Manuel Nieves-Cordones

Mamy-Jean De Dieu Andrianteranagna

Teresa Cuéllar

Isabelle Chérel

Rémy Gibrat

Martin Boeglin

Bertrand Moreau

Nadine Paris

Jean-Luc L Verdeil

Sabine Dagmar Zimmermann

M Andrianteranagna

I Gaillard

supports its function in massive potassium fluxes necessary for berry potassium loading and pulvinus-actuated leaf movements

Keywords: berry K+ loading, paraheliotropic movements, phloem, pulvinus, Shaker K+ channel, VvK3 berry K + loading, paraheliotropic movements, phloem, pulvinus, Shaker K + channel, VvK3.1 channel

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

that reflect their environment (e.g. climate and soils) and their particular varieties. In the past, the climatic characteristics of different wine growing regions were properly taken into account to best adapt the vines to their environment. However, climate change is now affecting viticulture with high temperatures and soil water deficits that largely constrain grapevine productivity. Berry properties such as color, flavor, and aroma components are altered by these excessive solar radiation and severe drought conditions. Importantly, the impacts of climate change also result in unbalanced wines with excessively low acidity [START_REF] Jaillon | The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla[END_REF][START_REF] Kodur | Effects of juice pH and potassium on juice and wine quality, and regulation of potassium in grapevines through rootstocks (Vitis): a short review[END_REF].

Grape acidity results from the ratio between free organic acids (i.e. malic and tartaric acids) and organic acids neutralized by potassium (K + ) ions. In grapevine berries, K + is involved as a major cellular counter-ion in the electrical neutralization of organic acids, in the control of pH, and in the fine-tuning of the acid-base balance of the flesh cells [START_REF] Mpelasoka | A review of potassium nutrition in grapevines with special emphasis on berry accumulation[END_REF][START_REF] Kodur | Effects of juice pH and potassium on juice and wine quality, and regulation of potassium in grapevines through rootstocks (Vitis): a short review[END_REF]. However, the combined effects of climate change have a reducing effect on the free organic acid content, by increasing the consumption of malic acid as a respiratory substrate [START_REF] Famiani | Malate as substrate for catabolism and gluconeogenesis during ripening in the pericarp of different grape cultivars[END_REF][START_REF] Rienth | Temperature desynchronizes sugar and organic acid metabolism in ripening grapevine fruits and remodels their transcriptome[END_REF] and favoring K + accumulation during the ripening period [START_REF] Kodur | Effects of juice pH and potassium on juice and wine quality, and regulation of potassium in grapevines through rootstocks (Vitis): a short review[END_REF][START_REF] Duchêne | Genetic variability of descriptors for grapevine berry acidity in Riesling, Gewürztraminer and their progeny[END_REF].

Excess K + then interacts with tartaric acid to form K + bitartrate, which precipitates during the winemaking process. This results in musts with low acidity and consequently wines that are too sweet, with strong sensations of wood and alcohol [START_REF] Mpelasoka | A review of potassium nutrition in grapevines with special emphasis on berry accumulation[END_REF].

Wine acidity, which normally allows flavor and aroma to develop during winemaking, is essential for the aging potential and also contributes to the red color of wine, through the degree of anthocyanin ionization [START_REF] Mpelasoka | A review of potassium nutrition in grapevines with special emphasis on berry accumulation[END_REF][START_REF] Davies | Transporters expressed during grape berry (Vitis vinifera L.) development are associated with an increase in berry size and berry potassium accumulation[END_REF].

The maintenance of berry quality can be achieved by controlling berry K + content at harvest. Nevertheless, this requires a better understanding of the molecular basis of K + accumulation throughout grape berry development, as well as improved identification of the different molecular actors involved in K + uptake and accumulation. In plants, K + accumulation is controlled by multigenic families that encode a broad spectrum of potassium transporters and channels as well as regulatory proteins (protein kinases, protein phosphatases, etc.) [START_REF] Dreyer | Potassium channels in plant cells[END_REF][START_REF] Sharma | The role of K(+) channels in uptake and redistribution of potassium in the model plant Arabidopsis thaliana[END_REF][START_REF] Ronzier | CPK13, a noncanonical Ca2+-dependent protein kinase, specifically inhibits KAT2 and KAT1 shaker K+ channels and reduces stomatal opening[END_REF][START_REF] Véry | Molecular biology of K+ transport across the plant cell membrane: what do we learn from comparison between plant species?[END_REF][START_REF] Lefoulon | The Arabidopsis AtPP2CA Protein Phosphatase Inhibits the GORK K+ Efflux Channel and Exerts a Dominant Suppressive Effect on Phosphomimetic-activating Mutations[END_REF]. Among the different K + transport systems that have been identified and characterized in grapevine [START_REF] Pratelli | A grapevine gene encoding a guard cell K(+) channel displays developmental regulation in the grapevine berry[END_REF][START_REF] Davies | Transporters expressed during grape berry (Vitis vinifera L.) development are associated with an increase in berry size and berry potassium accumulation[END_REF][START_REF] Cuéllar | A grapevine Shaker inward K(+) channel activated by the calcineurin B-like calcium sensor 1-protein kinase CIPK23 network is expressed in grape berries under drought stress conditions[END_REF][START_REF] Cuéllar | Potassium transport in developing fleshy fruits: the grapevine inward K(+) channel VvK1.2 is activated by CIPK-CBL complexes and induced in ripening berry flesh cells[END_REF], only two currently appear to be involved in the loading of this cation into grape berries during the ripening stage [START_REF] Hanana | A grape berry (Vitis vinifera L.) cation/proton antiporter is associated with berry ripening[END_REF][START_REF] Cuéllar | Potassium transport in developing fleshy fruits: the grapevine inward K(+) channel VvK1.2 is activated by CIPK-CBL complexes and induced in ripening berry flesh cells[END_REF]. The first transport system, VvK1.2 [START_REF] Cuéllar | Potassium transport in developing fleshy fruits: the grapevine inward K(+) channel VvK1.2 is activated by CIPK-CBL complexes and induced in ripening berry flesh cells[END_REF], is a voltage-gated inwardly rectifying K + -selective channel that belongs to the K + Shaker channel family. VvK1.2 is specifically expressed in the berry, strongly emerging in the plasma membrane of the flesh cells (mesocarp) and the perivascular cells at veraison (the onset of ripening). Its expression level then continuously increases during berry ripening and can be strongly increased further by mild drought stress. The expression pattern and transcriptional regulation of VvK1.2 both indicate that this channel likely plays a major role in K + uptake in flesh cells of ripening berries [START_REF] Cuéllar | Potassium transport in developing fleshy fruits: the grapevine inward K(+) channel VvK1.2 is activated by CIPK-CBL complexes and induced in ripening berry flesh cells[END_REF]. The second transport system, VvNHX1, is a vacuolar cation + /H + antiporter belonging to the NHX type of transporter families [START_REF] Hanana | A grape berry (Vitis vinifera L.) cation/proton antiporter is associated with berry ripening[END_REF]. VvNHX1 mediates Na + /H + and K + /H + coupled exchange, with a higher affinity for K + than Na + . Moreover, VvNHX1 expression is strongly increased starting from veraison and during berry maturation, indicating that this transporter may be responsible for vacuolar accumulation of K + at the inception of ripening, driving the uptake of water that generates vacuolar expansion [START_REF] Hanana | A grape berry (Vitis vinifera L.) cation/proton antiporter is associated with berry ripening[END_REF].

Here, we report the characterization of the grape Shaker K + channel VvK3.1. In plants, Shaker genes are known to encode highly K + -selective channels that are strongly voltageregulated. These channels are active at the plasma membrane and provide major pathways for bulk K + uptake or secretion in various tissues and cell types [START_REF] Sharma | The role of K(+) channels in uptake and redistribution of potassium in the model plant Arabidopsis thaliana[END_REF][START_REF] Véry | Molecular biology of K+ transport across the plant cell membrane: what do we learn from comparison between plant species?[END_REF]. We demonstrate that VvK3.1 is a weak inwardly rectifying K + channel that is only slightly activated by CBL-interacting Ser/Thr protein kinase (CIPK)/calcineurin Blike Ca 2+ sensor (CBL) complexes. This channel is highly expressed in all grapevine organs, with expression restricted to the phloem and phloem parenchyma. We found that VvK3.1 expression in the phloem continuously increases during berry ripening, suggesting a major role for this Shaker channel in the unloading of K + from phloem during grape maturation. This is a major concern for grapevines, as the fruit is energetically limited due to stomata disappearance after veraison. Unexpectedly, in addition to the phloem, VvK3.1 is expressed in particular structures localized at the joints of the petiole that display the histological features of a pulvinus, i.e. a motor organ known to be involved in plant leaf movements. Finally, the roles played by the VvK3.1 channel both in K + loading into berry tissues and in grapevine leaf movements are discussed.

Materials and Methods

Plant materials

Four-year-old grapevines (Vitis vinifera cv. Cabernet Sauvignon) were grown in field conditions within 70-l containers. The controlled watering process used to obtain plants with different water statuses has been described previously [START_REF] Cuéllar | A grapevine Shaker inward K(+) channel activated by the calcineurin B-like calcium sensor 1-protein kinase CIPK23 network is expressed in grape berries under drought stress conditions[END_REF][START_REF] Cuéllar | Potassium transport in developing fleshy fruits: the grapevine inward K(+) channel VvK1.2 is activated by CIPK-CBL complexes and induced in ripening berry flesh cells[END_REF].

Drought conditions were produced by subjecting plants to a reduced irrigation program over a period of 2 weeks, prior to measuring the leaf water potential (ψ) for at least 2 days before tissue collection [START_REF] Cuéllar | A grapevine Shaker inward K(+) channel activated by the calcineurin B-like calcium sensor 1-protein kinase CIPK23 network is expressed in grape berries under drought stress conditions[END_REF]. The ψ value ranged from -0.7 to -0.6 MPa in drought-treated plants, whereas ψ remained close to -0.2MPa at dawn for control plants that were not subjected to drought stress. All samples were collected at fruit set period with the exception of berry samples, which were harvested at various stages of grape development. Roots and leaves were collected from 2-month-old rooted canes planted in perlite [START_REF] Cuéllar | A grapevine Shaker inward K(+) channel activated by the calcineurin B-like calcium sensor 1-protein kinase CIPK23 network is expressed in grape berries under drought stress conditions[END_REF]. Samples were immediately frozen in liquid nitrogen before being used in the different experiments. For in situ hybridization and histology experiments, fresh organs were collected only in the control condition and were directly embedded in paraffin.

Isolation of cDNAs encoding VvK3.1, VvCIPK and VvCBL proteins

First strand cDNAs from grapevine were synthesized from total RNA prepared from postveraison berries using SuperScript III RT polymerase (Invitrogen 18080-051). VvK3.1 was cloned before the grape genome sequence was made available [START_REF] Jaillon | The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla[END_REF][START_REF] Velasco | A high quality draft consensus sequence of the genome of a heterozygous grapevine variety[END_REF] through a three-step strategy. First, two overlapping EST (expressed sequence tag) sequences (CF518215 and CF519070) corresponding to the 3' region of VvK3.1 cDNA were identified in the Vitis vinifera cv. Cabernet Sauvignon EST library (LIBEST_014375). These were used to design the cf-5' and cf-3' specific primers (Table S1), which amplify a 963 bp fragment. Next, the primers PTK25'-1 and PTK25'-3 were designed based on alignments of group 3 plant Shaker channels identified in different plant species (Arabidopsis thaliana, Solanum lycopersicum, Solanum tuberosum, Hordeum vulgare; http://biowed.pasteur.fr/seqanal/interfaces/clustalw.html). A VvK3.1 cDNA fragment encoding the conserved domains of the channel's hydrophobic core and its C-terminal domain was amplified through two successive PCR runs with PTK25-1/cf-3' and PTK25'- The corresponding full-length clone (GenBank/EMBL XM_002268888.3 ) was generated by nested PCR using primers flanking the 5' and 3' ends of the coding sequence (first step: full5'kt2f1/full3'KT2R1, second step: full5'KT2atgF2/fullKT2withtaaR2) before subcloning into the PCI vector and verification by sequencing of both strands.

VvCIPK03 (Gene ID: 100241657) and VvCIPK02/05 (Gene ID: 100261839), the closest relatives of A. thaliana CIPK6, were previously cloned [START_REF] Cuéllar | Potassium transport in developing fleshy fruits: the grapevine inward K(+) channel VvK1.2 is activated by CIPK-CBL complexes and induced in ripening berry flesh cells[END_REF]. VvCBL04 (Gene ID: 100246874), the closest relative of A. thaliana CBL4, was cloned through nested PCR using VvCBL04F1/VvCBL04R1 and VvCBL04F2/VvCBL04R2 from post-veraison berries. Sequencing of the PCR products confirmed that it corresponds to the expected cDNA.

Localization of VvK3.1 expression by mRNA in situ hybridization

In situ hybridization experiments were performed as described by [START_REF] Cuéllar | A grapevine Shaker inward K(+) channel activated by the calcineurin B-like calcium sensor 1-protein kinase CIPK23 network is expressed in grape berries under drought stress conditions[END_REF].

Briefly, VvK3.1 RNA probes were synthesized using the VvK3.1-specific primers K3.1-124-F/K3.1-124-R (Table S1). An 18S ribosomal probe was used as the control.

Sense and antisense probes were labelled with UTP-digoxigenin during the transcription step. Explants from roots, leaves, petioles and berries at different developmental stages were fixed in 4% paraformaldehyde and embedded in paraffin [START_REF] Cuéllar | A grapevine Shaker inward K(+) channel activated by the calcineurin B-like calcium sensor 1-protein kinase CIPK23 network is expressed in grape berries under drought stress conditions[END_REF].

Samples were cut into 8-µm sections and hybridized overnight before incubation for 1 h at 37°C in the presence of anti-DIG antibody conjugated with alkaline phosphatase (1/500 dilution; Roche, http://www.roche.com). Hybridization signals were detected using either the VectorBlue KIT III (Vector Laboratories, http://www.vectorlabs.com; positive signal is blue) or the BCIP/NBT substrate system (Dako; positive signal is purple). Slides were observed with a DM600 microscope (Qimaging, http://www.qimaging.com) and images were taken with a Qimaging Retiga2000R camera.

Histological staining of petiole cross sections

Petioles were fixed for 24 h in a solution containing 1% glutaraldehyde, 2% paraformaldehyde and 1% caffeine in 0.2 mM phosphate buffer at pH 7.2. Subsequently, the samples were dehydrated through an ethanol series, embedded in Technovit 7100 resin (Heraeus Kulzer), and cut into 3-µm semi-thin sections. These sections were stained with periodic acid-Schiff, which stains polysaccharides (i.e. walls and starch) in red, and naphthol blue-black, which stains both soluble and insoluble proteins in blue. Sections were observed by conventional light microscopy using a DM6000 Leica microscope and photographed.

Total RNA extraction and real-time quantitative RT-PCR analysis

Total RNA extractions from various tissues (leaves, stems, petioles, tendrils and berries at different developmental stages) were performed using the Plant RNeasy extraction kit (QIAGEN, Germany), using the modifications described in [START_REF] Cuéllar | A grapevine Shaker inward K(+) channel activated by the calcineurin B-like calcium sensor 1-protein kinase CIPK23 network is expressed in grape berries under drought stress conditions[END_REF]. Firststrand cDNAs were synthesized with SuperScript III reverse transcriptase (Invitrogen) and used as template for quantitative RT-PCR experiments, as previously described [START_REF] Cuéllar | A grapevine Shaker inward K(+) channel activated by the calcineurin B-like calcium sensor 1-protein kinase CIPK23 network is expressed in grape berries under drought stress conditions[END_REF][START_REF] Cuéllar | Potassium transport in developing fleshy fruits: the grapevine inward K(+) channel VvK1.2 is activated by CIPK-CBL complexes and induced in ripening berry flesh cells[END_REF]. Oligonucleotides were designed using Primer3 (http://frodo.wi.mit.edu/).

Primer pair sequences that are specific to VvK3.1 (K3.1-124-F/K3.1-124-R) and elongation factor 1-alpha (EF1-F/EF1-R; [START_REF] Cuéllar | A grapevine Shaker inward K(+) channel activated by the calcineurin B-like calcium sensor 1-protein kinase CIPK23 network is expressed in grape berries under drought stress conditions[END_REF] are provided in Table S1.

Functional characterization of VvK3.1

Oocyte handling and voltage clamp experiments were performed as previously described [START_REF] Cuéllar | A grapevine Shaker inward K(+) channel activated by the calcineurin B-like calcium sensor 1-protein kinase CIPK23 network is expressed in grape berries under drought stress conditions[END_REF][START_REF] Cuéllar | Potassium transport in developing fleshy fruits: the grapevine inward K(+) channel VvK1.2 is activated by CIPK-CBL complexes and induced in ripening berry flesh cells[END_REF] Pipettes were filled with solutions of 3 M KCl containing 100 (K100), 50 (K50) or 10 mM (K10) potassium gluconate, supplemented respectively with 0, 50 or 90 mM sodium gluconate in 1 mM CaCl 2 , 2 mM MgCl 2 , and 10 mM HEPES (pH 6.5 or 7.5) or 10 mM MES (pH 5.5). Inhibition by Cs + or Ba 2+ was assayed by addition of 10 mM CsCl or 30 mM BaCl to the K100 solution at pH 6.5. Voltage clamp protocols were applied with 1.4 s voltage pulses from +70 to -155 mV (or -170 mV), in -15 mV steps, followed by a 250-ms curves were obtained by plotting steady-state currents at the end of activating voltage pulses against corresponding applied membrane potentials, setting the current value at -140 mV in K100 at pH 6.5 to -1 for each oocyte. Voltage-pulse protocols, data acquisition and analysis were performed using the pClamp 10 program suite (Axon Instruments).

Results

Identification of the grapevine K + channel VvK3.1

Alignments of pore-forming and cyclic nucleotide-binding domains of K + channels cloned in plants were used to design adequate primers for identifying grapevine homologs of A.

thaliana Shaker K + channels. A 2,526 bp cDNA was obtained from the total RNA of postveraison Vitis vinifera (cv. Cabernet Sauvignon) berries, using a combination of reverse transcription (RT)-PCR amplifications and 5' RACE extension. The deduced amino acid polypeptide contains 841 residues and shares sequence homology with other plant Shaker channels. Phylogenetic analysis revealed that this grape Shaker, subsequently named

VvK3.1, is related to AKT2 and belongs to group III of the plant Shaker K + family (Fig. 1a). The corresponding polypeptide displays 68% amino acid sequence identity (ASI)

throughout the entire protein length with its A. thaliana counterpart, AKT2 [START_REF] Lacombe | A shaker-like K(+) channel with weak rectification is expressed in both source and sink phloem tissues of Arabidopsis[END_REF]. Of particular note is that group III is composed of a single member in both the A. thaliana and grapevine Shaker families. Sequence analysis of the VvK3.1 polypeptide identified the typical structural regions of Shaker subunits (Fig. 1b), with a short Nterminal cytosolic domain followed by 6 transmembrane segments. This includes a welldefined pore region between the fifth and the sixth transmembrane domain which contains the potassium-selective signature sequence "TXXTXGYGD" [START_REF] Dreyer | Single mutations strongly alter the K+-selective pore of the K(in) channel KAT1[END_REF]. The large cytosolic C-terminal part begins just after the end of the sixth transmembrane domain (S6) and successively contains a C-linker domain, a cyclic nucleotide-binding domain (CNBD), an ankyrin domain, and a K HA domain [START_REF] Ehrhardt | Association of plant K+(in) channels is mediated by conserved C-termini and does not affect subunit assembly[END_REF][START_REF] Sharma | The role of K(+) channels in uptake and redistribution of potassium in the model plant Arabidopsis thaliana[END_REF][START_REF] Nieves-Cordones | Distinct amino acids in the C-linker domain of the Arabidopsis K+ channel KAT2 determine its subcellular localization and activity at the plasma membrane[END_REF][START_REF] Véry | Molecular biology of K+ transport across the plant cell membrane: what do we learn from comparison between plant species?[END_REF].

The Shaker K + channel gene VvK3.1 is expressed in all tested grapevine organs

To evaluate the expression pattern of the VvK3.1 gene in various grapevine organs, realtime PCR was performed on total RNA extracted from leaves, stems, petioles, tendrils, detected in all analyzed samples (Fig. 2a). This result is in agreement with the previous observation that group III Shaker subunits (AKT2 branch) display the broadest expression spectrum [START_REF] Lacombe | A shaker-like K(+) channel with weak rectification is expressed in both source and sink phloem tissues of Arabidopsis[END_REF][START_REF] Ache | VFK1, a Vicia faba K(+) channel involved in phloem unloading[END_REF][START_REF] Langer | Poplar potassium transporters capable of controlling K+ homeostasis and K+-dependent xylogenesis[END_REF]. VvK3.1 expression in grape berries increased during grape development, reaching a maximal amount in ripe berries (Fig. 2b).

To check the VvK3.1 expression profile at different developmental stages, in situ hybridization analyses were conducted in flowers, as well as in berries at the fruit set and ripening (post-veraison) stages (Fig. 3). In flowers, VvK3.1 transcripts were detected in the phloem (Fig. 3a6). An intense signal was also observed in the inner part of the ovule, named the nucellus, where the embryo sac develops (Fig. 3a4). VvK3.1 expression was also detected to a lesser extent in the epidermis of ovarian locules and at the base of stigmas (Fig. 3a3-a6). During the fruit set stage in berries, VvK3.1 expression was mainly detected in the phloem and in the endosperm, a storage tissue that is derived from double fertilization (Fig. 3b2-b4). During the ripening stage (post-veraison period), VvK3.1 expression was strongly restricted to the phloem and perivascular cells (Fig. 3c3-c5). High levels of VvK3.1 transcripts were exclusively detected in the phloem of leaves (Fig. S1) and roots (Fig. S2).

Interestingly, aside from its expression in the phloem, VvK3.1 was identified in a structure located at both extremities of the petiole (Fig. 4a1-2, white arrows, and c3-6). When drought stress was applied by stopping plant irrigation, these particular zones of the petiole became curved, bringing the leaf parallel to the stem (Fig. 4a1-2). Histological examination of this part of the petiole (Fig. 4b1-3) revealed an asymmetrical organization with specific cell layers organized around the vascular systems at the abaxial face of the petiole (Fig. 4a4, abs). This organization is consistent with a pulvinus structure, as shown under higher magnification (Fig. 4b). In this zone we observed the presence of two different cell types: small cells with large pectin walls, in the outer cell layers (Fig. 4b2-3, red staining); and thin-walled large parenchyma cells containing a large vacuole, in the innermost cell layers (Fig. 4b-c). It is important to note that VvK3.1 expression is detected in both of these cell layers (Fig. 4c5). In plants, the pulvini are involved in leaf movements that occur in response to a variety of endogenous and exogenous stimuli [START_REF] Moran | Osmoregulation of leaf motor cells[END_REF], although so far this structure has not been described in grapevine. The response of grapevine to water stress is highly dependent on the variety and the rootstock, although a limited water stress is generally considered to be beneficial for berry quality. It has been demonstrated, from the start of the veraison stage, that grapevine is relatively tolerant to water deficit. Furthermore, regulated irrigation has been advantageously used to inhibit vine growth without any reduction in berry yield (Kennedy et al., 2000;[START_REF] Esteban | Effect of irrigation on changes in the anthocyanin composition of the skin of cv. Tempranillo (Vitis vinifera L) grape berries during ripening[END_REF]. Here, the effects of moderate drought stress (-0.7 to -0.6 MPa for at least 2 days before tissue harvesting) were reproduced in field conditions, as described above. Berries were sampled after flowering (AF) at three stages of development: post-fruit set stage (day 25 AF), veraison (day 60 AF), and during ripening (day 75 AF). VvK3.1 expression in berries was then investigated by real-time quantitative PCR (qPCR), and a slight increase in VvK3.1 transcript levels was observed upon drought stress (Fig. 5a), particularly in the early stages. Nevertheless, this induction of VvK3.1 transcript in response to drought stress was far less considerable than the quantity previously observed for VvK1.2 [START_REF] Cuéllar | Potassium transport in developing fleshy fruits: the grapevine inward K(+) channel VvK1.2 is activated by CIPK-CBL complexes and induced in ripening berry flesh cells[END_REF].

Effect of drought stress on

VvK3.1 transcript accumulation was also investigated by qPCR in leaves and roots harvested from water-stressed or control rooted canes. Drought stress, induced by stopping irrigation, was found to have different effects on VvK3.1 transcript levels in roots and leaves. Specifically, drought stress increased VvK3.1 transcript accumulation in leaves (Fig. 5b) but had no effect on the level of VvK3.1 in roots (Fig. 5c). In leaves, a significant (threefold) up-regulation in VvK3.1 was observed in response to water stress.

VvK3.1 behaves as a weak K + inward rectifier channel in Xenopus oocytes

Functional properties of the grapevine channel VvK3.1 were investigated in Xenopus laevis oocytes. Instantaneous and time-dependent currents (Fig. 6a) were reminiscent of those observed with the A. thaliana Shaker channel AKT2 previously characterized as a weak K + inward rectifier channel [START_REF] Philippar | Auxin-induced K+ channel expression represents an essential step in coleoptile growth and gravitropism[END_REF][START_REF] Lacombe | A shaker-like K(+) channel with weak rectification is expressed in both source and sink phloem tissues of Arabidopsis[END_REF][START_REF] Ache | VFK1, a Vicia faba K(+) channel involved in phloem unloading[END_REF][START_REF] Langer | Poplar potassium transporters capable of controlling K+ homeostasis and K+-dependent xylogenesis[END_REF]. The observed currents were dependent on external potassium concentrations and exhibited smaller amplitudes at reduced external K + concentrations (Fig. 6b). Reversal potentials were found to shift towards more hyperpolarized potentials in response to decreasing K + concentrations (Fig. S3a). VvK3.1 was inhibited in a voltagedependent manner by Cs + (Fig. 6c), with an increase in channel blocking at more negative potentials as previously described for AKT2 [START_REF] Lacombe | A shaker-like K(+) channel with weak rectification is expressed in both source and sink phloem tissues of Arabidopsis[END_REF]. Addition of 30 mM Ba 2+ also provoked a strong inhibition of currents (Fig. S3b), reminiscent of the complete AKT2 blocking by Ba 2+ [START_REF] Marten | AKT3, a phloem-localized K+ channel, is blocked by protons[END_REF]. shown similarly for A. thaliana AKT2 channel activity [START_REF] Lacombe | A shaker-like K(+) channel with weak rectification is expressed in both source and sink phloem tissues of Arabidopsis[END_REF], and in contrast to the behavior of inward rectifiers such as VvK1.2 [START_REF] Cuéllar | Potassium transport in developing fleshy fruits: the grapevine inward K(+) channel VvK1.2 is activated by CIPK-CBL complexes and induced in ripening berry flesh cells[END_REF]. Indeed, VvK3.1 activity was stimulated by an increase in external pH, suggesting an interference of protons with the channel permeation. Upon external acidification, the proportions of instantaneous as well as time-dependent currents were modified. For VvK3.1, inhibition of the time-dependent component was slightly stronger than that of the instantaneous current (Fig. 7b). This should result in a reduced inward rectification capacity of its residual current, whereas the opposite was found for AKT2 [START_REF] Lacombe | A shaker-like K(+) channel with weak rectification is expressed in both source and sink phloem tissues of Arabidopsis[END_REF].

Another level of Shaker K + channel activity regulation is based on CIPK-CBL interactions.

Indeed, the A. thaliana Shaker channel AKT2 was previously shown to be regulated by the CIPK6/CBL4 complex (calcineurin B-like (CBL)-interacting protein kinase 6 (CIPK) and Ca 2+-sensing CBL 4 partners) [START_REF] Held | Calciumdependent modulation and plasma membrane targeting of the AKT2 potassium channel by the CBL4/CIPK6 calcium sensor/protein kinase complex[END_REF]. Since the mechanisms of K + channel regulation involving CIPK-CBL complexes are conserved between A. thaliana and grapevine for other Shaker-type channels [START_REF] Cuéllar | A grapevine Shaker inward K(+) channel activated by the calcineurin B-like calcium sensor 1-protein kinase CIPK23 network is expressed in grape berries under drought stress conditions[END_REF][START_REF] Cuéllar | Potassium transport in developing fleshy fruits: the grapevine inward K(+) channel VvK1.2 is activated by CIPK-CBL complexes and induced in ripening berry flesh cells[END_REF], we also evaluated this regulation for VvK3.1. Thus, cDNAs from VvCIPK03, VvCIPK02/05 and VvCBL04, which are the closest grapevine relatives of A. thaliana AtCIPK6 and AtCBL4 (Fig. S4),

were cloned from post-veraison berries to assess whether this regulatory pathway is conserved in grapevine. When expressed without the channel, the VvCIPK03/VvCBL04

and VvCIPK02/05/VvCBL04 complexes did not elicit any significant currents in oocytes in comparison to water-injected oocytes (Fig. 8a, top). Conversely, even though the VvK3.1 channel was able to mediate K + currents when expressed alone (Fig. 8a, middle), co-expression with these two CIPK/CBL partners led to an increase in the current magnitude (Fig. 8a, bottom). Indeed, co-expression of VvK3.1 together with VvCIPK03/CBL04 significantly increased the K + current by about 60% at -140 mV, in comparison to the current recorded when the VvK3.1 channel was expressed alone in oocytes (Fig. 8b). The other tested CIPK-CBL complex, VvCIPK02/05/VvCBL04, provoked a weaker increase in K + current, by about 20% at -140 mV (Fig. 8b). Finally, it is worth noting that the relative proportions of the instantaneous and time-dependent components of the VvK3.1 currents were not altered by these two CIPK/CBL complexes (Fig. 8C). Similarly, when the AKT2 channel is co-expressed with AtCIPK3-AtCBL4 complex, no significant change in the proportion of the two current components is observed [START_REF] Held | Calciumdependent modulation and plasma membrane targeting of the AKT2 potassium channel by the CBL4/CIPK6 calcium sensor/protein kinase complex[END_REF]. In grapevine, VvK3.1 is the only subunit belonging to the third group of Shaker channels representing a subfamily of weak rectifiers. Similarly, this group contains a single subunit in A. thaliana, named AKT2. With 68% amino acid sequence identity, VvK3.1 is the closest relative of AKT2 in grapevine (Fig. 1). The Arabidopsis AKT2 channel is known to dominate the phloem K + conductance in both sieve and companion cells, where it has a major role in phloem K + loading and unloading [START_REF] Marten | AKT3, a phloem-localized K+ channel, is blocked by protons[END_REF][START_REF] Deeken | Developmental and light-dependent regulation of a phloem-localised K+ channel of Arabidopsis thaliana[END_REF][START_REF] Deeken | Loss of the AKT2/3 potassium channel affects sugar loading into the phloem of Arabidopsis[END_REF][START_REF] Lacombe | A shaker-like K(+) channel with weak rectification is expressed in both source and sink phloem tissues of Arabidopsis[END_REF][START_REF] Ache | VFK1, a Vicia faba K(+) channel involved in phloem unloading[END_REF][START_REF] Philippar | The K+ channel KZM1 mediates potassium uptake into the phloem and guard cells of the C4 grass Zea mays[END_REF][START_REF] Ivashikina | AKT2/3 subunits render guard cell K+ channels Ca2+ sensitive[END_REF][START_REF] Hafke | Functional sieve element protoplasts[END_REF]. When expressed in X. laevis oocytes, the AKT2 channel forms a weakly rectifying channel, thereby giving rise to two current components with different gating modes [START_REF] Marten | AKT3, a phloem-localized K+ channel, is blocked by protons[END_REF][START_REF] Lacombe | A shaker-like K(+) channel with weak rectification is expressed in both source and sink phloem tissues of Arabidopsis[END_REF][START_REF] Dreyer | A plant Shaker-like K+ channel switches between two distinct gating modes resulting in either inward-rectifying or "leak" current[END_REF]Michard et al., 2005a). This unique feature allows AKT2 to mediate inward as well as outward K + currents. As shown in Fig. 8, the VvK3.1 channel can also combine two gating modes: a time-dependent voltage-activated current, and an instantaneous ohmic current, like AKT2.

Although it has been described that AKT2 can switch between these gating modes via post-translational regulation (Michard et al., 2005a), the underlying mechanisms are still not fully understood [START_REF] Sandmann | The K (+) battery-regulating Arabidopsis K (+) channel AKT2 is under the control of multiple post-translational steps[END_REF].Two serine residues (S210 and S329), with one located in the S4-S5 linker and the other in the C-linker, have been identified as phosphorylation targets [START_REF] Dreyer | A plant Shaker-like K+ channel switches between two distinct gating modes resulting in either inward-rectifying or "leak" current[END_REF]Michard et al., 2005aMichard et al., , 2005b)). Moreover, the lysine (K197) located within the voltage sensor region enables AKT2 to sense its phosphorylation status and to change between the two modes (Michard et al., 2005b;[START_REF] Sandmann | The K (+) battery-regulating Arabidopsis K (+) channel AKT2 is under the control of multiple post-translational steps[END_REF]. Interestingly, these two serines and this lysine are present in the corresponding position in the VvK3.1 sequence, strongly indicating that VvK3.1 behaves similar to AKT2.

Moreover, in oocytes, the AKT2-like channels are inhibited upon external acidification [START_REF] Marten | AKT3, a phloem-localized K+ channel, is blocked by protons[END_REF][START_REF] Philippar | Auxin-induced K+ channel expression represents an essential step in coleoptile growth and gravitropism[END_REF][START_REF] Lacombe | A shaker-like K(+) channel with weak rectification is expressed in both source and sink phloem tissues of Arabidopsis[END_REF][START_REF] Langer | Poplar potassium transporters capable of controlling K+ homeostasis and K+-dependent xylogenesis[END_REF]. Our results demonstrate that the VvK3.1 channel displays a similar pH sensitivity, with one minor difference: the relative proportions of the instantaneous and time-dependent currents are differently disturbed as compared to AKT2 (Fig. 7). Indeed, external acidification results in an increased inward rectification of the AKT2 residual current [START_REF] Lacombe | A shaker-like K(+) channel with weak rectification is expressed in both source and sink phloem tissues of Arabidopsis[END_REF], whereas under the same conditions the instantaneous component prevails in the In addition, several reports have identified associations between AKT2 and different putative partners [START_REF] Chérel | Physical and functional interaction of the Arabidopsis K(+) channel AKT2 and phosphatase AtPP2CA[END_REF][START_REF] Held | Calciumdependent modulation and plasma membrane targeting of the AKT2 potassium channel by the CBL4/CIPK6 calcium sensor/protein kinase complex[END_REF][START_REF] Sklodowski | The receptor-like pseudokinase MRH1 interacts with the voltage-gated potassium channel AKT2[END_REF]. For example, the association of AKT2 with the AtCIPK6/AtCBL4 complex gives rise to an increase in macroscopic AKT2 currents, by enhancing the targeting of AKT2 to the plasma membrane [START_REF] Held | Calciumdependent modulation and plasma membrane targeting of the AKT2 potassium channel by the CBL4/CIPK6 calcium sensor/protein kinase complex[END_REF]. Since VvK3.1 appears to be closely related to AKT2, we assumed that a homologous regulatory network could control its activity at the cell membrane. In X. laevis oocytes, co-expression of VvCBL04 with VvCIPK03 or VvCIPK02/05, the closest relatives respectively of AtCBL4 and AtCIPK6 (Fig. S4) [START_REF] Cuéllar | Potassium transport in developing fleshy fruits: the grapevine inward K(+) channel VvK1.2 is activated by CIPK-CBL complexes and induced in ripening berry flesh cells[END_REF], produced an increase in the VvK3.1 current. This increase displayed a higher magnitude in the VvCIPK03-VvCBL04 pair (Fig. 8). Moreover, no significant effect on the relative proportions of the instantaneous and time-dependent currents was observed (Fig. 8c), similar to previous observations with AKT2 [START_REF] Held | Calciumdependent modulation and plasma membrane targeting of the AKT2 potassium channel by the CBL4/CIPK6 calcium sensor/protein kinase complex[END_REF].

Overall, the functional properties of VvK3.1 and AKT2 indicate that these two channels have very similar behaviors, providing additional support to the phylogenetic relationship between them and the group III plant Shaker K + channels. More generally, our results, together with those described earlier for VvK1.1 and VvK1.2 [START_REF] Cuéllar | A grapevine Shaker inward K(+) channel activated by the calcineurin B-like calcium sensor 1-protein kinase CIPK23 network is expressed in grape berries under drought stress conditions[END_REF][START_REF] Cuéllar | Potassium transport in developing fleshy fruits: the grapevine inward K(+) channel VvK1.2 is activated by CIPK-CBL complexes and induced in ripening berry flesh cells[END_REF], reinforce the observation that the mechanisms of K + channel regulation by CIPK-CBL complexes are conserved between A. thaliana and grapevine.

Role of the VvK3.1 channel in berry K + loading

Berry development consists of two successive growth periods. The first period comprises a rapid cell division phase followed by marked cell enlargement. This period is characterized by the synthesis and vacuolar storage of tartaric and malic acids. The second period commences with veraison, at the onset of ripening. At veraison, the berry starts to change color and soften, as it becomes a strong sink for water, K + , sugar and solute imports [START_REF] Davies | Transporters expressed during grape berry (Vitis vinifera L.) development are associated with an increase in berry size and berry potassium accumulation[END_REF][START_REF] Conde | Biochemical changes throughout grape berry development and fruit and wine quality[END_REF]. At this stage, berry loading is dependent on phloem sap flux, since the xylem has become non-functional [START_REF] Keller | Ripening grape berries remain hydraulically connected to the shoot[END_REF]Chatelet et al., 2008aChatelet et al., , 2008b;;[START_REF] Choat | Vascular function in grape berries across development and its relevance to apparent hydraulic isolation[END_REF][START_REF] Knipfer | Water Transport Properties of the Grape Pedicel during Fruit Development: Insights into Xylem Anatomy and Function Using Microtomography[END_REF]. Moreover, K + transport in the berry switches from the symplasmic to the apoplasmic mode [START_REF] Zhang | A shift of Phloem unloading from symplasmic to apoplasmic pathway is involved in developmental onset of ripening in grape berry[END_REF], meaning that unloaded solutes must cross plasma membranes at least twice before accumulating within the berry mesocarp. Previous research has posited that berry loading via an apoplasmic mode should reduce the transport of apoplasmic solutes out of the 14 berries [START_REF] Findlay | Solute accumulation by grape pericarp cells. IV. Perfusion of pericarp apoplast via the pedicel and evidence for xylem malfunction in ripening berries[END_REF][START_REF] Mpelasoka | A review of potassium nutrition in grapevines with special emphasis on berry accumulation[END_REF] and improve the control of efficient long-distance phloem transport from source leaves to ripening berries [START_REF] Zhang | A shift of Phloem unloading from symplasmic to apoplasmic pathway is involved in developmental onset of ripening in grape berry[END_REF].

Starting at veraison, the VvK3.1 channel is highly expressed in the berry phloem (Figs. 2 and3) and becomes the obvious candidate for loading K + into the berry. The unique functional characteristics of the VvK3.1 channel are indeed suited for this. In this scenario, the VvK3.1 channel would be switched from an inward-rectifying to a non-rectifying mode, so as to enable massive K + efflux, i.e. K + secretion into the berry mesocarp apoplasm.

Taking into account previous studies on AKT2 functioning [START_REF] Marten | AKT3, a phloem-localized K+ channel, is blocked by protons[END_REF][START_REF] Lacombe | A shaker-like K(+) channel with weak rectification is expressed in both source and sink phloem tissues of Arabidopsis[END_REF][START_REF] Dreyer | A plant Shaker-like K+ channel switches between two distinct gating modes resulting in either inward-rectifying or "leak" current[END_REF]Michard et al., 2005aMichard et al., , 2005b) ) and the fact that the AKT2 and VvK3.1 channels have very similar functional behaviors, VvK3.1 should be locked in the open state in its phosphorylated form across the entire physiological voltage range.

Therefore, this channel may drive the K + efflux that allows K + ions to move down their transmembrane concentration gradient (100 mM in the phloem cell cytoplasm and 1 mM in the apoplasm; [START_REF] Ache | VFK1, a Vicia faba K(+) channel involved in phloem unloading[END_REF][START_REF] Wada | Fruit ripening in Vitis vinifera: apoplastic solute accumulation accounts for pre-veraison turgor loss in berries[END_REF].

It is known that K + gradients play a major role in driving sugar, amino acid and water transport across plant cell membranes during phloem unloading [START_REF] Ache | VFK1, a Vicia faba K(+) channel involved in phloem unloading[END_REF][START_REF] Philippar | The K+ channel KZM1 mediates potassium uptake into the phloem and guard cells of the C4 grass Zea mays[END_REF]. However, the remarkable increase in solute import in berries at veraison signifies a challenge in terms of transport energization. Electrochemical gradients dependent on the H + -ATPase activity may be reduced in berries since, from the start of veraison, the stomata are no longer functional, photosynthesis has stopped, and ATP synthesis mostly depends on cellular respiration with malic acid as the substrate [START_REF] Kanellis | Grape[END_REF]. Under such circumstances, the recent concept of a K + battery for transmembrane transport processes seems appropriate, since it provides additional energy stored in the K + gradient between the phloem cytosol and the berry apoplasm [START_REF] Gajdanowicz | Potassium (K+) gradients serve as a mobile energy source in plant vascular tissues[END_REF][START_REF] Sandmann | The K (+) battery-regulating Arabidopsis K (+) channel AKT2 is under the control of multiple post-translational steps[END_REF][START_REF] Dreyer | The potassium battery: a mobile energy source for transport processes in plant vascular tissues[END_REF]. The K + battery also explains how an open AKT2-like channel, like VvK3.1, can drive K + ions from the cytosol to the apoplasm as well as reinforce the transmembrane electrical gradient by hyperpolarizing the plasma membrane potential. This electrical gradient can compensate for the reduced pH gradient present under energy limitation. Furthermore, it can be used to retrieve sucrose from the apoplasm and to maintain sucrose levels in the phloem vessels until reaching the different sites of unloading.

In grapevine, sucrose is used for long-distance transport in the phloem from source leaves to grape berries, and sugar loading into berries is performed from veraison onward and during the ripening period [START_REF] Kuhn | Sucrose transporters of higher plants[END_REF][START_REF] Lecourieux | An update on sugar transport and signalling in grapevine[END_REF]. Anatomical sections of grape berries reveal a large network of vascular bundles with a central capillary bundle and many major and minor peripheral capillary bundles that irrigate the berry [START_REF] Zhang | A shift of Phloem unloading from symplasmic to apoplasmic pathway is involved in developmental onset of ripening in grape berry[END_REF]. This observation strongly suggests that sucrose retrieval could be performed over the entire length of the grape berry's vascular bundle (from the different sites of phloem unloading), in order to distribute the large amount of sucrose to all parts of the berry mesocarp apoplasm.

The main advantage of this K + battery mechanism is that it does not require ATP consumption [START_REF] Dreyer | The potassium battery: a mobile energy source for transport processes in plant vascular tissues[END_REF]. Thus, it is tempting to speculate that the non-rectifying mode of VvK3.1 contributes to K + secretion into the grape berry apoplasm, in addition to switching on the K + battery to allow sucrose retrieval in energy-limited conditions. At this stage, the inwardly rectifying VvK1.2 channel [START_REF] Cuéllar | Potassium transport in developing fleshy fruits: the grapevine inward K(+) channel VvK1.2 is activated by CIPK-CBL complexes and induced in ripening berry flesh cells[END_REF] that is highly expressed in flesh cells and perivascular cells surrounding the vascular bundles would then contribute to the berry loading mechanisms. In particular, this Shaker channel is strongly activated by interacting with specific VvCIPK/VvCBL pairs. This can allow rapid absorption of K + by perivascular cells and then by flesh cells to keep the apoplasmic K + concentration at low levels, despite the efflux from the phloem stream. Indeed, since the transmembrane gradient of K + can be maintained between phloem cells and the berry apoplast, the phloem stream flux towards the sink may be stimulated and employment of the K + battery process for sucrose retrieval may persist over a long period of time. This latter process should also allow saving ATP, which in grape berry is locally produced by cell respiration, for other transport mechanisms driven by ATP-consuming H + -ATPase.

VvK3.1 and K + transport in the grapevine pulvinus

In addition to the phloem, VvK3.1 is also strongly expressed in a second tissue located at the two ends of the petiole (Fig. 4). The careful histological examination of this zone (Fig.

4. a1-2) enabled us to identify this as a specialized motor organ known as the pulvinus.

Nastic, paraheliotropic and diaheliotropic movements are mediated by this organ, allowing it to play a key role in leaf movements [START_REF] Pastenes | Paraheliotropism can protect water-stressed bean (Phaseolus vulgaris L.) plants against photoinhibition[END_REF]. Because grapevine is a major crop in both warm and drought-prone areas, this plant has the ability to perform solar tracking in order to reduce leaf temperature and transpirational water loss, and protect its photosynthetic activity. In this context, several studies on the impacts of excess light on the photosynthesis rate in grapevine leaves have been conducted [START_REF] During | CO2 assimilation and phosphorespiration of grapevine leaves: Responses to light and drought[END_REF][START_REF] Iandolino | Simulating three-dimensional grapevine canopies and modelling their light interception characteristics[END_REF][START_REF] Keller | Partitioning of assimilates[END_REF]. High irradiance induces photodamage to photosystem II (PSII)

F o r P e e r R e v i e w
16 in leaves [START_REF] Adir | Photoinhibition -a historical perspective[END_REF][START_REF] Pastenes | Paraheliotropism can protect water-stressed bean (Phaseolus vulgaris L.) plants against photoinhibition[END_REF][START_REF] Takahashi | Different thermal sensitivity of the repair of photodamaged photosynthetic machinery in cultured Symbiodinium species[END_REF] and leads to photoinhibition, which can limit plant photosynthesis activity, growth and productivity.

Several mechanisms have been identified to protect or repair PSII under conditions of excess light energy [START_REF] Müller | Non-photochemical quenching. A response to excess light energy[END_REF][START_REF] Takahashi | Impairment of the photorespiratory pathway accelerates photoinhibition of photosystem II by suppression of repair but not acceleration of damage processes in Arabidopsis[END_REF][START_REF] Takahashi | Different thermal sensitivity of the repair of photodamaged photosynthetic machinery in cultured Symbiodinium species[END_REF][START_REF] Huang | Cyclic electron flow plays an important role in photoprotection for the resurrection plant Paraboea rufescens under drought stress[END_REF].

Among them, leaf movements have been observed in several plant species in response to direct sunlight [START_REF] Takahashi | Photoprotection in plants: a new light on photosystem II damage[END_REF]. In a light-avoiding movement known as paraheliotropism, the grapevine leaves orient at an angle parallel to the sun's rays [START_REF] Iandolino | Simulating three-dimensional grapevine canopies and modelling their light interception characteristics[END_REF][START_REF] Keller | Partitioning of assimilates[END_REF]. This movement helps to prevent photodamage of PSII and minimizes photoinhibition [START_REF] Pastenes | Leaf movements and photoinhibition in relation to water stress in field-grown beans[END_REF][START_REF] Takahashi | Different thermal sensitivity of the repair of photodamaged photosynthetic machinery in cultured Symbiodinium species[END_REF].

Paraheliotropic movements are also observed when environmental conditions change, e.g.

when exposed to excess light, high temperatures, or drought stress [START_REF] During | CO2 assimilation and phosphorespiration of grapevine leaves: Responses to light and drought[END_REF][START_REF] Pastenes | Leaf movements and photoinhibition in relation to water stress in field-grown beans[END_REF][START_REF] Iandolino | Simulating three-dimensional grapevine canopies and modelling their light interception characteristics[END_REF].

These grapevine leaf movements are governed via common plant mechanisms based on differentially changing cell turgor within the pulvinus. Substantial information about pulvinus structure and function has been gained by studying the nyctinastic leguminous tree Samanea saman [START_REF] Satter | The effects of blue and far red light on rhythmic leaflet movements in samanea and albizzia[END_REF]Gorton, 1987aGorton, , 1987b;;[START_REF] Yu | Extracellular protons inhibit the activity of inwardrectifying potassium channels in the motor cells of Samanea saman pulvini[END_REF][START_REF] Yu | Phosphorylation of SPICK2, an AKT2 channel homologue from Samanea motor cells[END_REF]. In this species, two groups of specialized motor cells arranged in two opposite zones can be distinguished in the pulvinus: the flexor cell, located in the adaxial side; and the extensor cell, located in the abaxial side [START_REF] Uehlein | Aquaporins and plant leaf movements[END_REF]. Similar to the guard cells, solute content and ion composition (which are mainly dependent on potassium) induce osmotic water fluxes in the motor cells, allowing them to change their volume and shape according to turgor changes. Accordingly, uptake of K + can lift the leaves through swelling of the abaxial extensor cells, concomitant with shrinking of the adaxial flexor cells (Fig. 4a). Upon redistribution of potassium ions, the opposite effect takes place: the extensor cells shrink and the flexor cells swell, lowering the position of the leaves (Fig. 4b) (Gorton, 1987a and b;[START_REF] Satter | Potassium flux and leaf movement in Samanea saman. I. Rhythmic movement[END_REF]. Moreover, our in situ hybridization experiments detected an intense expression of VvK3.1 in this structure, which was restricted to the abaxial side of the pulvinus, where the extensor cells are usually found (Fig. 4c3).

Taking into account the unique functional features of VvK3.1, we propose that this channel mediates both inward and outward K + currents to allow for the influx or efflux of potassium involved in either the swelling or shrinking of extensor cells. The activity of VvK3.1-like channels has already been recorded in Samanea saman motor cells, with results that support our hypothesis [START_REF] Moshelion | Diurnal and circadian regulation of putative potassium channels in a leaf moving organ[END_REF][START_REF] Yu | Phosphorylation of SPICK2, an AKT2 channel homologue from Samanea motor cells[END_REF]. Interestingly, pH values recorded in the Samanea saman extensor apoplast have been observed to range 17 from 6.5 to 7.2 [START_REF] Lee | Effects of white, blue, red light and darkness on pH of the apoplast in the Samanea pulvinus[END_REF], which would be compatible with a significant

VvK3.1 activity in extensor cells. Since VvK3.1 is only expressed in extensor cells, two alternative hypotheses may be considered for the mechanism that takes place in flexor cells. The first one is that the elasticity of pectin walls in the parenchyma cells of the flexor tissue is sufficient to facilitate and accommodate leaf movements. The second hypothesis speculates that there are other transport systems involved in K + conductance in grapevine flexor cells [START_REF] Yu | Extracellular protons inhibit the activity of inwardrectifying potassium channels in the motor cells of Samanea saman pulvini[END_REF][START_REF] Moshelion | Diurnal and circadian regulation of putative potassium channels in a leaf moving organ[END_REF].

Here, we have provided evidence that the VvK3.1 channel, in addition to its role in grapevine pulvinus functioning, may play a key role in berry loading during grape berry ripening by controlling phloem K + transport. In grape berries, veraison occurs at a time of profound developmental changes. The shift from the symplasmic to the apoplasmic mode of phloem unloading modifies the translocation pathways. The VvK3.1 and VvK1.2 [START_REF] Cuéllar | Potassium transport in developing fleshy fruits: the grapevine inward K(+) channel VvK1.2 is activated by CIPK-CBL complexes and induced in ripening berry flesh cells[END_REF] channels are both expressed in perivascular cells (Fig. S5), and are likely involved in the reorganization of the K + transport mechanisms that take place during the loading of ripening berries. Moreover, the expression of these two channels is upregulated upon drought stress exposure, suggesting a compensatory measure to control K + transport, thereby preventing the decrease of water availability. Our results strongly indicate that a better understanding of the mechanisms regulating K + transport during berry maturation will likely help to stimulate advances in our knowledge of grapevine drought stress adaptation, berry development, and the determinants of fruit acidity and quality. S1: Primers used in this study.

   5' RACE was performed to obtain the 5' end of VvK3.1 cDNA, using the primers AS5, AS6 and AS8.

  -40 mV at a holding potential of 0 mV. Normalized current-voltage (I-V)

  at different stages of development. High VvK3.1 transcript levels were

  external solution reduced VvK3.1 current amplitudes (Fig.7a), as

Fig. 1 :Fig. 2 :

 12 Figure legends

Fig. 3 :

 3 Fig. 3: In situ localization of VvK3.1 transcripts in flowers and berries at fruit set and ripening stages Longitudinal and equatorial sections were hybridized with VvK3.1 RNA sense probe (left column: a1, a2, b1, c1, c2) and antisense probe (right columns). Sections hybridized with the sense probe (negative control) did not show any significant signal. A positive blue signal was observed in flowers (a), as well as in berries at fruit set (b) and at post-veraison (c), when the sections were hybridized with VvK3.1 antisense probe. (a) Flowers: signals were detected in the phloem, ovary and ovule. In the ovary, a blue color was observed in the epidermis bordering the ovarian locule. In the ovule, a strong signal was detected in the nucellus. (b) Berries at fruit set: blue signals were observed in the endosperm and the phloem.(c) Berries during ripening: an intense signal was detected in the vascular bundles, where the expression of VvK3.1 is restricted to phloem and perivascular cells.

Fig. 4 .

 4 Fig. 4. Expression of VvK3.1 in pulvinus structures located in the petiole (a) Anatomical position of the pulvinus and cross sections. A vine branch was photographed before (a1) and after (a2) the onset of drought stress (brought on by stopping irrigation). Under drought stress, the petiole position dropped, becoming almost perpendicular to the stem. The positions of pulvinus structures (white arrows) are clearly visible, with one located toward the stem side and the other toward the leaf side. Cross sections of the petiole were observed by stereomicroscope (a3) or light microscope after histological staining (a4). (b) Histological staining of petiole cross sections. Three-micrometer semi-thin sections were stained with naphthol blue-black and periodic acid-Schiff. Phloem elements are distinguishable by their dark purple cell walls stained by periodic acid-Schiff (b3). The pulvinus is composed of thin-walled parenchyma with intercellular spaces arranged around the vascular bundle (b2, b3).

  In situ hybridizations with the VvK3.1 RNA sense probe (left column) and antisense probe (right column) are shown. A negative control was performed with the VvK3.1 sense probe and no signal was observed (c1 and c2). When the cross sections were hybridized with VvK3.1 antisense probe, intense signals (blue color) were detected in the phloem sap (c3, c4 and c6) and in the abaxial side of the pulvinus structure (c3, c5 and c6). No signal was observed on the adaxial side.Ad.s, adaxial side; Ab.s, abaxial side; Ep, epiderm; ICL, innermost cell layer; OCL, outer cell layer; Ph, phloem; Pul, pulvinus; VB, vascular bundles; Xy, xylem.

Fig. 5 .Fig. 6 .

 56 Fig. 5. Regulation of VvK3.1 expression in response to drought stress VvK3.1 transcript accumulation was analyzed by real-time quantitative PCR on total RNA. (a) Berries were collected from field-grown 4-year-old plants under controlled irrigation. Drought stress was applied by decreasing the level of irrigation for 15 days before berries were collected. At this point, the leaf water potential was then in the range of -0.7 to -0.6 MPa in drought stressed plants, compared with -0.2 MPa in control plants under standard irrigation. Absolute transcript levels of the control (white bars) or water-stressed berries (grey bars) were normalized using EF1-alpha transcript signals. (b) Leaves and (c) roots were collected from rooted canes. The plants were subjected to drought stress by stopping irrigation for 10 days. Data are expressed relative to VvK3.1 transcript accumulation in plant material collected at t = 0. All data presented are the means (± SE) of two biological replicates.

Fig. 7 :Fig. 8 :

 78 Fig. 7: The grapevine channel VvK3.1 is strongly regulated by pH (a) VvK3.1 currents are regulated by external pH. Current-voltage curves are shown for normalized VvK3.1 currents in 100 mM K + at pH 5.5, 6.5 and 7.5 (n=15 ± SD). Currents were normalized for each oocyte, setting the current value at -140 mV in K100 pH 6.5 to -1. Note that the currents are reduced by external acidification. (b) Proportion (i.e. percentage scale) of the time-dependent (grey) and instantaneous (black) currents as a function of external pH. (n=15 ± SD). Oocytes were injected withVvK3.1 cRNA and steady-state currents were recorded at -140 mV before analysis. Note that the proportion of instantaneous current is stronger at pH 5.5 than at pH 7.5.

Fig. S1 :

 S1 Fig. S1: Localization of VvK3.1 transcript in leaves by in situ hybridization.

Fig. S2 :

 S2 Fig. S2: Localization of VvK3.1 transcripts in roots by in situ hybridization.

Fig. S3 :

 S3 Fig.S3: Reversal potential and Ba 2+ inhibition.

Fig. S4 :

 S4 Fig. S4: Phylogenetic relationships of CIPKs and CBLs in Arabidopsis and grapevine.

Fig. S5 :Fig. 3 :Fig. 8 :

 S538 Fig. S5: Expression of VvK3.1 and VvK1.2 in perivascular cells as revealed by in situ hybridization

Manuscript submitted to New Phytologist for review

Acknowledgements:

This work was supported by SweetKaliGrape ANR (ANR-33 14-CE20-0002-02). M.N.-C. received a grant from the Marie Curie program (FP7-IEF, grant no. 272390), and M.A. received a scholarship from the Conabex Commission of Madagascar.