RhSUC1 partial (R. hybrida, HO762757), RhSUC2 (HQ403679), RhSUC3 partial (CF349302), RhSUC4 partial (HO762758), StSUT1 (Solanum tuberosum, CAA48915), StSUT2 (AAP43631), StSUT4 (AAG25923), VfSUT1 (Vicia fava, CAB07811), VvSUC11 (Vitis vinifera, AAF08329), VvSUC12 (AAF08330), VvSUC27 (AAF08331), ZmSUT1 (Zea mays, NP_001104840), ZmSUT2 (AAS91375), ZmSUT4 (AAT51689). Bars indicate the evolutionary distance. [START_REF] Girault | Sugars are under light control during bud burst in Rosa sp[END_REF]. Light-induced bursting buds displayed a high import of sucrose and glucose, and an increase in transcript accumulation and enzymatic activity of vacuolar acid invertase [START_REF] Girault | Sugars are under light control during bud burst in Rosa sp[END_REF]. Taken together, these results suggest that bud outgrowth in the light is closely linked to the mobilization of sugars. This is in agreement with our findings, supporting a possible role of sucrose transport in the photocontrol of bud burst. Here, in vitro experiments show that buds require the import of exogenous metabolizable sugars (mainly sucrose) to develop under light (Fig. 1; Table 1). Similarly, an exogenous source of metabolizable sugars promotes in vitro development of palm bud [START_REF] Al-Khateeb | Regulation of in vitro bud formation of date palm (Phoenix dactylifera L.) cv. Khanezi by different carbon sources[END_REF]. The optimal sugar concentration for in vitro bud development (250 mm in Rosa, Table 1) closely matches those found in the phloem sap of several species [START_REF] Verscht | Sugar concentrations along and across the Ricinus communis L. hypocotyl measured by single cell sampling analysis[END_REF][START_REF] Nadwodnik | Subcellular concentrations of sugar alcohols and sugars in relation to phloem translocation in Plantago major, Plantago maritima, Prunus persica,andApium graveolens[END_REF]. Organogenesis remains constant during sugar starvation, while bud elongation is highly diminished (Table 1), suggesting that this latter could be the main sugar-dependant process.

Other evidence indicates that Rosa buds do not store enough sugars to promote their own development, and thus need to import soluble sugars. Growing buds for 3 d in the absence of sugar caused a drop in their sugar content. In contrast, buds grown in the presence of sugars develop well and show a similar metabolizable sugar content as the control, supporting their high metabolic activity. Such elevated metabolic activity in the outgrowing bud has been previously reported in other ligneous species such as peach tree and walnut [START_REF] Marquat | Dormancy in vegetative buds of peach: relation between carbohydrate absorption potentials and carbohydrate concentration in the bud during dormancy and its release[END_REF]Maurel et al. 2004a;[START_REF] Decourteix | Sucrose (JrSUT1) and hexose (JrHT1 and JrHT2) transporters in walnut xylem parenchyma cells: their potential role in early events of growth resumption[END_REF]. Furthermore, we demonstrated that this import is an active, light-and energy-dependant sucrose absorption occurring during bud outgrowth (Table 2). These modifications are in accordance with an increase in plasmalemma ATPase activity [START_REF] Aue | Change in plasma membrane ATPase activity during dormancy release of vegetative peach-tree buds[END_REF][START_REF] Alves | Plasma membrane H + -ATPase, succinate and isocitrate dehydrogenases activities of vessel-associated cells in walnut trees[END_REF]) and active sugar absorption [START_REF] Marquat | Dormancy in vegetative buds of peach: relation between carbohydrate absorption potentials and carbohydrate concentration in the bud during dormancy and its release[END_REF]Maurel et al. 2004b) in the bud or neighbouring stem region. In walnut stem, the expression of the putative sucrose transporter JrSUT1 is correlated a bud sink strength increase during outgrowth [START_REF] Decourteix | Sucrose (JrSUT1) and hexose (JrHT1 and JrHT2) transporters in walnut xylem parenchyma cells: their potential role in early events of growth resumption[END_REF][START_REF] Bonhomme | Carbohydrate uptake from xylem vessels and its distribution among stem tissues and buds in walnut (Juglans regia L.)[END_REF]. Our data clearly show that Rosa bud is a sink organ requiring importation of metabolizable sugars, at least sucrose, to ensure correct outgrowth in light.

Rosa bud contains several sucrose transporter isoforms

We have identified four putative sucrose transporters (RhSUC1, RhSUC2, RhSUC3 and RhSUC4) confirming that they are encoded by a small multigenic family. Depending on the genus, the number of isoforms is different: two in tobacco [START_REF] Bürkle | The H + -sucrose cotransporter NtSUT1 is essential for sugar export from tobacco leaves[END_REF][START_REF] Lemoine | Identification of a pollen-specific sucrose transporter-like protein NtSUT3 from tobacco[END_REF], three in tomato [START_REF] Barker | SUT2, a putative sucrose sensor in sieve elements[END_REF][START_REF] Weise | A new subfamily of sucrose transporters, SUT4, with low affinity/high capacity localized in enucleate sieve elements of plants[END_REF], four in grapevine [START_REF] Davies | Three putative sucrose transporters are differentially expressed in grapevine tissues[END_REF][START_REF] Ageorges | Identification and functional expression in yeast of a grape berry sucrose carrier[END_REF], five in rice [START_REF] Aoki | The sucrose transporter gene family in rice[END_REF][START_REF] Hirose | Disruption of a gene for rice sucrose transporter, OsSUT1, impairs pollen function but pollen maturation is unaffected[END_REF], seven in Hevea [START_REF] Dusotoit-Coucaud | Molecular characterisation of the sucrose transporter HbSUT1B in relation to rubber production in latex cells[END_REF][START_REF] Tang | The sucrose transporter HbSUT3 plays an active role in sucrose loading to laticifer and rubber productivity in exploited trees of Hevea brasiliensis (para rubber tree)[END_REF] and nine in Arabidopsis (Sauer 2007 for review).

The isolated putative sucrose transporters share a high sequence identity with those from herbaceous and ligneous plants (Fig. 2), particularly RhSUC4 with apple tree (77%) and other isoforms with Hevea (66-77%). Furthermore, the four putative sucrose transporters are spread within three phylogenetic clades defined by [START_REF] Sauer | Molecular physiology of higher plant sucrose transporters[END_REF] that groups all known dicotyledonous sequences. Group 2 contains high affinity, low capacity transporters implicated in phloem loading and sucrose import in sink cells. RhSUC1 and RhSUC2 are homologs of these transporters. RhSUC3 is similar to group 3 transporters that contain low affinity and high capacity transporters found in sieve elements and in several sink tissues where they may mediate sucrose efflux during phloem unloading and act as sucrose sensors. RhSUC4 belongs to group 4, containing tonoplastic porters mainly found in sink tissues [START_REF] Sauer | Molecular physiology of higher plant sucrose transporters[END_REF]).

RhSUC2 could be implied in bud sucrose loading during light-controlled bud burst

Many sucrose transporters are expressed in various sink tissues where they are implicated in several different physiological processes such as tuberization [START_REF] Kühn | The sucrose transporter StSUT1 localizes to sieve elements in potato tuber phloem and influences tuber physiology and development[END_REF], fruit formation [START_REF] Davies | Three putative sucrose transporters are differentially expressed in grapevine tissues[END_REF][START_REF] Ageorges | Identification and functional expression in yeast of a grape berry sucrose carrier[END_REF][START_REF] Hackel | Sucrose transporter LeSUT1 and LeSUT2 inhibition affects tomato fruit development in different ways[END_REF][START_REF] Zhang | Functional characterization of a LAHC sucrose transporter isolated from grape berries in yeast[END_REF], seed formation [START_REF] Weber | A role for sugar transporters during seed development: molecular characterization of a hexose and a sucrose carrier in fava bean seeds[END_REF][START_REF] Baud | The AtSUC5 sucrose transporter specifically expressed in the endosperm is involved in early seed development in Arabidopsis[END_REF][START_REF] Hackel | Sucrose transporter LeSUT1 and LeSUT2 inhibition affects tomato fruit development in different ways[END_REF][START_REF] Zhou | A suite of sucrose transporters expressed in coats of developing legume seeds includes novel pH-independent facilitators[END_REF]), latex synthesis (Dusotoit-Coucaud et al. 2009, 2010;[START_REF] Tang | The sucrose transporter HbSUT3 plays an active role in sucrose loading to laticifer and rubber productivity in exploited trees of Hevea brasiliensis (para rubber tree)[END_REF]) and pollen development [START_REF] Lemoine | Identification of a pollen-specific sucrose transporter-like protein NtSUT3 from tobacco[END_REF][START_REF] Stadler | The AtSUC1 sucrose carrier may represent the osmotic driving force for anther dehiscence and pollen tube growth in Arabidopsis[END_REF][START_REF] Takeda | Sugar transporters involved in flowering and grain development of rice[END_REF]. In Rosa, quantitative and semi-quantitative RT-PCR showed that three of the four putative sucrose transporters (RhSUC2, RhSUC3 and RhSUC4) are expressed in the bud (Figs 3 & 4), with high accumulation of RhSUC2 in bursting buds (Fig. 3). Furthermore, it is the only sucrose transporter whose expression was correlated with light-induced bud break in beheaded plants. This treatment induces the outgrowth of axillary buds in the presence of light but not in dark [START_REF] Khayat | Environmental factors involved in the regulation of sprounting of basal buds in rose plants[END_REF][START_REF] Girault | Light controls shoot meristem organogenic activity and leaf primordia growth during bud burst in Rosa sp[END_REF]. Under light conditions, RhSUC2 transcript level increases within 48 h and remains high during the entire 96 h time course relative to dormant bud (dark condition). In contrast, we found no correlation between the expression profiles of RhSUC3 and 4, and bud outgrowth potential in the presence of light, reinforcing the potential role of RhSUC2 in light-induced bud break. In light, the accumulation of RhSUC2 transcript in the bud is concomitant with enzymatic vacuolar acid invertase activity [START_REF] Girault | Sugars are under light control during bud burst in Rosa sp[END_REF], which is a major component of organ sink strength [START_REF] Nägele | Mathematical modeling of the central carbohydrate metabolism in Arabidopsis reveals a substantial regulatory influence of vacuolar invertase on whole plant carbon metabolism[END_REF]) and cell elongation [START_REF] Morris | Invertase activity in sinks undergoing cell expansion[END_REF][START_REF] Sturm | Invertases. Primary structures, functions, and roles in plant development and sucrose partitioning[END_REF]. Within this context, the imported sucrose can contribute to cellular growth processes, such as carbon skeleton and energy source, and also as osmotically active molecules required for cell expansion. Likewise, the sucrose imported into the bud or its cleavage products (hexoses) may be a signal molecule to regulate genes involved in development (see [START_REF] Gibson | Control of plant development and gene expression by sugar signaling[END_REF] for review).

The transport activity of RhSUC2 was tested in baker's yeast. We found that the heterologous expression of RhSUC2 demonstrated that it encodes a functional, high affinity sucrose/H+ co-transporter. Its transport activity is sensitive to PCMBS (thiol inhibitor) as reported for several sucrose transporters [START_REF] Sauer | SUC1 and SUC2: two sucrose transporters from Arabidopsis thaliana; expression and characterization in baker's yeast and identification of the histidine-tagged protein[END_REF][START_REF] Ludwig | Plant sucrose-H + symporters mediate the transport of vitamin H[END_REF][START_REF] Meyer | AtSUC3, a gene encoding a new Arabidopsis sucrose transporter, is expressed in cells adjacent to the vascular tissue and in a carpel cell layer[END_REF][START_REF] Barth | PmSUC3: characterization of a SUT2/SUC3-type sucrose transporter from Plantago major[END_REF] and quite specific to sucrose because only maltose appears as a competitor for sucrose transport (Table 3). Interestingly, the sucrose transport activity of RhSUC2 was repressed by sucrose itself, at concentration (5 mm) close to the Km value. Such regulation has already been observed in yeast, where the HXT2 hexose transporter expression is repressed by high level of glucose, at transcriptional and posttranslational levels [START_REF] Wendell | Expression of high-affinity glucose transport protein Hxt2p of Saccharomyces cerevisiae is both repressed and induced by glucose and appears to be regulated posttranslationally[END_REF]. Physiological significance of such regulation of RhSUC2 deserves to be clarified in the near future.

Taken together, these results strongly suggest that RhSUC2 can play a central role in sucrose influx into outgrowing buds. Sink organs actively import sugars such as sucrose, but also as hexose or polyols [START_REF] Gao | Cloning, expression, and characterization of sorbitol transporters from developing sour cherry fruit and leaf sink tissues[END_REF][START_REF] Büttner | The monosaccharide transporter (-like) gene family in Arabidopsis[END_REF][START_REF] Sauer | Molecular physiology of higher plant sucrose transporters[END_REF][START_REF] Dusotoit-Coucaud | Cloning and characterization of a new polyol transporter (HbPLT2) in Hevea brasiliensis[END_REF]. In vegetative buds of peach tree, physiological data indicated the existence of an active uptake of glucose that is concomitant with the stimulation of cell wall invertase (Maurel et al. 2004a), and such co-existence was reported in many sink organs [START_REF] Sherson | Roles of cell wall invertases and monosaccharide transporters in the growth and development of Arabidopsis[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]. Potential involvement of hexose transporters in lightinduced bud break in Rosa could not be ruled out, even if no cell wall invertase induction occurred concomitantly with bud burst [START_REF] Girault | Sugars are under light control during bud burst in Rosa sp[END_REF].

Numerous Rosaceae transporters contain both sucrose and sorbitol transporters that play different roles in sink organs [START_REF] Gao | Cloning, expression, and characterization of sorbitol transporters from developing sour cherry fruit and leaf sink tissues[END_REF][START_REF] Dusotoit-Coucaud | Cloning and characterization of a new polyol transporter (HbPLT2) in Hevea brasiliensis[END_REF]). However, it does not seem to be the case in Rosa, because sorbitol was not detected in phloem sap, in stems or buds [START_REF] Zimmermann | List of sugars and sugar alcohols in sieve-tube exudates[END_REF][START_REF] Girault | Sugars are under light control during bud burst in Rosa sp[END_REF]. Furthermore, exogenous sorbitol (100 mm) induces only a limited bud burst and prevents it at higher concentrations (Table 1). It seems therefore unlikely that polyol transporters are implicated in sugar importation during bud burst.

RhSUC2 could be regulated by the combined effect of light and beheading

Beheading transiently induces RhSUC2 expression in buds after 24 h, independently from light (Fig. 5). This may be caused by a wound effect or to hormonal induced by apical dominance suppression. Previous work demonstrated that wounding [START_REF] Meyer | Wounding enhances expression of AtSUC3, a sucrose transporter from Arabidopsis sieve elements and sink tissues[END_REF]) and different hormones such as ethylene [START_REF] Chincinska | Sucrose transporter StSUT4 from potato affects flowering, tuberization, and shade avoidance response[END_REF][START_REF] Dusotoit-Coucaud | Molecular characterisation of the sucrose transporter HbSUT1B in relation to rubber production in latex cells[END_REF], 2010;[START_REF] Tang | The sucrose transporter HbSUT3 plays an active role in sucrose loading to laticifer and rubber productivity in exploited trees of Hevea brasiliensis (para rubber tree)[END_REF], gibberelins [START_REF] Chincinska | Sucrose transporter StSUT4 from potato affects flowering, tuberization, and shade avoidance response[END_REF] and cytokinins [START_REF] Harms | Isolation and characterization of P-type H + -ATPase genes from potato[END_REF] all affect different kinds of sucrose transporters at the transcriptional level. In our system, beheading is used to suppress apical dominance, via a reduction of the auxin flux [START_REF] Shimizu-Sato | Auxin-cytokinin interactions in the control of shoot branching[END_REF]. Auxin application (1 and 10 mm) prevents stimulation of RhSUC2 expression under light (Fig. 7), which was correlated with the absence of bud break in vitro (data not shown; [START_REF] Prasad | Does auxin play a role in the release of apical dominance by shoot inversion in Ipomoea nil[END_REF]. This finding suggests that the effect of light on RhSUC2 expression was negatively affected by auxin and could occur in the absence of apical dominance.

The effect of light on RhSUC2 expression is in agreement with previous work implying several sucrose transporters. In rice, OsSUT1 mRNA accumulates in light, but is absent in dark [START_REF] Matsukura | Sugar uptake and transport in rice embryo. Expression of companion cell-specific sucrose transporter (OsSUT1) induced by sugar and light[END_REF]. In carrot, the levels of DcSUT1 transcripts were light dependent, with high levels during the day and low levels during the night [START_REF] Shakya | Characterization of source-and sink-specific sucrose/H + symporters from carrot[END_REF]. In potato, StSUT4-RNAi transgenic plants resulted in an early flowering, high tuber production and a reduced sensitivity towards light enriched in far-red wavelengths -therefore mimicking shading [START_REF] Chincinska | Sucrose transporter StSUT4 from potato affects flowering, tuberization, and shade avoidance response[END_REF]. In potato model, [START_REF] Kühn | Sucrose transporters of higher plants[END_REF] have proposed that StSUT4 expression is co-regulated by light via phytochrome B.

In conclusion, we show that photocontrol of bud burst is highly correlated with sugar import in buds. This process requires an active sucrose transport, probably mediated by RhSUC2. This finding supports a new function of sucrose transporter in plant physiology. Moreover, this work lays the foundation to deepen the molecular mechanisms by which light regulates RhSUC2 expression and therefore to better understand RhSUC2 involvement in the modulation of plant shape by light condition.

Figure 1 .

 1 Figure 1. Effect of in vitro sugar feeding and plant beheading on bud burst after 6 d under light. Dormant buds before treatment served as control (a). In planta buds after beheading (b). Buds grown in vitro without sugar (c), with 250 mm mannitol (d), sorbitol (e) or sucrose (f). The horizontal white bars = 2 mm.

Figure 2 .

 2 Figure 2. Effect of in vitro sugar feeding on bud sugar content, before treatment and after 72 h under light. Buds were grown in vitro without sugar, with 250 mm mannitol or sucrose. Dormant buds before treatment served as control. Sucrose (a), glucose (b), fructose (c) and starch (d) contents were determined. Each value is a mean of three independent biological replicates. Error bars represent standard errors. Asterisks indicate significant differences with the control (Student's t-test, P < 0.05).

Figure 3 .

 3 Figure 3. Phylogenetic tree of plant sucrose transporters. Confirmed or predicted plant sucrose transporter complete protein sequences

Figure 4 .Figure 5 .

 45 Figure 4. Transcript level of Rosa hybrida putative sucrosetransporter genes in several organs. Total RNA was isolated from different tissues: dormant buds (DB), bursting buds (BB), flower buds (FB), young leaves (YL), mature leaves (ML), stems (S) and roots (R). Transcript levels were subsequently estimated by semi quantitative RT-PCR using gene-specific primers. RhSAND1, a stably expressed gene, was used as a control. Experiment was repeated twice.

Figure 6 .

 6 Figure 6. Characterization of the sucrose transport activity of RhSUC2 in baker's yeast Saccharomyces cerevisiae SUSY7. (a) Time course of sucrose uptake (pH 4.5, 0.5 mm sucrose). (b) pH dependence of sucrose uptake (0.5 mm sucrose). (c) Sucrose concentration-dependent sucrose uptake (pH 4.5). Bars represent standard errors (n = 3). (d) Lineweaver-Burk transformation of data presented in (c). Regression line is shown for data obtained at sucrose concentration below 2.5 mm. Kinetic parameters are Km = 2.99 mm and Vmax = 21.41 nmol sucrose mg -1 protein min -1 .

Figure 7 .

 7 Figure 7. Effect of auxin treatments on RhSUC2 transcript level in buds. Plants at visible flower bud (VFB) stage were used for treatments. Buds were grown in vitro in the presence of basic Murashige and Skoog media (MS) alone or added with 0.1, 1 or 10 mm of N(-1-naphtyl) acetic acid (NAA) (MS + NAA) during 72 h under light. Dormant buds before treatment served as control (C). Transcript levels were estimated by quantitative RT-PCR using gene-specific primers. Gene transcript level is expressed relatively to control and normalized to RhSAND1 transcript level. Each value is a mean of three independent biological replicates. The error bars represent standard errors. Asterisks indicate significant differences with the control (Student's t-test, P < 0.05).

Table 1 .

 1 Bud burst, length and leaf-like organ number before treatment, after 6 d of in vitro sugar feeding or plant beheading,

	under light
		Light
		Bud burst
	Treatment	percentage	Bud length (mm)	Number of leaf-like organ	Number of plants
	Before treatment	0	1.8 Ϯ 0.3 (a)	9.3 Ϯ 0.7 (a)	18
	Stem portions grown in vitro with:
	0mm sugar	19	3.3 Ϯ 1.4	12 Ϯ 0.6	16
	100 mm
	Mannitol	0	1.9 Ϯ 0.7 (a)	10 Ϯ 0.9 (a)	16
	Sorbitol	81	5.3 Ϯ 1.6 (b)	12 Ϯ 1.0	16
	Sucrose	89	10 Ϯ 3.4 (b)	13 Ϯ 0.9 (b)	18
	250 mm
	Mannitol	0	2.1 Ϯ 0.4 (a)	11 Ϯ 0.8	16
	Sorbitol	0	2.1 Ϯ 0.6 (a)	11 Ϯ 0.8	14
	Sucrose	100	9.5 Ϯ 1.4 (b)	15 Ϯ 0.7 (b)	16
	500 mm
	Mannitol	6	1.6 Ϯ 0.3 (a)	9.4 Ϯ 1.7 (a)	16
	Sorbitol	0	1.3 Ϯ 0.2 (a)	9.9 Ϯ 0.8 (a)	16
	Sucrose	75	5.1 Ϯ 2.7	13 Ϯ 2.0	16
	Beheaded plants	90	7.5 Ϯ 2.8 (b)	12 Ϯ 0.8	30

Plants before treatment are used as controls. Bud length and leaf-like organ values are means Ϯ standard errors of all measurements done for each condition. Letters indicate a significant difference lower (a) or higher (b) compared to buds of stem portions grown without sugars (P < 0.01, Student's t-test).

Table 2 .

 2 Bud sucrose accumulation after plant beheading and

	growth under light or darkness
		Total sucrose accumulation
	Treatment	(mBq per bud)
	Before beheading (control)	4.62 Ϯ 0.76 (a)
	Beheaded plants exposed
	96hto:
	Light	11.93 Ϯ 0.98 (b)
	Darkness	6.80 Ϯ 2.31 (a)
	Plants at visible flower bud (VFB) stage were beheaded, their
	leaves were removed and plants were treated under light or dark-
	ness for 96 h. Untreated plants were used as control before treat-
	ment. Buds were incubated 2 h in the presence of 1 mm [U-14C]
	sucrose (18.5 kBq mL -1 ). Letters indicate stastitical groups (Stu-
	dent's t-test, P < 0.05). Each value is a mean of three independent
	biological replicates Ϯ SE.

Table 3 .

 3 Effect of competing sugars and inhibitors on sucrose

	uptake by RhSUC2 expressed in baker's yeast SUSY7
	Treatment	% Activity
	Competing sugar
	Control	100
	25 mm Sucrose	21 Ϯ 6.6*
	25 mm Glucose	140 Ϯ 7.2*
	25 mm Fructose	157 Ϯ 4.4*
	25 mm Raffinose	103 Ϯ 3.9
	25 mm Maltose	43 Ϯ 2.2*
	Inhibitors
	Control	100
	50 mm CCCP	25 Ϯ 2.5*
	100 mm PCMBS	9 Ϯ 2.0*

* represents a significant difference between the control and the treatment (Student's t-test, P < 0.001).

ACKNOWLEDGMENTS

This research was in part funded by a PhD grant from the 'Ministère de l' Enseignement Supérieur et de la Recherche'. We thank Doris Rentsch for providing the yeast pDR296 plasmid. We thank S. Chalain, B. Porcheron, M. Laffaire, B. Dubuc, A. Lebrec and L. Hamama for their technical assistance. We are truly grateful to Pr Eric Davies (NC State University) for English revision of this manuscript.


L Plantier
email: lise.plantier@freescalecrolles.st.com

Y Le Friec

A Humbert

G Imbert

E Sabouret

M Sardo

Vivette Girault

D Delille

Sophie Jullian

K Junker

V Pantel

L Arnal

J Arnaud

Torres

Material and electrical characterization of TMS-based silicidation of the Cu-dielectric barrier interface for electromigration improvement of 65 nm interconnects

Keywords: CuSiN, Silicide, Electromigration, TMS

Introduction

The continuous dimensional scaling of interconnect technologies is associated with an increase of the signal propagation delay in the interconnects and a loss of intrinsic reliability. Indeed, for the latter, keeping the same current leads to higher current density through lines thus increasing electromigration phenomenon. The interface between copper and its overlying dielectric barrier becomes the primary contributor to EM degraded performances [START_REF] Lu | Proceedings of the IITC[END_REF][START_REF] Li | [END_REF][4]. Cu interconnect reliability can be improved by engineering interface composition to reduce surface diffusion of Cu.

Surface treatments have been employed to modify interfaces in an effort to increase electromigration performances [START_REF] Hatano | Proceedings of the IITC[END_REF][START_REF] Lloyd | Proceeding of the 2002 International Integrated Reliability Workshop[END_REF]. Copper silicide formation, obtained by means of a self-aligned process done prior to dielectric barrier deposition, has been demonstrated to enhance the EM lifetime [1]. In previous studies, silane has commonly been used as the silicon source for such a reaction. In this study, tri-methyl silane (TMS) based copper silicidation is evaluated as an alternative to the silane process [1]. Indeed, as TMS dissociation is more difficult to achieve due to the high energy of the carbon-silicon bond, less silicide is formed, limiting the resistance increase associated with the process [1].

In the current integrations, ultra low k (ULK) films are capped with a SiO 2 layer to protect the ULK film from the patterning steps and to prevent the plasma damage that can occur during the treatment prior to SiCN dielectric barrier deposition [START_REF] Humbert | [END_REF]. Removal of this layer could provide further capacitance reduction, since the capacitance of interconnects increases in the presence of the SiO 2 layer. In this study, the impact of the silicidation process on the ULK surface has been characterized.

Experimental details

Silicide formation was considered as a pre-treatment to dielectric barrier deposition. All the process steps were performed in a standard PECVD chamber using 300 mm silicon substrates. The silicide formation was accomplished using a three-step approach [1], as illustrated in Fig. 1. After copper surface cleaning by CuO x reduction in a NH 3 based plasma, the silicide phase is formed by adsorption of the Si-based precursor, TMS (SiH(CH 3 ) 3 ) at the Cu grain surface and boundaries. A subsequent plasma-based nitridation step may follow to stabilize the compound by forming a CuSiN-like phase. Dilution conditions of the nitridation step were varied according to the values listed in Table 1. The process temperature was kept constant at 335 °C.

The film stacks used for the unpatterned Cu and ULK (porous SiOC:H k = 2.5) substrates and C065 patterned wafers are shown in Fig. 2. The patterned stack has a typical 65 nm integration scheme with a SiOCH (k = 3.0) interlayer dielectric. SIMS, AFM and light scattering measurements were performed following the CuSiN formation. Some of the samples were then capped with 30 nm of SiCN, for adhesion measurements using the four-point bending technique. FT-IR spectroscopy and dielectric constant measurements were performed following the silicidation process on the ULK surface. Patterned wafers with copper silicided interfaces were evaluated and compared to standard processed samples by means of electrical characterization and electromigration performance assessment at the package level under down-stream applied stress current.

Results and discussion

Unpatterned wafer characterization

Surface roughness was seen to increase during the silicidation process using both AFM and optical microscopy. These techniques illustrate the differences within wafer process uniformity. Light scattering effects or ''haze'' mapping provide a complete map of the relative surface roughness, as seen in Fig. 3. A correlation with local AFM measurements (10 • 10 lm box) was established. One AFM example of central roughness measurement is given in Fig. 3a, additional measurements have been done correlated to difference of roughness between center and edge as seen with light scattering technique. The main advantage of light scattering in this study was the quick information regarding the overall relative roughness of the wafer surface.

Results show that the silicidation obtained with TMSbased chemistry followed by pure NH 3 nitridation is nonuniform, with a rough surface in the center of the wafer. TMS chemistry followed by NH 3 /N 2 nitridation shows a more uniform surface, with lower global roughness.

Nevertheless, the SIMS depth profile in Fig. 4 shows the formation of a Cu x Si y C z O-N like layer at the copper's top surface. The carbon-contamination originates from the use of a TMS precursor. The relative thickness of the layer was observed to vary by a factor of 3 from the edge to the center with a lack of Cu detection at the wafer edge. The 3 nm thick layer observed in the center is believed to be sufficient to achieve EM enhancements with minimal copper consumption and therefore limited resistance impact. Adhesion measurements were performed using a fourpoint bend technique, on samples with Si/TaN-Ta/Cu/ SiCN stacks such as the one described in Fig. 5. Strain energy release rate (G) results of several samples are presented in Table 2.

When silicidation is performed without any nitridation step, the adherence matches the reference value of strain energy release rate.

For the TMS process, whatever the silicide nitridation performed, results show a 30-50% lower adhesion compare to the reference process of a Cu-silicide treated interface without silicidation. However, when silicidation without the subsequent nitridation step is performed, the adhesion matches the reference value of strain energy release rate. The adhesion degradation with nitridation is likely associated with nitrogen and hydrogen penetration to the Cu/ CuSi x interface. Follow-up analysis will be used to study potential SiN formation at the interface and process optimization to control the N penetration. Although previous studies [8] indicate adhesion improvement with CuSi x formation, no such improvement was seen here relative to the reference. However, it is important to note that the reference include an NH 3 -based plasma treatment already optimized for adhesion.

The dielectric constant (k) measurements of the ULK substrate (Fig. 2c), including a standard reference, were performed using a mercury probe (see Table 3). Measurements were performed directly on ULK surface as capping SiO 2 layer removal is consider to provide global ILD capacitance reduction. All the processes strongly degrade k, especially the NH 3 /N 2 nitridation process. In this latter case, the surface leakage is so high that capacitance measurement is not able to provide any values. When the CuSiN pure NH 3 nitridation process is used, helium pre-treatment seems to limit the nitridation's impact on the ULK surface.

Chemical bonding information was provided by FT-IR spectroscopy. The main change was observed for the 3000-3500 cm À1 wavelength region as shown in Fig. 6. Indeed, intensity of Si-OH and H-OH links increases when CuSiN processes are applied. Nevertheless, there is a difference between CuSiN process with pure NH 3 and with NH 3 /N 2 nitridation. After both processes, there is a transformation from a hydrophobic surface (standard ULK) to a hydrophilic one but with a higher susceptibility to moisture absorption in the case of CuSiN process with NH 3 /N 2 nitridation. He pre-treatment decreases the hydrophilic nature of the surface but is not sufficient to prevent a dielectric constant shift. The difference of peak intensity could be related to the nitridation plasma used during CuSiN formation. NH 3 partial pressure is higher for pure NH 3 nitridation. The content of NH 3 (3%) provided during the NH 3 /N 2 nitridation might not be sufficient to inhibit the hydrophilic nature of the surface.

Electrical characterization of patterned wafers

Line resistance and current leakage values were evaluated (Fig. 7). The TMS process with pure NH 3 nitridation considerably degrades the electrical performance of the circuit, by the formation of a leakage path along the upper dielectric surface. An example can be seen in TEM image of Fig. 8 in which a clear residue is observed on the dielectric surface at metal level where CuSiN has been formed. This effect is not visible at the other standard metal levels. Moreover, the edge-center effect observed on unpatterned physical characterizations has been confirmed by a wafer mapping of the chips, showing high leakage results in the center compared to the acceptable leakage results for the edge chips.

The TMS process with NH 3 /N 2 nitridation conserves the electrical properties of the interconnects. No improvements of these properties were expected as the CuSiN formed has higher resistivity compared to copper. Indeed, EM reliability demonstrates the potential improvement brought by the TMS-based silicidation plus NH 3 /N 2 nitridation compared to the standard SiCN deposition process: 40% longer failure times are observed, if one takes into account the late failures of each distribution only (standard, TMS-based, see Fig. 9). Other reports [START_REF] Dumont-Girard | Proceedings of the IITC[END_REF] have indicated MTF increase of two times with silicidation treatments. The more modest improvements described in this report may be attributable to a combination of the following: (a) structures using 65 nm node design rules have increased via sensitivity as compared to previously published results on 90 nm based structures thereby diminishing the surface treatment impact, and (b) silicidation treatment process designed to minimize resistance increase with tradeoffs to interface improvement. The EM data points were collected for 0.10 lm wide lines tested at package level under accelerated conditions corresponding to a temperature of 300 °C and a current density of 1.5 MA/cm 2 . The failure criterion was set to 10% of the resistance increase. The early failures were associated with void excursions in the Cu metallization. It is important to note that although comparable adhesion was observed for the two splits, MTF showed a difference. This calls into question the proposed correlation between MTF and adhesion put forth by Lloyd [START_REF] Lloyd | Proceeding of the 2002 International Integrated Reliability Workshop[END_REF]. However it is important to note that the accuracy of the four-point bend test is limited in the extreme of ''good'' adhesion (>20 J/ m 2 ) due to a mixture of interfacial failure modes and fracture in the epoxy.

Conclusion

A process optimization has been completed to improve the copper dielectric interface by forming of a CuSiN layer between copper and dielectric. This layer is a TMS-based copper silicidation providing the advantages of improved process control and Si depth penetration as compared to standard silane sources [1]. The nitridation step was identified as a key predictor of electrical performance, since pure NH 3 -based nitridation leads to leakage pathways on the dielectric surface. Furthermore, an optimized regime was identified on patterned structures to minimize the impact on line resistance and current leakage while achieving the first improvement in terms of electromigration robustness. However, plasma treatment studies performed on ULK films have shown deterioration of the material integrity. Multi-step plasma treatments are required to create a protective skin on the ULK prior to the CuSiN treatment, in order to achieve the electromigration benefits without a capacitance increase.

Fig. 1 .

 1 Fig. 1. CuSiN formation process [1].

Fig. 2 .

 2 Fig. 2. Stack descriptions for (a) patterned and unpatterned wafers generated for experiment, (b) with Cu substrate and (c) ULK substrate.

Fig. 3 .Fig. 4 .Fig. 5 .

 345 Fig. 3. (a) AFM surface roughness, (b) light scattering mapping, for TMS with pure NH 3 nitridation process and (c) light scattering mapping, for TMS with NH 3 /N 2 nitridation process.

Fig. 6 .

 6 Fig.6. FT-IR spectrum of all the conditions tested. Si-OH Peak[9].

Fig. 7 .Fig. 8 .

 78 Fig. 7. Leakage current results. Y-axis is cumulative percentage.

Table 2

 2 Values of strain energy release rate G (J/m 2 ) at Cu/cap interface; average wafer values

	Treatment TMS pure	TMS	(No CuSiN)	TMS
		NH 3		NH 3 /N 2	standard NH 3 /N 2	silicidation
		nitridation	nitridation	treatment + SiCN	only + SiCN
		+ SiCN		+ SiCN
	G (J/m 2 )	17			13	26	23
	Avg
	G st. dev.	7			4	13	12
	Table 3
	Treatments were applied on a ULK substrate
	After		No	CuSiN	CuSiN	He + CuSiN
	treatment on	treat.	process with	process with	process with pure
	ULK			NH 3 /N 2	pure NH 3	NH 3 nitridation
	substrate			nitridation	nitridation
	k value at the	2.44 Non-measurable	4.57	4.01
	center			(excessive surface
				leakage)
	k values were obtained using a Hg probe (S = 0.0214 cm 2 ).