Arkadiusz Frąckowiak

Boleslaw Barszcz

Iwona Olejniczak

Mikolaj Tomasik

Natalia Jarzyniak

Roman Swietlik
email: swietlik@ifmpan.poznan.pl

Pascale Auban-Senzier

Marc Fourmigué

Olivier Jeannin

Franck Camerel
email: franck.camerel@univ-rennes1.fr

Bolesław Barszcz

Mikołaj Tomasik

Roman Świetlik

Mixed valence

may come

Introduction

Most molecular conductors based on cation radical salts are built from the association in the crystal of partially oxidized tetrathiafulvalene (TTF) derivatives (TMTTF, TMTSF, BEDT-TTF, …) and small inorganic counter ions such as Br -, PF 6 -, I 3 -,… or more complex metallate anions such as AuI 2 -, Cu(NCS) 2 -or Cu[N(CN) 2 ]Br -… Besides such inorganic counter ions, organic anions such as carboxylates or sulfonates have been also successfully investigated in conducting cation radical salts with TTF derivatives. 1 One can mention aliphatic sulfonates such as F 3 C-SO 3 -, 2,3 or F 5 SCH 2 CF 2 SO 3 -, 1,4 aromatic sulfonates (C 6 F 5 -SO 3 -, C 6 H 5 -CH 2 -SO 3 -, 5 HO 2 C-p-C 6 H 4 -SO 3 -, 6 N,N'-disulfo-1,4-benzoquinonediimine, 7 1,1'-ferrocene-disulfonate), 8 or sulfonate anions substituted with aminoxyl radical species. [9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26] Aromatic disulfonate salts (Scheme 1) were also used for that purpose, involving 1,5naphtalenedisulfonate, 5 2,6-naphthalene-and 2,6-anthracenedisulfonate (Scheme 1). 27 One interesting peculiarity of these conducting TTF salts with sulfonate anions is the recurrent presence of C-H•••O 3 S hydrogen bond interactions between the TTF hydrogen atoms (activated upon TTF oxidation) 28 and the negatively charged oxygen atoms of the sulfonate moiety. Such hydrogen bonds involving carbon-bonded hydrogen atoms, albeit weak, have been shown to play a crucial role in the organic solid state, 29 and particularly in molecular conductors. 28, 30-32 We have for example shown that the BEDT-TTF salt with 2,6-anthracene-bis(sulfonate) exhibits a metal-insulator transition at T MI = 85 K which finds its origin in minute modifications of such C-H•••O interactions, leading to a charge ordering process and gap opening. 27 Sulfonate anions can also act as halogen bond (rather than hydrogen bond) acceptors and cation radical salts with iodinated tetrathiafulvalenes were also reported, exhibiting strong C TTF -I•••O 3 S-R halogen bonds. 33 In the course of our investigations of such complex sulfonate anions, [35][36][37][38][39] we developed recently a platinum(II) chelate of 5-sulfo-8-quinolinol, [Pt(qS) 2 ] 2-, which can be associated through the socalled ionic self-assembly (ISA) approach, with various ammonium cations such as (H 2n+1 C n ) 2 Me 2 N + (n = 12, 16, 18) to afford phosphorescent crystalline or liquid crystalline phases. 40 Its availability prompted us to use this planar bis-sulfonate as counter ion in cation radical salts with TTF derivatives. We describe here two phases we obtained concomitantly in the presence of TMTTF, with different degrees of charge transfer, as demonstrated here from combined structural and spectroscopic (IR, Raman) investigations.

Experimental

Materials. The phosphonium salt, [PPh 4 ] 2 [[Pt(qS) 2 ] as well as the potassium and the sodium derivatives were prepared as previously reported. 27 TMTTF was prepared as previously described. 41 1,1,2trichloroethane (TCE) was purchased form TCI Europe and used as received.

Electrocrystallization. Two-compartment electro-crystallization cells, equipped with Pt wires (diameter 1 mm, length 2 cm), were filled with TMTTF (10 mg) in the anodic compartment and a solution of [PPh 4 ] 2 [[Pt(qS) Crystallography. X-ray crystal structure collections were performed on an APEXII Bruker-AXS diffractometer equipped with a CCD camera and a graphite-monochromated Mo-Kα radiation source (λ = 0.71073 Å) at 150 K. Details of the structural analyses are summarized in Table 1. Absorption corrections were performed with SADABS. Structures were solved by direct methods using the SIR97 program, 42 and then refined with full-matrix least-squares methods based on F 2 (SHELXL-97) 43 with the aid of the WINGX program . 44 All non-hydrogen atoms were refined with anisotropic atomic displacement parameters. H atoms were finally included in their calculated positions. Crystallographic data were deposited as CCDC 1997195 (Phase A) and 1997196 (Phase B).

Resistivity measurements. Gold contacts were evaporated on the single crystals and gold wires (17 μm diameter) were glued with silver paste on those contacts. Because of very high resistance values, two-probe DC measurements were performed under vacuum, applying a constant voltage of 0.2 V and measuring the current using a Keithley 6487 Picoammeter / Voltage Source. Low temperature was provided by a home-made cryostat equipped with a 4 K pulsetube using a cernox in good thermal contact with the samples as thermometer.

Infrared and Raman spectroscopy. To assign vibrational bands of [Pt(qS) 2 ] 2-anions, we have performed IR and Raman spectra of two salts [Pt(qS) 2 ]K 2 and [Pt(qS) 2 ]Na 2 (Cf Figures S3-S4 and Table S1 in ESI). Their Raman spectra were measured for powders using green light (λ=514.5 nm) from an argon-ion laser and IR spectra were recorded for powders dispersed in KBr pellets. Additionally, quantum chemical calculations of normal modes of [Pt(qS) 2 ] 2- anions, were performed with Gaussian 03 program (Gaussian, Inc., Pittsburgh, PA). 45 The equilibrium geometry with no symmetry constrains and harmonic vibrational transitions were evaluated with the B3LYP density functional theory using a LanL2DZ basis set. Then, we calculated the IR and Raman scattering intensities and modelled respective vibrational spectra of [Pt(qS) 2 ] 2-anions.

Room temperature Raman spectra of (TMTTF) 3 [Pt(qS) 2 ] crystals were measured in a backward scattering geometry using Labram HR Horiba 800 Jobin Yvon with a He-Ne laser (λ = 632.8 nm) and an argon-ion laser (λ=514.5 nm). The exciting laser beam was polarized but the polarization of scattered light was not studied. The spectra were recorded with the spectral resolution of 2 cm -1 . To avoid sample overheating, the power of the laser beam was reduced down to about 0.05-0.1 mW. Polarized reflectance measurements of (TMTTF) 3 [Pt(qS) 2 ] single crystals were performed in the frequency range from 700 to 12000 cm -1 using Fourier transform FT-IR Bruker Equinox 55 spectrometer equipped with a Bruker microscope Hyperion 2000. The optical conductivity spectra were determined by Kramers-Kronig analysis of the reflectance data. For low temperature IR measurements (down to T = 50 K), the samples were mounted in the Oxford Instruments continuous flow helium cryostat.

Results

Crystal structures

The electrocrystallization of TMTTF was performed in 1,1,2trichloroethane (TCE), in the presence of the tretraphenylphosphonium salt of [Pt(qS) 2 ] 2-, (Ph 4 P) 2 [Pt(qS) 2 ] as electrolyte. 40 It afforded simultaneously on the anode crystals with two different habits, named hereafter as Phase A and Phase B. Phase A crystallizes in the triclinic system, space group P-1, with one TMTTF and one solvent molecule (1,1,2-trichloroethane) in general position and one [Pt(qS) 2 ] 2-complex on inversion centre, hence a (TMTTF) 2 [Pt(qS) 2 ](TCE) 2 stoichiometry, implying that each TMTTF molecule is in its monocationic state, TMTTF +• . This is also confirmed by the intramolecular bond lengths within the TTF core (Table 2), with a particularly long central C=C bond at 1.395(4) Å. As shown in Figure 1, the TMTTF +• cations associate into face-toface, almost eclipsed dicationic dyads, alternating with the [Pt(qS) 2 ] 2-complex along the (a-b) direction. We note also the presence of a short Cl

H 20 •••O 4 : 2.247(2) Å, C 20 -H 20 •••O 4 : 150.3(2)°; H 9C •••O 2 : 2.361(2) Å, C 9 -H 9C •••O 2 : 174.4(2)°; H 10A •••O 2 : 2.470(2) Å, C 10 -H 10A •••O 2 : 167.2(2)°.
Phase B crystallizes in the monoclinic system, space group C2/m, with the platinum complex on a 2/m site, lying flat on the mirror plane, one TMTTF on another 2/m site, but with the mirror plane perpendicular to the molecule and along its long axis and noted Mol. 1, and a second TMTTF molecule on a mirror plane, also perpendicular to the molecule and along its long axis, noted Mol.

B. H 3A •••O 2 : 2.400 (3) Å, C 3 -H 3A •••O 2 : 156.9(2)°, H 9C •••O 3 : 2.405(2) Å, C 9 -H 9C •••O 3 : 148.2(2)°.
The presence in Phase B of trimeric dicationic (TMTTF) 3 2+ moieties raises questions about the actual charge distribution within the three TMTTF molecules. Only a few TMTTF salts have been reported in the literature, which exhibit the same trimeric motifs with the charge +2. They are obtained upon association with dianionic counter ions, such as [Ti 2 F 10 ] 2-50 or [Re 2 Cl 8 ] 2-. 51 In both salts, the charge is distributed non-uniformly among TMTTF molecules: for the Ti 2 F 10 salt (+0.5, +1, +0.5) and the Re 2 Cl 8 salt (0, +1, +1). This charge distribution within the trimer can be evaluated in a more quantitative way by using the linear relationship between the charge and a structural parameter δ defined as δ = (b+c) -(a+d) where a-d are intramolecular bond lengths (see Table 2). Such correlations were already established for BEDT-TTF, TTF and TMTSF, 52 but not TMTTF. Using the reference compounds collected in Table 2 (See Figure S2 in ESI), we found a linear relation which writes as ρ = A -Table 2. Averaged intramolecular bond lengths in the TTF core in the two salts and in reference compounds. The δ parameter used to estimate the TMTTF charge (ρ calcd ) is defined as (b+c)-(a+d) where a-d are averaged intramolecular distances (see text for details).

B × δ with A = 6.3382 and B = 7.5911. As shown in Table 2, the inversion centred Mol. 1 is found indeed with a calculated charge close to +1 (1.08) while the charge of the outer Mol. 2 is close to +0.5 (0.39) and the sum for the three molecules (1.08 + 2 × 0.39 = 1.86) close to the +2 charge imposed by the stoichiometry. To address this charge distribution in more details, we have performed extensive IR and Raman investigations of the trimeric phase, as reported below.

Raman spectra of (TMTTF) 3 [Pt(qS) 2 ]

Room temperature Raman spectra of the salt (TMTTF) 3 [Pt(qS) 2 ] 2 for two perpendicular polarizations of the exciting laser beam (λ = 514.5 nm), i.e. parallel and perpendicular to the stacking c-axis, are shown in Figure 5. The observed vibrational features of the TMTTF molecules in (TMTTF) 3 [Pt(qS) 2 ] 2 have been assigned with the help of experimental and calculated IR and Raman spectra of the [Pt(qS) 2 ] 2-dianion (Figures S3,S4, and Table S1 in ESI) since the spectra are dominated by vibrational bands related to [Pt(qS) 2 ] 2-anions. Additionally, in the spectrum some bands attributed to TMTTF molecules can be clearly identified. The observed vibrational bands together with proposed assignments are collected in Table 3.

To determine the charge distribution among molecules in the TMTTF trimers, we focused our attention on vibrational features observed in the frequency ranges of C=C stretching and C-S ring breathing vibrations since these modes are most sensitive to the charge residing on donor molecules. The neutral TMTTF 0 has two Raman active C=C stretching modes ν 3 (a g ) = 639 cm -1 and ν 4 (a g ) =1538 cm -1 and one IR active mode ν 28 (b 1u ) = 1627 cm -1 . 53,54 When the ionization degree increases, the positions of these modes shift linearly towards lower wavenumbers: for the fully ionized TMTTF + molecule the shift equals to 72 cm -1 for ν 3 (a g ), 120 cm -1 for ν 4 (a g ), and 80 cm -1 for ν 28 (b 1u ). However, in Raman spectra only the ν 3 (a g ) mode can be used for determination of the charge density since the ν 4 (a g ) mode is nearly independent on charge due to strong coupling with electrons in TMTTF stacks (or clusters). 55 Recently, it has been shown that two C-S stretching ring breathing modes, i.e. symmetric ν 10 (a g ) = 494 cm -1 and antisymmetric ν 35 (a g ) = 439 cm -1 (for TMTTF 0 ), can be also useful for determination of the charge distribution in TMTTF stacks. 56 On the ionization of TMTTF form 0 to +1 the ν 10 (a g ) mode shifts linearly by 29 cm -1 towards higher wavenumbers.

The Raman spectra of (TMTTF) 3 [Pt(qS) 2 ] for polarization E ┴ c within the frequency region 1200 -1700 cm -1 are displayed in 1244, 1328 (1333), 1374, 1411 (1413), 1464, and 1500 cm -1 , one can also distinguish two bands at 1453 and 1591 cm -1 that we assign to the C=C stretching modes of TMTTF ν 4 (a g ) and ν 3 (a g ), respectively. Assuming the liner dependence of band position against the charge on molecule, the band wavenumber ν 3 (a g ) = 1591 cm -1 corresponds to the charge +0.66, 54 however this value is probably not very reliable due to strong overlap with a band of [Pt(qS) 2 ] 2-. If the charge distribution inside TMTTF trimers is non-uniform, the ν 3 (a g ) mode should be split but an eventual splitting is probably hidden in the broad vibrational feature centered at 1591 cm -1 . On the other hand, the band position ν 4 (a g ) = 1453 cm -1 corresponds to the charge +0.71. This value is to be expected considering that, due to strong coupling with electrons, the band provides information about an average charge density in TMTTF trimer. The band at 1385 cm -1 is attributed to the ν 6 (a g ) mode (symmetrical vibration of CH 3 groups). More reliable information about the charge distribution can be obtained from the analysis of the ring breathing ν 10 (a g ) mode.

In

a (Å) b (Å) c (Å) d (Å) δ param. ρ cryst ρ calcd Ref.
(TMTTF)(ClO 4 ) 2

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the frequency range 280 -700 cm -1 (Figure 6b) we identify three bands at 507, 514, 520 cm -1 assigned to the ν 10 (a g ) mode of TMTTF molecules with various charges (see also Figure 7) and a band at 562 cm -1 attributed to the ν 9 (a g ) mode (S-C-CH 3 stretching). 53,54 The position of the ν 10 (a g ) mode also depends linearly on charge, therefore the three ν 10 (a g ) peaks correspond to TMTTF molecules with charges +0.45, +0.69, +0.90, respectively, 56 which is consistent with the initial assignment, based on the crystallographic investigations, of +0.5, +1, +0.5 within the trimer. Additionally, one can find bands assigned to the dianion at 308, 467 and 659 cm -1 which show a considerable intensity enhancement for the excitation λ = 514.5 nm. Room temperature Raman spectra of the salt (TMTTF) 3 [Pt(qS) 2 ] within the region of C=C (a) and stretching (b) modes for the green (λ = 514.5 nm) and red (λ = 632.8 nm) excitations. The electrical vector of the laser beams is perpendicular to the c-axis.

Infrared reflectance of (TMTTF) 3 [Pt(qS) 2 ]

Figure 8 presents the room temperature reflectance spectra of (TMTTF) 3 [Pt(qS) 2 ], recorded for two perpendicular polarizations of the incident IR beam, and corresponding optical conductivity spectra determined by Kramers-Kronig analysis. The optical response is one-dimensional in agreement with interactions calculated on basis of the crystal structure. For polarization parallel to TMTTF stacks E║c the spectrum displays a broad electronic absorption (centred at about 3750 cm -1 ) associated with intra-trimer charge-transfer excitations. Below 1700 cm -1 , the spectra for both polarizations show many vibrational features related with both the donor and acceptor modes (Table 3). The strongest bands for polarization E║c are a consequence of coupling of the totally symmetric a g modes of TMTTF with charge transfer transitions.

In order to shed more light on both vibrational and electronic features of the (TMTTF) 3 [Pt(qS) 2 ] spectra and eventual phase transitions, polarized reflectance measurements as a function of temperature have been performed. Temperature dependence of the IR reflectance and the optical conductivity spectra for polarization E ║ c are shown in Figure S5a in ESI and Figure 9, respectively. On temperature decreasing the charge-transfer band slightly shifts towards higher wavenumbers and it is better seen that this band consists of two components at least. At T = 50 K, one can distinguish two bands centred at 3950 and 4650 cm -1 which should be related to intra-trimer charge-transfer transitions. Since interactions between

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The vibrational spectra for polarization E ║ c are dominated by a strong and broad vibrational feature between 1300 and 1420 cm -1 which is a result of electron-molecular vibration (EMV) coupling of the ν 4 (a g ) mode (Figure 10 and Figure S5b in ESI). The apparent doublet structure of this band is a consequence of the strong antiresonance centred at 1370 cm -1 related to the ν 6 (a g ) mode (symmetrical vibration of CH 3 groups). Such antiresonance superimposed on the C=C vibronic feature is common not only for TMTTF salts but also for salts formed by other TTF derivatives. The

- - 1639 - 1627 - - - 1538 - - - - - - - - - - 1183 - - - 1092 - - 934 935 - - - - - 560 - - 494 - - - - - - - 1567 - 1547 - - - 1418 - - - - - - - - - - 1185 - - - 1104 - - 941 935 - - - - - 566 - - 523 - - - - - ? [Pt(qS) 2 ] 2- ν 3 (a g ) and [Pt(qS) 2 ] 2- ν 28 (b 1u ), q=+0.84 ν 28 (b 1u ), q=+0.64 ν 28 (b 1u ), q=+0.54 [Pt(qS) 2 ] 2- [Pt(qS) 2 ] 2- ν 4 (a g ) [Pt(qS) 2 ] 2- [Pt(qS) 2 ] 2- ν 6 (a g ) dip IR [Pt(qS) 2 ] 2- [Pt(qS) 2 ] 2- [Pt(qS) 2 ] 2- [Pt(qS) 2 ] 2- [Pt(qS) 2 ] 2- [Pt(qS) 2 ] 2- [Pt(qS) 2 ] 2- ν 48 (b 2u ) [Pt(qS) 2 ] 2- [Pt(qS) 2 ] 2- [Pt(qS) 2 ] 2- ν 7 (a g ) [Pt(qS) 2 ] 2- [Pt(qS) 2 ] 2- ν 8 (a g ) ν 32 (b 1u ) ? [Pt(qS) 2 ] 2- [Pt(qS) 2 ] 2- [Pt(qS) 2 ] 2- [Pt(qS) 2 ] 2- ν 9 (a g ) [Pt(qS) 2 ]
2ν 10 (a g ), q=+0.90 ν 10 (a g ), q=+0.69 ν 10 (a g ), q=+0.45 2-modes is given in ESI a excitation λ = 633.8 nm b doublet: the band 1385 cm -1 is more intense for excitation 457 nm and the band 1374 cm -1 for excitation 514.5 nm

[Pt(qS) 2 ] 2- [Pt(qS) 2 ] 2- [Pt(qS) 2 ] 2- [Pt(qS) 2 ] 2- * Ref.

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c observed for excitation λ = 457 nm effect of EMV coupling of the ν 3 (a g ) mode is seen as an antiresonance dip at 1597 cm -1 , similarly as in other TMTTF salts. 54,57 This dip is not useful for precise determination of the charge density on TMTTF because due to EMV coupling its position does not exhibit linear dependence on charge. Moreover, in the case of (TMTTF) 3 [Pt(qS) 2 ], the dip can be distorted by the neighbouring band at 1584 cm -1 assigned to the dianion. Three weak bands at 1584, 1576 and 1551 cm -1 are related to the IR active antisymmetric C=C stretching ν 28 (b 1u ) mode; these bands correspond to TMTTF molecules with charges +0.54, +0.64 and +0.84, respectively. It is commonly assumed that the ν 28 (b 1u ) mode is the best mode for evaluation of the charge density on TMTTF since it is not coupled with electrons. 54 However, for most of TMTTF salts, its observation is rather difficult because of polarization along long molecular axis which is usually nearly perpendicular to the best developed crystal face. Nevertheless, the specific arrangement of TMTTF molecules in (TMTTF) 3 [Pt(qS) 2 ] against the crystal face (010) allows detection of the ν 28 (b 1u ) peaks not only for E ║ c but also for E ┴ c, though their intensities are weak (Figure 11 and Figure S6 in ESI). All the vibrational bands observed in IR spectra of the (TMTTF) 3 [Pt(qS) 2 ] salt together with their proposed assignments are listed in Table 3. On temperature decreasing the intensity of both charge-transfer and vibrational features increases gradually without any indication of phase transition or any visible modification of charge density distribution in TMTTF trimers. The only important change is observed for components of the EMV-activated ν 8 (a g ) mode that is the totally symmetric vibration of CH 3 groups. 53,54 As seen in Figure 12, at ambient temperature, one can distinguish a doublet structure related to the ν 8 (a g ) mode at 928 and 950 cm -1 . On closer examination one can also distinguish a band at about 920 cm -1 , nevertheless its weak temperature dependence indicates that it is not related with the EMV coupling effect, i.e. it cannot be attributed to the ν 8 (a g ) mode. On cooling the higher frequency component splits, so that at T = 50 K a triplet at 926, 945 and 956 cm -1 is observed. The ν 8 (a g ) mode is involved -as results from crystal structure datain hydrogen bonding with anions. These hydrogen bonds are surely responsible for the doublet structure at ambient temperature and then, when the crystal contracts by temperature lowering, small changes of these bonds yield the additional splitting.

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Discussion of IR and Raman spectra

In general, charge-transfer (CT) salts containing trimers of electron acceptors (or donors) with two unpaired electrons (or holes) are scarce. The most extensively studied was the salt Cs 2 (TCNQ) 3 (where TCNQ is the electron acceptor tetracyano-pquinodimethane). 58 In this 2:3 salt the TCNQ molecules form strongly trimerized one-dimensional stacks, nevertheless the centrosymmetric trimers cannot be considered as well-isolated. 59 Similar trimerized TCNQ stacks were also discovered in the salt MT 2 (TCNQ) 3 ˑH 2 O (where MT is S-methyl thiouronium). 60 Recently, a new charge-transfer salt ([4-damp]) 2 [Cl 4 Q] 3 (4-damp = 4-dimethylamino-N-methylpyridinium and Cl 4 Q = tetrachloroquinone) with well-isolated centrosymmetric trimer dianions (Cl 4 Q) 3

2-was described. 61 It is important to note that in these compounds the electron density in trimers is not uniformly distributed among molecules. In Cs 2 (TCNQ) 3 the charges are -0.14e and -0.93e on centric and non-centric molecules, respectively. 62 On the other hand, as estimated by analysis of TCNQ bond lengths, in MT 2 (TCNQ) 3 ˑH 2 O the charge on the centric (-0.9) is greater than the charge on non-centric (-0.4) molecules. 63 Similarly, in ([4damp]) 2 [Cl 4 Q] 3 the charge in trimers is distributed according to the pattern -0.59/-0.76/-0.59 (close to the -2 charge imposed by the stoichiometry). 61 In TTF salts with metal chloride dianions, described by the formula (TTF) 14 (MCl 4 ) 4 (M = Co, Mn, Zn), two sets of orthogonal TTF trimers with two holes exist in the monoclinic unit cell: two centric trimers are perpendicular to the other two non-centric ones -they alternate in both crystallographic directions (the charge distribution in trimers was not determined in these materials). 64 There are also two above mentioned 3:1 salts of TMTTF with the dianions [Ti 2 F 10 ] 2-and [Re 2 Cl 8 ] 2-, with doubly charged TMTTF trimers and non-uniform charge distribution. 50,51 The optical properties of trimerized salts were extensively studied both experimentally and theoretically. Though, the first estimation of charge distribution comes from analysis of the bond lengths of donors (or acceptors), more accurate results can be obtained by analysis of the charge sensitive vibrational bands observed in Raman and IR spectra. The analysis of intra-trimer and eventually in some cases also inter-trimer CT excitations allows for evaluation of the electron interactions (Hubbard parameters U and V) and transfer integrals. The vibrational part of IR spectra is dominated by bands activated as a result of EMV-coupling. 58 The theory of optical properties of a quasi-isolated trimer with uniform charge distribution was developed by Yartsev and applied for IR spectra of Cs 2 (TCNQ) 3 . 65 In the linear approximation, the electrons couple with the totally symmetric a g modes of TCNQ molecules. The EMV coupling was described by introducing the internal trimeric modes of vibration. There exist three trimeric modes. Two of the modes are Raman active: the mode corresponding to in-phase vibrations of a g modes of three monomers and the mode corresponding to in-phase vibrations of the non-centric molecules together with out-of-phase vibration of the centric molecule. The third trimeric mode is IR active: the non-centric molecules perform out-of-phase a g vibrations. Subsequently, to model the influence of counter-ions, Yartsev extended the theory to an arbitrary trimer with non-uniform charge distribution showing that, in this case, the EMV features should exhibit a doublet structure. 66 On the other hand, Painelli et al. analysed the spectra of Cs 2 (TCNQ) 3 in terms of a model based on vibronic Mulliken theory by considering both intraand inter-trimer CT excitations and assuming that both excitations linearly couple with the totally symmetric a g modes of TCNQ molecule. 62 An important result of these considerations is that the EMV coupling is responsible for perturbation of the Raman active a g modes (frequency shift). The two CT transitions have also influence on the vibronic IR modes, i.e. the vibronic absorptions are more shifted towards lower frequencies and their intensities are smaller in comparison with the analogous effects expected for one intra-trimer transition only. 62 The salt (TMTTF) 3 [Pt(qS) 2 ] provides an opportunity for extending the above-mentioned studies for isolated TMTTF trimers with two charges. Our spectroscopic data confirm the results of Xray studies that the charge is not distributed uniformly among molecules in trimers. As seen in Table 4, there exist a small difference between the charge distribution determined from the ν 10 (a g ) and ν 28 (b 1u ) modes observed in Raman and IR spectra, respectively. Probably more reliable is ν 28 (b 1u ) since this mode is more sensitive to the charge density. On the other hand, the totally symmetrical ν 10 (a g ) mode can be less reliable due to possible influence of the EMV-coupling effect. Generally, the charge distribution in conducting stacks is dependent not only on intratrimer electronic interactions but also on the interactions with the acceptor. The anions exert Coulomb interactions over the cloud of holes in stacks formed by TMTTF molecules. Moreover, as discussed in Introduction, the cloud of holes can be also distorted by the donor-acceptor hydrogen bonding. 28,[30][31][32] These interactions with [Pt(qS) 2 ] 2-, but first of all the hydrogen bonding, should be directly responsible for the non-uniform charge distribution in trimers.

The TMTTF trimers are well isolated from each other, therefore only intra-trimer CT excitations are important for analysis of spectroscopic data. In this case the Raman active a g modes are not perturbed by inter-trimer CT and their positions well correspond to the charge density on molecules. In IR spectra, the a g modes are activated by EMV coupling which has to be considered within the model of vibrations of TMTTF trimers (trimeric modes). As emphasized above, only one of the three trimeric modes is IR active (out-of-phase oscillations of non-centric molecules). However, according to the theory of EMV coupling in the trimer with nonuniform charge distribution, 66 the IR bands should exhibit a doublet (or even triplet) structure. Such doublet structure is only clearly observed for the ν 8 (a g ) mode and below about 200 K the doublet transforms into triplet (Figure 12). A small modification of the hydrogen bonding interactions between donor and acceptor is responsible for this change. It is possible that the strong band related to the C=C stretching ν 4 (a g ) shows also multiplet structure (Figure 10) but, due to huge deformation by anti-resonance, it is difficult to confirm. This deformation makes also difficult to model this vibronic feature with the trimeric model of EMV coupling.

Conclusions

Electrocrystallization of TMTTF in the presence of the dianionic [Pt(qS) 2 ] 2-platinum complex afforded simultaneously two phases. Phase A with isolated dicationic (TMTTF) 2 2+ species is insulating and characterized by C-H hydrogen bonds with the anionic sulfonate moieties. Phase B is a mixed valence salt formulated as (TMTTF) 3 [Pt(qS) 2 ], organized into strongly trimerized TMTTF chains, with a notably higher conductivity (σ RT = 4 ×10 -5 S cm -1 ). Charge distribution within the TMTTF trimers was evaluated from the intramolecular bond distances and IR and Raman spectra. The three methods showed a similar charge distribution, described by +0.5/+1/+0.5 motif, which is not modified at lower temperatures. Small differences between the charges estimated from IR and Raman data can be associated with various charge sensitivity of modes selected for charge evaluation and/or a small contribution of the EMV coupling. While spectroscopic studies of the EMV coupling phenomena in trimers were essentially concentrated on salts of radical anions of TCNQ, the trimeric structure found here in (TMTTF) 3 [Pt(qS) 2 ] provides an opportunity for extending these studies to radical cation salts. In that respect, the doublet (or triplet) structure expected in the IR spectra of such trimers with non-uniform charge distribution is clearly identified in the EMV-activated ν 8 (a g ) mode associated with the totally symmetric vibration of methyl groups. The doublet structure is related with weak C-H•••O hydrogen bonds between radical cation and the dianionic [Pt(qS) 2 ] 2- platinum complex which is responsible for non-uniform charge density distribution inside TMTTF trimer. The transformation of the doublet into triplet structure below about 200 K gives evidence of small modification of the donor-acceptor hydrogen bonding interactions.

Scheme 1 .

 1 Scheme 1. Aromatic disulfonate derivatives used as counter ions in conducting salts.

1 .Figure 1 .

 11 Figure 1. Phase A: (a) Overlap between TMTTF +• radical cations; (b) detail of one (a,b) plane. Hydrogen atoms and TCE molecules have been omitted for clarity.

Figure 2 .

 2 Figure 2. C-H hydrogen bonds with the anionic sulfonate moieties in Phase A. H 20 •••O 4 : 2.247(2) Å, C 20 -H 20 •••O 4 : 150.3(2)°; H 9C •••O 2 : 2.361(2) Å, C 9 -H 9C •••O 2 : 174.4(2)°; H 10A •••O 2 : 2.470(2) Å, C 10 -H 10A •••O 2 : 167.2(2)°.

  2. Altogether, it corresponds to a 0.25 [Pt(qS) 2 ], 0.25 Mol.1 and 0.5 Mol.2 moieties in the asymmetric unit, that is a (TMTTF) 3 [Pt(qS) 2 ] stoichiometry, with three TMTFF molecules for one dianion. In the structure, the TMTTF are organized into trimers, with inversioncentred Mol. 1 sandwiched between two Mol. 2. These trimers stack along the c axis while they are fully isolated from each other in the (a,b) plane. Comparison of the intramolecular bond lengths within the TTF core in Mol. 1 and Mol. 2 (Table 2) tends to indicate that the central, inversion centred Mol. 1 bears a tentative +1 charge while the charge of the outer Mol. 2 is close to +0.5. Note that this tentative charge distribution is associated with a planar geometry for Mol. 1 while Mol. 2 adopts a boat conformation with a folding of one dithiole ring by 7.6(1)°. As shown in Figure 4, we also note a set of weak C-H•••O hydrogen bonding interactions, with the 𝑹𝑹 𝟐𝟐 𝟏𝟏 (𝟕𝟕) cyclic motif but with slightly longer H•••O distances. At variance with Phase A, Phase B is semiconducting (Figure S1 in ESI), with σ RT value of 4 ×10 -5 S cm -1 and an activation energy of 1620 K (0.14 eV).

Figure 3 .

 3 Figure 3. Projection views of the unit cell of Phase B: (a) along c direction, (b) along b direction. Hydrogen atoms were omitted for clarity.

Figure 4 .

 4 Figure 4. C-H hydrogen bonds with the anionic sulfonate moieties in Phase B. H 3A •••O 2 : 2.400 (3) Å, C 3 -H 3A •••O 2 : 156.9(2)°, H 9C •••O 3 : 2.405(2) Å, C 9 -H 9C •••O 3 : 148.2(2)°.

Figure 5 .

 5 Figure 6a. Apart from numer ous bands related to the accept or molec ule at

  Figure 6.Room temperature Raman spectra of the salt (TMTTF) 3 [Pt(qS) 2 ] within the region of C=C (a) and stretching (b) modes for the green (λ = 514.5 nm) and red (λ = 632.8 nm) excitations. The electrical vector of the laser beams is perpendicular to the c-axis.

Figure 7 .Figure 8 .

 78 Figure 7. Raman bands related to the ν 10 (a g ) mode (excitation λ = 632.8 nm). Dashed lines show a band deconvolution by using the Lorentz functions.

53 , 54 *

 5354 * assignment of the [Pt(qS) 2 ]

Figure 9 .Figure 10 .Figure 11 .

 91011 Figure 9. Temperature dependence of the optical conductivity of (TMTTF) 3 [Pt(qS) 2 ] for polarization E ║ c.

Figure 12 .

 12 Figure 12. Temperature dependence of the optical conductivity of (TMTTF)3[Pt(qS)2] within the region of ν 8 (a g ) mode (polarization E║ c).

  2 ] (50 mg) in 1,1,2-trichloroethane (5 mL + 25 μL H 2 O) as electrolyte in both compartments. Water was added to improve the solubility of the sulfonate derivative. Electrocrystallizations were performed at 20 °C with a constant current of 0.5 μA during 11 days. (TMTTF) 2 [Pt(qS) 2 ]•(TCE) 2 (black prism; Phase A) and (TMTTF) 3 [Pt(qS) 2 ] (black plate; Phase B) were obtained on the anode.

Table 1 .

 1 Crystallographic data

		(TMTTF)2[Pt(qS)2](TCE)2	(TMTTF)3[Pt(qS)2]
	Formula	C42H40Cl6N2O8PtS10	C48H46N2O8PtS14
	FW (gmol -1 )	1429.14	1422.79
	System	triclinic	monoclinic
	Space group	P-1	C2/m
	a (Å)	9.8814(4)	22.6764(15)
	b (Å)	12.1332(4)	9.9976(6)
	c (Å)	12.3986(5)	12.7217(9)
	α (deg)	90.2050(10)	90.00
	β (deg)	113.0050(10)	110.900(3)
	γ (deg)	109.1680(10)	90.00
	V (Å 3 )	1277.47(9)	2694.4(3)
	T (K)	150(2)	150(2)
	Z	1	2
	Dcalc (gcm -1 )	1.858	1.754
	µ (mm -1 )	3.522	3.201
	Total refls	19611	11442
	Abs corr	multi-scan	multi-scan
	Tmin, Tmax	0.810, 0.869	0.794, 0.968
	θmax (°)	27.525	27.498
	Uniq refls	5787	3259
	Rint	0.0397	0.0396
	Uniq refls [I > 2σ(I)]	5510	3056
	R1	0.0294	0.0308
	wR2 (all data)	0.0577	0.0593
	GOF	1.048	1.017
	Res. dens. (e Å -3 )	-0.764, +1.04	-0.984, +0.78

Table 3

 3 Vibrational bands of the (TMTTF) 3 [Pt(qS) 2 ] salt at T=295 K.

	Infrared (cm E ║c	(TM TTF) 3 [Pt(qS) 2 ] -1 ) Raman (cm Excit. λ = 514.5 nm -1 ) E ┴ c E ║ c E ┴ c	TMTTF 0 exp. * (cm -1 )	+ TMTTF exp.* (cm -1 )	Assignment **
	1653	1655	-	-
	1647	-	-	-
	1597	-	1593	1591
	1584	1585	-	-
	1576	1574	-	-
	1551	1551	-	-
	1500	1499	1500	1500
	1460 1420-1300	1457 -	1465 1453 a	1464 1453
	-	1410	-	1411
	≈ 1370 ---	-1389 1368 -1319	-1385 b 1374 b 1333 a 1319	-1385 b b 1374 a 1328 1321 c
	-	1305	-	-
	-	-	1244	1244
	1230	1234	-	-
	1212	1207	-	-
	1191	1192	-	-
	1161	1161	1167	1167
	-	1144	1151	-
	-	-	1124	1124
	1091	1090	-	-
	1056	1053	-	-
	1033	1032	1034	1035
	940	940	950, 928	-
	925	920	-	933
	839	839	-	-
	-	815	818	819
	759	759	764	764
	717	714	712	713
	669	668	659	659
	-	-	562	562
	----	----	535 520 a 514 a 507 a	-520 a a 514 507 a
	-	-	467	467
	-	-	307	308
	-	-	247	248
	-	-	157	157

Table 4 .

 4 Charge distribution in TMTTF trimers at RT as estimated by various methods.

		Charge (e)
	X-ray	Raman	IR
	(Table 2)	ν 10 (a g )	ν 28 (b 1u )
	+0.5	+0.45	+0.54
	+1.0	+0.90	+0.84
	+0.5	+0.69	+0.64

Conflicts of interest

There are no conflicts to declare.


In grapevine as in many crops, soluble sugar content is a major component of yield and economical value. This paper identifies and characterizes a GSK3 protein kinase, cloned from a cDNA library of grape (Vitis vinifera L.) Cabernet Sauvignon berries harvested at the ripening stage. This gene, called VvSK1, was mainly expressed in flowers, berries and roots.

In the berries, it was strongly expressed at post-véraison, when the berries accumulate glucose, fructose, and abscisic acid (ABA). In grapevine cell suspensions, VvSK1 transcript abundance is increased by sugars and ABA. In transgenic grapevine cells overexpressing VvSK1, the expression of four monosaccharide transporters (VvHT3, VvHT4, VvHT5 and VvHT6) was up-regulated, the rate of glucose uptake was increased 3-5 fold, and the amount of glucose and sucrose accumulation was more than doubled, while the starch amount was not affected. The present work provides the first example of the control of sugar uptake and accumulation by a sugar inducible protein kinase.

INTRODUCTION

Efficient and coordinated transport of assimilates is essential for plant growth, development and productivity. Plant growth and plant productivity are not limited by photosynthesis per se, but rather by the ability of the plant to export and to partition its assimilates in a proper way. Increased crop yield are paralleled by a better partitioning of assimilates between the harvestable part of the plant and the rest of the plant [START_REF] Gifford | Photosynthesis, carbon partitioning, and yield[END_REF][START_REF] Gifford | Crop productivity and photoassimilate partitioning[END_REF]Hoffmann-Thoma, 1996). Long-distance and intercellular transport of sugars is mediated by proton-coupled sucrose transporters (belonging to the disaccharide transporter family), and hexose and polyol transporters (belonging to the monosaccharide transporter family). Many of these transporters have been identified in a wide range of species [START_REF] Lemoine | Sucrose transporters in plants: update on function and structure[END_REF][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][START_REF] Büttner | The monosaccharide transporter(-like) gene family in Arabidopsis[END_REF]). Disaccharide transporter genes belong to small multigenic families (e.g. 9 members in Arabidopsis and 4 in tomato) [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][START_REF] Hackel | Sucrose transporter LeSUT1 and LeSUT2 inhibition affects tomato fruit development in different ways[END_REF] and can be clustered into 4 groups regarding the homologies of the encoded protein sequences. The Arabidopsis genome has 53 homologous sequences encoding putative monosaccharide transporters, which are distributed into 7 distinct clusters [START_REF] Büttner | The monosaccharide transporter(-like) gene family in Arabidopsis[END_REF].

Control of the expression of these transporters in specific cell types at precise stages of development allows fine-tuning in the plant of its carbon distribution. Although many sugar transporters have now been identified and characterized, little is known about their regulation.

Upregulation of a glucose transporter gene has been observed after treatment of Chenopodium rubrum cells with cytokinin (Ehness and Roitsch, 1997). In Arabidopsis, the monosaccharide transporter gene, AtSTP4, may be induced by fungal and bacterial elicitors (Truernit et al., 1996;[START_REF] Fotopoulos | The monosaccharide transporter gene, AtSTP4, and the cell-wall Invertase, Atbfruct1, are induced in Arabidopsis during infection with the fungal biotroph Erysiphe cichoracearum[END_REF], whereas the fungal elicitor cryptogein rapidly and completely blocks glucose uptake in tobacco cells [START_REF] Bourque | The elicitor cryptogein blocks glucose transport in tobacco cells[END_REF].

Accumulating evidence indicates that sugars may control the expression and activity of their own transporters. The ability to sense altered sugar concentration is important and allows the plant to regulate its metabolism and compartmentation processes in order to face the demands of sink organs [START_REF] Roitsch | Source-sink regulation by sugar and stress[END_REF]. There is some discrepancy about the effect of sugars on the control of these transporters. In Chenopodium rubrum cell suspensions, monosaccharide transporter genes are constitutively expressed and not regulated by sugar [START_REF] Roitsch | Expression of a sugar-transporter gene family in a photoautotrophic suspension culture of Chenopodium rubrum L[END_REF]. Sucrose was reported to repress the sugar beet sucrose transporter BvSUT1 both at transcriptional and translational levels [START_REF] Chiou | Sucrose is a signal molecule in assimilate partitioning[END_REF]Vaughn et al., 2002;[START_REF] Ransom-Hodgkins | Protein phosphorylation plays a key role in sucrose-mediated transcriptional regulation of a phloem-specific proton-sucrose symporter[END_REF], whereas it up-regulates OsSUT1 in rice [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]. Down-regulation of monosaccharide transport in response to glucose supply was also observed in suspension-cultured cells of olive [START_REF] Oliveira | Utilization and transport of glucose in Olea europaea cell suspensions[END_REF] and in peach tree buds [START_REF] Maurel | Sorbitol uptake is regulated by glucose through the hexokinase pathway in vegetative peach-tree buds[END_REF]. By contrast, the expression of VvHT1 in grape cell suspensions is induced by low concentration of sucrose [START_REF] Atanassova | Sugar-regulated expression of a putative hexose transport gene in grape[END_REF] and repressed at high sucrose concentration [START_REF] Conde | Pathways of glucose regulation of monosaccharide transport in grape cells[END_REF], suggesting that the effect of sugars on transporter genes depend on sugar concentration and on the physiological status of the cells.

In contrast to the situation in bacteria, yeast and mammals, where sugar signaling pathways are extensively studied (Johnston, 1999), signaling cascades involved in response to sugar in plants are still poorly understood. However, the combination of mutant isolation and pharmacological approaches reveals the involvement of protein kinases (PKs), protein phosphatases, and other signal transduction mediators such as Ca 2+ and calmodulin [START_REF] Smeekens | Sugar-induced signal transduction in plants[END_REF]. Among the PKs identified in sugar sensing and signaling in plants, the importance of SNF1 (Sucrose Non Fermenting)-like protein kinase complexes and interacting proteins has been well established [START_REF] Rolland | Sugar sensing and signaling in plants: conserved and novel mechanisms[END_REF]. In addition, the involvement of other PKs, like CDPKs (Calcium Dependent PK) [START_REF] Ohto | Sugar-induced increase of calcium-dependent protein kinases associated with the plasma membrane in leaf tissues of tobacco[END_REF] and MAPKs (Mitogen Activating PK) (Ehness et al., 1997) has also been suggested.

Glycogen synthase kinase 3 (GSK3), is a constitutively active serine/threonine kinase involved in diverse physiological activities and functions ranging from metabolism, cell cycle, gene expression and development [START_REF] Rayasam | Glycogen synthase kinase 3: more than a namesake[END_REF]. In mammalian cells, GSK-3 is an important signaling player that is ubiquitously expressed and has been implicated in the regulation of glucose transport and metabolism [START_REF] Grimes | The multifaceted roles of glycogen synthase kinase 3beta in cellular signaling[END_REF]. The few existing reports about regulation of glucose uptake by GSK-3 have produced different findings in different cell systems [START_REF] Nikoulina | Inhibition of glycogen synthase kinase 3 improves insulin action and glucose metabolism in human skeletal muscle[END_REF]Wieman et al., 2007) implicating GSK-3 in either insulin signaling or in non-insulin stimulated condition in cell types that are not insulin sensitive. GSK-3/SHAGGY-like kinases (GSKs) are a large gene family in plants (Jonak and Hirt, 2002). Recent functional studies indicate that plant GSKs are involved in diverse important processes including hormone signaling, development and stress responses [START_REF] Dornelas | Arabidopsis thaliana SHAGGY-related protein kinases (AtSK1 1 and 12) function in perianth and gynoecium development[END_REF]Jonak et al., 2000;[START_REF] Piao | Constitutive over-expression of AtGSK1 induces NaCl stress responses in the absence of NaCl stress and results in enhanced NaCl tolerance in Arabidopsis[END_REF][START_REF] Choe | Arabidopsis brassinosteroid-insensitive dwarf12 mutants are semidominant and defective in a glycogen synthase kinase 3beta-like kinase[END_REF][START_REF] Li | Regulation of brassinosteroid signaling by a GSK3/SHAGGY-like kinase[END_REF][START_REF] Perez-Perez | The UCU1 Arabidopsis gene encodes a SHAGGY/GSK3-like kinase required for cell expansion along the proximodistal axis[END_REF]Vert andChory, 2006, Kempa et al., 2007).

In this work, we report the involvement of VvSK1 (Vitis vinifera Shaggy Kinase), a GSK-3/SHAGGY-like kinase in the regulation of sugar accumulation in grape cell suspension in response to sugar. This GSK3-like kinase, whose expression is increased by sugars, probably acts through the regulation of monosaccharide transporter gene expression. The analysis of its gene expression during grape berry development also suggests that it may be involved in the control of sugar accumulation during berry ripening.

RESULTS

Isolation and Sequence Analysis of VvSK1

Total RNA extracted from Cabernet Sauvignon berry (CSB) cells treated or not with 58 mM sucrose were 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). Among the genes that were significantly up-regulated after 1 h of sucrose treatment, a gene corresponding to the EST sequence (TC46745) showed homology to the GSK3 family and was selected for further investigation. This choice was directed by previous data showing that members of the GSK3 family are involved in the regulation of carbohydrate metabolism in eukaryotic cells [START_REF] Embi | Glycogen synthase kinase-3 from rabbit skeletal muscle. Separation from cyclic-AMP-dependent protein kinase and phosphorylase kinase[END_REF]Woodgett and Cohen, 1984;Kempa et al., 2007). The full-length cDNA was amplified by PCR using a cDNA library prepared from post-véraison Cabernet Sauvignon berries and it was named VvSK1 (Vitis vinifera Shaggy-like Kinase 1; GenBank accession XP_0022721 12; GSVIVT00026235001). VvSK1 encodes a protein of 468 amino acids that contains the 11 highly conserved domains characteristic of kinases, including the tyrosine residue in the T-loop. VvSK1 exhibits high amino acid sequence similarities with GSKs from others species: 87% with NtSK91 (tobacco), 87% with LpSK6 (tomato), 87% with PSK6 (Petunia Hybrida), and 86% with AtSK8 (Arabidopsis) (Fig. 1).

Analysis of the Vitis vinifera genome (Jaillon et al., 2007) allowed the identification of 6 additional GSK3 members that were numbered from 2 to 7. Interestingly, VvSK1 only shares 44-67% identity with other members of grape GSK family. Based on protein sequence homology, the VvSKs were grouped into four classes (I-IV) (Fig. 2). VvSK1

Expression of VSK1 in response to sugar and ABA treatments

The expression profile of VvSK1 was assessed by RNA blot using RNA samples extracted from CSB suspension cells collected 0, 1, 2, 4, 6 and 8 h after supply of 58 mM sucrose as well as from control cells without sugar supply. VvSK1 transcripts accumulate from 1 h until the end of the experiment (Fig. 3A). To exclude an osmotic effect of sucrose, CSB suspension cells were treated with the same concentration of mannitol or PEG 600. None of these chemicals affected VvSK1 expression, indicating that the expression profile previously observed is not due to an osmotic stress but rather to a sucrose specific effect (Fig. 3B).

In order to elucidate the signaling pathway involved in the transcriptional regulation of this gene, we tested different sugar analogs on CSB suspension cells (Fig. 3C). Cells were treated with 3-OMG (which is absorbed but poorly phosphorylated) and 2-DOG (which is absorbed, phosphorylated but not further metabolized), or with 58 mM sucrose in the presence of the hexokinase (HXK) inhibitor mannoheptulose (MHL) [START_REF] Gibson | Plant sugar-response pathways. Part of a complex regulatory web[END_REF]. Blockage of HXK activity in cells treated with 10 mM MHL resulted in repressed levels of VvSK1 transcripts.

Addition of 58 mM 3-OMG has no effect on VvSK1 expression. By contrast, addition of 58 mM 2-DOG up-regulates VvSK1 as was observed after sucrose treatment. Taken together, these results suggest that sucrose up regulates VvSK1 expression through an HXK-dependent pathway.

Hormones play a crucial role during grape berry development. More particularly, endogenous ABA levels increase after véraison, and treatments that delay this increase delay the onset of ripening [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] Deluc | All rights reserved. pathways affecting important flavor and quality traits in grape berries of Cabernet Sauvignon and Chardonnay[END_REF]. In order to investigate a potential effect of ABA on VvSK1 expression, CSB cell suspensions were treated with 20 tM ABA and collected 0, 2, 4, 6, 8 and 12 h later. VvSK1 expression was monitored by quantitative RT-PCR. The results revealed that ABA increased the transcript abundance of VvSK1 by two fold within 2 h and this amount of VvSK1 transcripts was maintained for at least 10 h after ABA supply (Fig. 3D).

Relative expression profile of VvSK1 in grapevine organs and berry tissues

The relative transcript abundance of VvSK1 was determined in different grapevine organs by RT-PCR with RNA extracted from Cabernet Sauvignon roots, stems, leaves, flowers and mature berries. VvSK1 was predominantly expressed in roots, inflorescences and mature berries, whereas it was hardly detectable in stems and leaves (Fig. 4A). VvSK1 transcript accumulation was also assessed during berry development. VvSK1 was present in green berries, but the accumulation of the transcript increased mainly after véraison stage and was maintained until the harvesting stage (Fig. 4B). This expression profile is in agreement with microarray data that were previously obtained from Shiraz and Chardonnay developing berries (David Glissant and Serge Delrot, unpublished data).

The level of expression of VvSK1 in the different berry compartments at véraison (seeds, pulp and skin) was also analyzed by RT-PCR. As shown in Figure 3C, VvSK1 showed ubiquitous expression in the berry even though expression levels seemed higher in skin and pulp than in seeds.

Overexpression of VvSK1 in grapevine cells affects sugar transport

In order to elucidate the function of VvSK1, transgenic grape cells overexpressing HAtagged VvSK1 under the control of the CaMV 35S promoter were prepared. After stabilization of the cell suspension by subculture in glycerol-maltose-NOA culture medium supplemented with paromomycin and cefotaxime, the expression of VvSK1-HA was tested by PCR and western blotting. A DNA fragment of 1408 bp corresponding to VvSK1 was strongly amplified by VvSK1 specific primers, in the cells expressing the 35S:: VvSK1-HA construct, in comparison with cells expressing the empty vector (Fig. 5A). In addition, the recombinant protein VvSK1-HA with a molecular weight of 57.2 kDa was detected in a western blot with an anti-HA antibody (Fig. 5B). To confirm that VvSK1 is a functional protein kinase, we performed a phosphorylation assay using VvSK1 produced in Escherichia coli as a glutathione S-transferase (GST) fusion protein. VvSK1 phosphorylated myelin basic protein as an artificial substrate (Fig. 5C) showing that in addition to being functional, VvSK1 exhibits a basal constitutive activity.

The expression level of different hexose transporter genes in the 35S:: VvSK1-HA grape cells was measured in order to gain greater insights into sugar accumulation during grape berry development. Seven plasma membrane MST homologues (VvHTs) and a plastidic Glucose transporter (pGLT) have been cloned from grape berries (Hayes et al., 2007;Vignault et al., 2006). Specific primers were designed for the sequences of VvHT1 (accession no. AJ001061),

VvHT2 (AY663846), VvHT3 (AY538259), VvHT4 (AY538260), VvHT5 (AY538261), VvHT6
(AY861386), and VvpGLT (AY608701) and used to test the corresponding transcript amounts in 41B cells overexpressing VvSK1 (Fig. 6A). Compared to control cells, overexpression of VvSK1 increased the accumulation of VvHT3, VvHT4, VvHT5 and VvHT6 transcripts when compared to control cells whereas VvHT1, VvHT2, and pGLT transcripts were not significantly affected. Although transcript abundance was increased for all four hexose transporters, they were up-regulated in a slightly different way. Thus, VvHT3 and VvHT4 transcript abundance was increased by 1.7 fold in the VvSK1 overexpressing line, whereas it was increased by 3.4 and 2.8 fold for VvHT5 and VvHT6, respectively. Interestingly, VvHT3, VvHT4, VvHT5 and VvHT6 expression was increased after supply of 58 mM sucrose. Their transcripts accumulated within 1 h and peaked 6 h after sucrose addition (Fig. 6B). These results suggest that sucrose might affect the expression of these 4 VvHTs through a signaling cascade involving VvSK1.

As a consequence, VvSK1 might also affect sugar transport activities in grape cells. To test this hypothesis, uptake of D-[ 14 C]-glucose was compared in control cells and in cells overexpressing VvSK1. The rate of glucose accumulation by 41B cells overexpressing VvSK1 was 3-5 fold higher than in control cells during the time course studied (Fig. 7).

Analysis of sugar content in grape cells overexpressing VvSK1

The stimulation of hexose transporters gene expression in 41B cells overexpressing VvSK1 and the enhanced ability of these cells to accumulate glucose indicated that their sugar content might be altered by VvSK1 expression. Soluble sugars (glucose, fructose and sucrose), and starch contents were assayed seven days after subculture (Fig. 8). Comparison of the sugar profiles showed that 41B cells overexpressing VvSK1 displayed a 2-fold increase in glucose and a 3.3-fold increase in sucrose content as compared to PFB8 control cells. By contrast, starch amounts were not significantly different between both cell lines. Fructose amounts were too small to be quantified and therefore could not be compared. For this experiment, 3 independent flasks of 41 B cell suspension cultures for each condition were used.

DISCUSSION

Sugars provide the reduced carbon needed for the structural materials of the cell, storage compounds and all major primary and secondary metabolites. They also influence plant growth and development by acting as regulatory signals, which affect the expression of various genes involved in many processes of the plant life cycle [START_REF] Koch | Carbohydrate-modulated gene expression in plants[END_REF]Jiang and Carlson, 1997;[START_REF] Smeekens | Sugar regulation of gene expression in plants[END_REF][START_REF] Roitsch | Source-sink regulation by sugar and stress[END_REF][START_REF] Sheen | Sugars as signaling molecules[END_REF]. It is therefore important to identify the molecular players that may directly or indirectly control sugar transport in plants.

Although many sugar transporters have now been identified, little is known about sugar transport regulation at the molecular level [START_REF] Delrot | Regulation of sugar, amino acid and peptide plant membrane transporters[END_REF].

Protein kinases are major components of intracellular signal transduction enabling plant cells to rapidly respond to the environment. The present paper shows that VvSK1 is a sugar and ABA induced GSK3 protein kinase that increases the transcript abundance of several monosaccharide transporters and the soluble sugar content of grape cells. MsK4, a GSK3 homologue from group IV, controls carbohydrate metabolism in response to salt stress (Kempa et al., 2007). MsK4 is a starch-associated protein kinase that, when over-expressed, modulates carbohydrate metabolism. Additionally, MsK4 acts as a signaling component in the high-salinity response, linking stress signaling to metabolic adaptation.

The Vitis GSK3 phylogenic tree revealed that VvSK1 was less related to the other VvSKs than to homologues from other species. Indeed, VvSK1 shared highest similarities with GSK3s from Solanacea NSK91, NSK59, LpSK6 (87%, 87%, 86.6%), PSK6 and PSK7 (87% and 84.3%) or from Arabidopsis ASK2 () and ASK8 () (78% and 86%). These GSK3s, which belong to the same clade (group III), are preferentially expressed in flowers [START_REF] Tichtinsky | Figure 1. Sequence alignment of VvSK1 with other GSK3 like PK from various plant species. Identical residues are shown in black, conserved residues in dark gray, and similar residues in light gray. GeneBank accession numbers are as follows : VvSK1 (XP_002272112)[END_REF][START_REF] Charrier | Expression profiling of the whole Arabidopsis shaggy-like kinase multigene family by real-time reverse transcriptase polymerase chain reaction[END_REF]Wilson et al., 2004). Among these, ASK(AtSK3-2) was shown to be involved in the control of cell elongation in flower development and in response to light [START_REF] Claisse | The Arabidopsis thaliana GSK3/Shaggy like kinase AtSK3-2 modulates floral cell expansion[END_REF]. Our data showed that VvSK1 was not only expressed in flowers, but also in two other major sink organs: roots and berries, indicating that its role was not only restricted to flower development in grapevine (Fig. 4A). In berries, VvSK1 transcript amounts transiently decreased after the green stage and increased again after véraison stage (Fig. 4B), when both sugars and ABA accumulate in this organ [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].

Our experiments showed that both sugar and ABA could increase the transcript abundance of VvSK1 in grape cell suspensions (Fig. 3A and3D). It is well known that sugar signaling does not operate alone but rather is integrated in cellular regulatory networks. Most notably, there is a tight interaction between sugar and hormonal signaling, particularly for ABA [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]Léon and Sheen, 2003 ;[START_REF] Rolland | Sugar sensing and signaling in plants: conserved and novel mechanisms[END_REF]. Also, it is known that ABA enhances sugar accumulation in crop sink organs [START_REF] Kobashi | Effect of abscisic acid (ABA) on sugar accumulation in the flesh tissue of peach fruit at the start of the maturation stage[END_REF] and data from the literature show that both sugar and ABA act synergistically [START_REF] Smeekens | Sugar-induced signal transduction in plants[END_REF]Léon and Sheen, 2003 ;[START_REF] Rolland | Sugar sensing and signaling in plants: conserved and novel mechanisms[END_REF]Ramon et al., 2008). In peach fruit flesh, sugar accumulation is stimulated by ABA that in turn stimulates uptake of sugars [START_REF] Kobashi | Effect of abscisic acid (ABA) on sugar accumulation in the flesh tissue of peach fruit at the start of the maturation stage[END_REF]. ABA induces the expression of VvHT1 [START_REF] Atanassova | Sugar-regulated expression of a putative hexose transport gene in grape[END_REF] and acts synergistically with physiological concentrations of monosaccharides to transiently induce VvHT1 transcription. VvHT1 responsiveness to both signals involves the presence in its promoter of a specific overlapping configuration of the two sugar response cis-elements. This configuration seems a target for the fixation and the action of VvASR, Vitis vinifera Abscisic acid, Stress, Ripening-induced) protein (also called VvMSA, 'akir et al., 2003). VvASR expression is strongly up-regulated by the combined effect of sugars and ABA ('akir et al., 2003). More recently, it was shown that ABA increases invertase activities and amounts during grape development in correlation with soluble sugar concentration [START_REF] Pan | Abscisic acid activates acid invertases in developing grape berry[END_REF].

VvSK1 was induced by addition of exogenous sucrose and this induction was not due to an osmotic effect (Fig. 3C). This indicates that VvSK1 was involved downstream of sugar perception. In plants, two signaling pathways relaying sugar sensing have been described. The first one is HXK-dependent and the second is independent of HXK activity [START_REF] Smeekens | Sugar-induced signal transduction in plants[END_REF]. To investigate the role of HXK in the sucrose induction of VvSK1, we tested the effect of glucose analogs and an inhibitor of HXK activity. The results showed that the HXK inhibitor MHL blocked VvSK1 induction by sucrose, whereas 3-OMG had no effect and 2-DOG stimulated VvSK1 expression in grape cells (Fig. 3B). This indicates that an HXKdependent pathway may be involved in the sugar control of VvSK1 expression in grape cells VvSK1 over-expression in grape cell suspension affects the level of expression of some sugar transporters genes expressed in grape berries (Fig. 6A). The VvHTs whose expressions were affected by VvSK1 over-expression were also affected at the transcriptional level by sugars and ABA (Fig. 6B and data not shown). This result was in accordance with the observation that VvSK1 over-expression increased by more than 3-fold the uptake rate of radiolabeled glucose in comparison to control cells (Fig. 7). The involvement of VvSK1 in the control of sugar compartmentation was further strengthened by the fact that cells overexpressing VvSK1 accumulate twice more glucose and three times more sucrose than the control, whereas the starch content remains unaffected. Sugar levels of suspension cells significantly differ from those in berries, where monosaccharide concentrations can reach 1.5 M whereas sucrose is poorly detectable [START_REF] Fillion | Cloning and expression of a hexose transporter gene expressed during the ripening of grape berry[END_REF][START_REF] Liu | Sugar and acid concentrations in 98 grape cultivars analyzed by principal component analysis[END_REF]. In this context, the role of VvSK1 on sugar transport and accumulation in grape berries has to be further investigated by altering VvSK1 expression in berries.

In this work, VvSK1 was identified as a potential regulator of hexose uptake and sugar accumulation in grapevine cells. As a functional PK, VvSK1 can regulate its targets by direct phosphorylation. Protein phosphorylation is one of the most important and best characterized post-translational protein modification regulating cellular signaling processes. In plants, previous work suggested that protein phosphorylation plays a direct role in the regulation of sugar transporter transcription and sugar transport activity (Roblin et al. 1998;[START_REF] Ransom-Hodgkins | Protein phosphorylation plays a key role in sucrose-mediated transcriptional regulation of a phloem-specific proton-sucrose symporter[END_REF]. Key enzymes from carbon metabolism may also be directly regulated by phosphorylation (Sugden et al., 1999;Ikeda et al., 2000). Furthermore, in yeast, the transcription factor RGT1 that functions as a repressor in the absence of glucose becomes a transcriptional activator required for maximal induction of HXT1 gene expression, upon phosphorylation in the presence of high concentrations of glucose [START_REF] Mosley | Glucose-mediated phosphorylation converts the transcription factor Rgt1 from a repressor to an activator[END_REF].

Therefore, one can hypothesize that VvSK1 can affect sugar uptake and accumulation in grapevine through the regulation of hexose transporter expression. This regulation might be mediated through VvSK1 phosphorylation events affecting the activity and/or stability of specific transcription factors, sugar transporters and/or of enzymes from sugar metabolism.

This still has to be demonstrated.

In grapevine, one of the main features of berry development is the accumulation of hexose sugars (i.e., glucose and fructose), which begins at véraison and continues throughout the ripening process. In addition to participating in the regulation of sugar homeostasis in grape cells, VvSK1 may play a role in grape berry development by affecting sugar compartmentation. This hypothesis is further supported by the capacity of VvSK1 to up regulate several VvHT gene expression and more particularly that of VvHT3 and VvHT6.

VvHT6 transcript abundance correlates well with hexose accumulation in the berries, and was recently proposed to play a significant role in hexose accumulation during berry ripening [START_REF] Deluc | Transcriptomic and metabolite analyses of Cabernet Sauvignon grape berry development[END_REF]. If VvHT6, which is highly homologous to the tonoplast glucose transporter AtTMT2 (Wormit et al., 2006) acts as a tonoplast sugar transporter, it would only favor indirectly glucose uptake across the plasma membrane, by decreasing the cytosolic glucose concentration. VvHT3 transcript accumulates in young berries but also later during the phase of sugar storage (Hayes et al., 2007). VvHT4 expression shows little change throughout berry development whereas VvHT5 transcript amounts increased by 3 fold at 14 weeks post-flowering. As both genes are very weakly expressed in Cabernet Sauvignon berry flesh, these transporters, unlike VvHT3 and VvHT6, may not contribute significantly to sugar import during berry development (Hayes et al., 2007). Nevertheless, the authors did not rule out the possibility that these VvHTs could be better expressed in more specific pulp cell types. Furthermore, as VvSK1 expression in not restricted to the berry, the role of the kinase in the regulation of these 2 VvHTs in other plant organs is possible if they are expressed in the same organs.

In grape berries, like in other sink organs, sugar accumulation significantly impacts crop yield and economical value. To our knowledge, this work provides the first demonstration that soluble sugar content may be significantly affected by manipulation of a single gene involved in signaling pathways. VvSK1 transgenic cells may provide a useful model to investigate the metabolic and physiological consequences of a deregulation of sugar uptake and accumulation. Analysis of the effect of glucose analogs and the involvement of ABA, place VvSK1 downstream to the HXK-dependent sugar signaling cascade, ABA being involved downstream of HXK1 (Zhou et al., 1998;[START_REF] Arenas-Huertero | Analysis of Arabidopsis glucose insensitive mutants, gin5 and gin6, reveals a central role of the plant hormone ABA in the regulation of plant vegetative development by sugar[END_REF]. The expression profiles of VvSK1 in sink organs (roots and fruits) and during berry development indicate a key role of this PK in the maturation process that still remains to be demonstrated in planta. Overall, our data provide a first trail for the identification of potential signaling components affecting sugar transport and accumulation during grape berry development and ripening.

MATERIALS AND METHODS

Isolation of grapevine Shaggy like kinase cDNA

VvSK1 was isolated from an expressed sequence tag (EST; accession number TC46745 (new TC92799)) collected at the DFCI Vitis vinifera Gene Index (VvGI) and containing the entire open reading frame (ORF). For expression analysis, the entire clone was generated from a cDNA library of grape Cabernet Sauvignon berries (ripen stage) by PCR using synthetic oligonucleotide primers designed to begin and end at the start and stop codons of the ORF (forward primer TACCCGGGACTCGAGATGAATGTGATGCGTCGTCTCAAGA GCATT ; reverse primer ATGGATCCTCAGCGGCCGCCTTTCCTTGCATGCTCAGGAA TGAGA). The VvSK1 cDNA was cloned into the pGEM-T Easy vector (Promega) for DNA sequencing prior to -HA tagging and subcloning into the vector pFB8 downstream of the 35S promoter of cauliflower mosaic virus.

RNA isolation and cDNA production

After collection, the cells were immediately frozen in liquid nitrogen and stored at -80 °C until RNA extraction. The samples were ground under liquid nitrogen with a mortar and pestle and RNA was isolated as previously described (Reid et al. 2006). Briefly, the extraction buffer contained 300 mM Tris HCl (pH 8.0), 25 mM EDTA, 2 M NaCl, 2% CTAB (Cetyltrimethylammonium bromide), 2% PVPP, 0.05% spermidine trihydrochloride and 2% b-mercaptoethanol. Fifteen mL of pre-warmed (65°C) extraction buffer was added to the ground tissue. Samples were incubated at 65°C for 10 min, and then extracted twice with chloroform. The final aqueous layer was centrifuged at 30,000 g for 20 min at 4 °C, then 0.1 vol of 3 M NaOAc (pH 5.2) and 0.6 vol of isopropanol were added to the supernatant and stored overnight at -80°C. Samples were pelleted by centrifugation (3500g, 30 min) the next day and resuspended in 0.7 mL TE (pH 7.5). To selectively precipitate the RNA, 0.3 vol of 8 M LiCl was added and the sample was stored overnight at 4 °C. RNA was pelleted the next day, washed with ice cold 70% EtOH, air dried, and dissolved in DEPC-treated water. RNA isolation was followed by DNaseI treatment. Total RNA was quantified and the quality was evaluated on 1% agarose gels.

For RT-PCR analysis, 2 tg of total RNA were reverse transcribed with oligo(dT)12-18 in a 20 tL reaction mixture using the Moloney murine leukemia virus reverse transcriptase (Promega) according to the manufacturer's instructions. After heat inactivation of the reaction mixture, PCR was performed using 2.5 tL of the first-strand cDNA sample with 20 pmol of the primers in a 50 tL reaction.

Real-time PCR expression analysis was performed by using a commercial IQ SYBR-Green Supermix kit (Bio-Rad) with the CFX96 Real-Time PCR Detection system (Bio-Rad). Gene

V v H T 2 -R e v , T C G G G C T T T A C G G A G A G A G ; V v H T 3 -F o r , G C T A C T C T C T A C T C G T C C G C T T T G ; a n d V v H T 3 -R e v ,
CCCGTCATTGCTTCCGAACTTG; VvHT4-For, GGGCTGGCGAGTTTCTCTAG; and

V v H T 4 -R e v , A G T T C T G C T T G G A C A T C G T T T G ; V v H T 5 -F o r , C A G G C T G T T C C A C T G T T C T T A T C G ; a n d V v H T 5 -R e v , AGTTAGGAGGACCGCAGGAATC; VvHT6-For, TTTATTTGCATGAGGAGGGAGTC; a n d V v H T 6 -R e v , G A G C A G C A G C C T G G A T A T A A T C ; V v p G L T -F o r , C A G T G G T T A T G G G A G C C G A G T T T G ; a n d V v p G L T -R e v ,
TCCACCAGAAGCTCTAGCCTTGAC.

Grapevine transformation and culture conditions

Grapevine transformations were made in 41B rootstock (V. vinifera cv. Chasselas Ñ V. (XP_002276754, GSVIVT00020713001), VvSK5 (XP_002270455, GSVIVT00035848001), VvSK6 (XP_002281788, GSVIVT00018774001), VvSK7 (XP_002263398, GSVIVT00035285001), ASK1-alpha (At5g26751), ASK2-beta (At3g61160), ASK3-gamma (At3g05840), ASK4-delta (At1g57870), ASK5-epsilon (At5g14640), ASK6-zeta (At2g30980), ASK7-BIN2-eta (At4g18710), ASK8-theta (At4g00720), ASK9-iota (At1g06390), and ASK10-kappa (At1g09840).

(

  XP_002272112) belongs to class III. VvSK2 (GSVIVT00028178001), VvSK3 (GSVIVT00022544001) and VvSK4 (GSVIVT00020713001) are related to members of class I. VvSK5 (GSVIVT00035848001) and VvSK6 (GSVIVT00018774001) are related to class II, and VvSK7 (GSVIVT00035285001) to class IV.

  transcripts were quantified upon normalization to EF1and actin as internal standards, by comparing the cycle threshold of the target gene with those of standard genes. All biological samples were tested in triplicate, and SE values of means were calculated using standard statistical methods. Specific oligonucleotide primer pairs were designed with Beacon Designer 7 © software (Premier Biosoft International). Specific annealing of the oligonucleotides was controlled on dissociation kinetics performed at the end of each PCR run. The efficiency of each primer pair was measured on a PCR product serial dilution. The p r i m e r s u s e d f o r a m p l i f i c a t i o n w e r e a s f o l l o w s : V v S K 1 -F o r , GGTCGATCAAGAAGCAGAACAG and VvSK1 -Rev, GCAGCAGTTTCCTGTGATGTAG ; V v H T 1 -For , CGTTGTTCACAT CGTCGCT TTATC ; and V v H T 1 -Rev, GAGCAGTCCTCCGAATAGCATTG ; VvHT2-For, GGCATAGGGGTGGTGGTAG; and

Figure 1 .

 1 Figure 1. Sequence alignment of VvSK1 with other GSK3 like PK from various plant species. Identical residues are shown in black, conserved residues in dark gray, and similar

Figure 2 .

 2 Figure2. Phylogenetic relationship between GSK3 proteins from grape and Arabidopsis. A phylogenetic tree was constructed using MEGA version 4[START_REF] Tamura | MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0[END_REF] using the Minimum Evolution method with 2.000 bootstrap replicates. Names and GenBank accession numbers are as follows: VvSK1 (XP_002272112, GSVIVT00026235001), VvSK2 (XP_002279596, GSVIVT00028178001), VvSK3 (XP_002280673, GSVIVT00022544001), VvSK4 (XP_002276754, GSVIVT00020713001), VvSK5 (XP_002270455, GSVIVT00035848001), VvSK6 (XP_002281788, GSVIVT00018774001), VvSK7 (XP_002263398, GSVIVT00035285001), ASK1-alpha (At5g26751), ASK2-beta (At3g61160), ASK3-gamma (At3g05840), ASK4-delta (At1g57870), ASK5-epsilon (At5g14640), ASK6-zeta (At2g30980), ASK7-BIN2-eta (At4g18710), ASK8-theta (At4g00720), ASK9-iota (At1g06390), and ASK10-kappa (At1g09840).

Figure 6 .Figure 7 .Figure 8 .

 678 Figure 6. Over-expression of VvSK1 modifies the transcription of some grape monosaccharide transporter genes. A, Quantitative RT-PCR analysis of monosaccharide transporters in control (PFB8, grey bars) and 35S::VvSK1-HA (black bars) grape cells. Expression levels of VvHTs were evaluated in transgenic 41B cells. B, Kinetic of expression of VvHT3 (), VvHT4 (), VvHT5 (), and VvHT6 () assessed by quantitative RT-PCR using total RNA extracted from CSB cells treated with 58 mM sucrose. Elongation factor VvEF1γ and actin were used as internal controls. All data are means of three replicates, with error bars indicating SE. The primer sequences are given in materials and methods.

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ACKNOWLEDGEMENTS

The authors thank Christian Kappel for assistance in bioinformatics, Messaoud Meddar for preparing the cell culture media, Ghislaine Hilbert and Christelle Renaud for help in sugar content analysis, Prof Heribert Hirt for critical reading of the manuscript and Prof. Grant Cramer for improving the style of the manuscript.

berlandieri). An embryogenic cell suspension culture was initiated as described previously (Coutos-Thévenot et al. 1992a,b). This cell suspension was subcultured weekly in 25 mL of glycerol-maltose culture medium (Coutos-Thevenot et al., 1992b) supplemented with synthetic auxin (naphthoxy acetic acid at 1 mg L -1 ) in the dark. Embryogenic cells were transformed using an Agrobacterium cocultivation method [START_REF] Mauro | High efficiency regeneration of grapevine plants transformed with the GFLV coat protein gene[END_REF] and, after selection, the transgenic cells were subcultured in the same condition in a medium supplemented with paromomycin at 2 tg mL -1 (final concentration) and cefotaxime at 200 tg mL -1 (Duchefa).

Western blot analysis

Western blotting was performed with 30 tg of protein extracts separated by SDSpolyacrylamide-gel electrophoresis, immunoblotted to poly(vinylidene difluoride) membranes (Millipore) and probed with -HA antibody (Sigma) in Tris-buffered saline pH 7.4 containing 0.05% Tween 20. Peroxidase-conjugated goat anti-rabbit IgG was used as secondary antibody and the reaction was revealed by fluorography (ECL; Amersham Biosciences).

In vitro kinase Assay

The open reading frame of VvSK1 was cloned into GST fusion protein expression vector pGEX4T-1 (Pharmacia). Expression of the GST fusion protein and affinity purification were performed as described previously (Kiegerl et al., 2000). Kinase reactions were performed in 15 µL of kinase buffer (20 mM HEPES, pH 7.4, 10 mM MgCl2, 5 mM EGTA, and 1 mM DTT) containing 5 µg of GST fusion protein, 5 µg of myelin basic protein, 0.1 mM ATP, and 2 µCi of [-32P]ATP. The protein kinase reactions were performed at room temperature for 30 min, and the reactions were stopped by adding 4x SDS loading buffer and heating for 5 min at 95°C. The phosphorylation of myelin basic protein was analyzed by autoradiography after separation by 12.5% SDS-PAGE.

Glucose uptake studies

Transgenic cells overexpressing VvSK1 were collected 10 days after subculture and washed 3 times with culture medium lacking sugar at pH 5.0. Cells were then resuspended in the same fresh medium and equilibrated for 2 h at 120 rpm. Uptake was initiated by addition of D-[ 14 C]-glucose (final concentration: 0.133 Mbq/g FW). One mL aliquotes of the cell suspension were collected after 0, 0.5, 2 and 4 h of incubation with the radioactive sugar. The reaction was stopped by addition of 2 x 5 mL of culture medium without sugar and the cells