Villette J , Cuéllar T , Zimmermann SD , Verdeil JL , Gaillard I .

	Fig 1. The phylogenetic relationships of VvK5.1. An unrooted phylogenetic tree was constructed with 16 polypeptide Shaker K+ channel sequences belonging to the outwardly rectifying potassium channel subfamily. VvK5.1 (XP_010660282.1) is the grapevine Shaker channel. Other sequenced species close to VvK5.1 are AtSKOR (At3g02850) and AtGORK (At5g37500) from Arabidopsis thaliana; SlSKOR1 (XP_004240037.1) and SlSKOR2 (XP_004250206.1) from tomato (Solanum lycopersicum); CcSKOR1 (XP_006421368.1) and CcSKOR2 (XP_006427880.1) from clementine (Citrus clementina); CisSKOR1 (XP_006464550.1) and CisSKOR2 (XP_015389397.1) from orange tree (Citrus sinensis); CmSKOR1 (XP_008460504.1) from melon (Cucumis melo); CusSKOR1 (XP_004140369.2) from cucumber (Cucumis sativa); MnSKOR1 (XP_010108959.1) from blackberry (Morus notabilis); MdSKOR1 (XP_008343075.1) and MdSKOR2 (XP_008381509.1) from apple (Malus domestica); and PtSKOR1 (XP_006372521.1) and PtSKOR2 (XP_002305894.2) from poplar (Populus trichocarpa). Bootstrap values are reported next to the nodes of the tree. Branch length is proportional to the evolutionary distance between the outward rectifying potassium channels of different dicotyledon species.
	Fig 2. Functional characterization of the grapevine channel VvK5.1 by heterologous expression in Xenopus laevis oocytes. (A) Representative current traces in response to voltage–clamp pulses from –110 mV to +40 mV that depend on varying external K+ concentrations at pH 7.4 as indicated. Time-dependent outwardly rectifying currents are activated upon depolarization. (B) Current–voltage curves for mean currents mediated by VvK5.1 that depend on K+ concentrations; n=10 ±SE. Note the shift in the activation potential at the reduced external K+. (B1) VvK5.1 reversal potentials (Erev) were determined from tail current analysis, indicating a shift in dependence on external K+ concentrations as shown; n=10 ±SE. The line in the insert corresponds to the predicted Nernst potentials of K+. Tail currents were analysed at voltage pulses ranging from –110 mV to +40 mV, following activation of the channel at +30 mV. (C) Current–voltage curves for mean currents mediated by VvK5.1 that depend on K+ concentrations; n=8 ±SE. (C1) The maximal currents at +50 mV decreased between 10 mM and 0.1 mM K+, despite the increase in driving force.
	Fig 3. Functional properties of the grapevine channel VvK5.1 when expressed in Xenopus laevis oocytes. (A) Dependence of VvK5.1-normalized mean currents upon external pH in 10 mM K+; n=8 ±SE. Normalization was conducted by setting the currents at +50 mV in the standard solution (pH 6.5) to 100%. The current is reduced upon acidification of the external bath solution. (B) Inhibition of VvK5.1-mediated normalized currents by 10 mM BaCl (n=11 ±SD) or TEA (n=5 ±SD) in an external solution of 10 mM K+ at pH 7.4. Inhibition is shown as a percentage of control currents (n=16).
	Fig 4. VvK5.1 transcript levels in grapevine organs and during berry development. RT-qPCR was performed on first-strand cDNAs synthesized from total RNAs of different organs. (A) VvK5.1 transcript levels in roots from rooted canes, or in vegetative organs (stems, leaves, tendrils, and stalks) collected at fruit set (15 d after flowering), flowers, or in berries at three different developmental stages (fruit set, veraison, and ripeness). Vegetative organs, flowers, and berries were collected from grapevines grown in open field conditions under standard irrigation. (B) VvK5.1 transcript levels of berries collected at different developmental stages in field conditions. The fruit set, veraison, and ripening phases are indicated. Note that VvK5.1 expression suddenly and strongly increased in grape berries at veraison. The mean values and SE of two biological replicates are presented.
	Fig 5. In situ localization of VvK5.1 transcripts in flowers. Longitudinal sections of flowers were hybridized with a VvK5.1 RNA sense probe as negative control (left column: A, D) or antisense probe (two right columns: B, C, E, F). Sections hybridized with the sense probe did not show any signal. Sections hybridized with the VvK5.1 antisense probe showed positive blue signals in the stigmas, the transmitting tract (B and C), and the ovule (E, F). In the ovule, blue signal was observed in the nucellus (F). Nu, nucellus; O, ovary; Ov, ovule; S, stigmas; St, style; Tt, transmitting tract.
	Fig. 6. In situ localization of VvK5.1 transcripts in berries during ripening. Longitudinal and equatorial sections were hybridized with the VvK5.1 RNA sense probe as negative control (left column: A, D, G, J) or VvK5.1 RNA antisense probe (right column: B, C, E, F, H, I, K, L). Sections probed with sense probes did not show any significant signal. Sections hybridized with RNA antisense probe showed positive blue signals in ripening berries. Intense blue signals were specifically found in the phloem (B, C, E), perivascular cells (B, C, E), and, to a lesser extent, in the epicarp cells (F). DAPI staining was performed after in situ hybridization of a longitudinal section revealed a vascular bundle (H), in order to identify companion cells via their nucleus and to distinguish them from enucleated phloem sieve tubes. The section stained with DAPI was observed by fluorescence microscopy to localize cell nuclei (I), and by DIC microscopy to visualize the cell walls (K). A zoomed composite picture (L) merging (H), (I), and (K) was constructed using Image J to help localize phloem companion cells (CCs) and enucleated sieve tubes (STs). As an example, the locations of two CCs are indicated by double arrows in (H), (I), (K), and (L). Note that these two cells display a positive blue signal after in situ hybridization (H and L). CC, companion cells; CVB, central vascular bundle; Ep, epicarp; VB, vascular bundle; Mes, mesocarp; Ph, phloem; PVC, perivascular cells; ST, sieve tubes; Xy, xylem.
	Fig. 7. In situ localization of VvK5.1 transcripts in roots of rooted canes. Equatorial sections were hybridized with VvK5.1 RNA sense probe as negative control (left column: A, D, G) or with VvK5.1 RNA antisense probe (two right columns: B, C, E, F, H, I). Sections hybridized with VvK5.1 sense probe did not show any blue staining at the different magnifications (A, D, G). In contrast, positive signals were observed in the stele with VvK5.1 RNA antisense probe (B, C, E, H). The blue signals were located in the phloem in the small (SC) and large parenchyma cells (LPC) of the pericycle (C, E, H). A weaker signal was also detectable in the phloem. (E) and (H) were observed by fluorescence microscopy after DAPI staining to visualize nuclei and the different cell density of the pericycle (F, I). Rh, rhizodermis; C, cortex; En, endoderm; P, Pericycle; PM, medullary parenchyma; Xy, xylem; Ph, phloem; SC, small cells; LPC, large parenchyma cells.
	Fig. 8. Lateral root primordium-specific activity of the VvK5.1 promoter. Tissue-specific activity of the VvK5.1 promoter was investigated by histochemical analysis of GUS staining (blue colour) in transgenic Arabidopsis seedlings expressing GUS under control of the VvK5.1 promoter region. This activity was observed in 6- (A) and 10-day- (B–G) old seedlings grown in vitro in a growth chamber (16 h light photoperiod, 140 μM photons m−2 s−1, 21 °C ,and 70% humidity during both light and darkness) on half-strength Murashige and Skoog (MS/2) medium, supplemented with hygromycin (25 mg l−1). (A) A full view of the whole plantlet. Lateral primordia locations are indicated by arrows. (B–G) A coordinated cell division programme produces the root primordium, shown from the two-cell stage (B) to the stage where the lateral root primordium emerges at the root surface (G). Note that in each stage of the cell division programme, an intense blue colour is present within the most central cells of the primordium, which are known to undergo further cell divisions. In the stages where the primordium emerges at the root surface (F and G), the most intense blue colour is located within the root apex and the root cap.

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