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

Authors

  • Teresa Cuéllar,

    1. UMR1083, Sciences pour l’Œnologie, INRA, 2 Place Viala, 34060 Montpellier Cedex 1, France
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    • Present address: UPR28 Génetique Palmier, CIRAD, Avenue Agropolis, 34398 Montpellier Cedex 5, France.

  • François Pascaud,

    1. Biochimie et Physiologie Moléculaire des Plantes, CNRS-INRA-SupAgro Montpellier-Université de Montpellier, 2 place Viala, 34060 Montpellier Cedex 1, France
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  • Jean-Luc Verdeil,

    1. Développement et Amélioriation des Plantes. CIRAD, TA 80 / 03, Avenue Agropolis, 34398 Montpellier Cedex 5, France
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  • Laurent Torregrosa,

    1. Diversité et Adaptation des Plantes Pérennes Cultivées, INRA-Supagro Montpellier, 34060 Montpellier Cedex 01, France
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  • Anne-Françoise Adam-Blondon,

    1. Génomique Végétale, CNRS-INRA-UEVE, 2, Rue Gaston Crémieux, CP5708, 91057 Evry Cedex, France
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  • Jean-Baptiste Thibaud,

    1. Biochimie et Physiologie Moléculaire des Plantes, CNRS-INRA-SupAgro Montpellier-Université de Montpellier, 2 place Viala, 34060 Montpellier Cedex 1, France
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  • Hervé Sentenac,

    1. Biochimie et Physiologie Moléculaire des Plantes, CNRS-INRA-SupAgro Montpellier-Université de Montpellier, 2 place Viala, 34060 Montpellier Cedex 1, France
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  • Isabelle Gaillard

    Corresponding author
    1. UMR1083, Sciences pour l’Œnologie, INRA, 2 Place Viala, 34060 Montpellier Cedex 1, France
    2. Biochimie et Physiologie Moléculaire des Plantes, CNRS-INRA-SupAgro Montpellier-Université de Montpellier, 2 place Viala, 34060 Montpellier Cedex 1, France
      For correspondence (fax +33 4 67 52 57 37; e-mail gaillard@supagro.inra.fr).
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For correspondence (fax +33 4 67 52 57 37; e-mail gaillard@supagro.inra.fr).

Summary

Grapevine (Vitis vinifera), the genome sequence of which has recently been reported, is considered as a model species to study fleshy fruit development and acid fruit physiology. Grape berry acidity is quantitatively and qualitatively affected upon increased K+ accumulation, resulting in deleterious effects on fruit (and wine) quality. Aiming at identifying molecular determinants of K+ transport in grapevine, we have identified a K+ channel, named VvK1.1, from the Shaker family. In silico analyses indicated that VvK1.1 is the grapevine counterpart of the Arabidopsis AKT1 channel, known to dominate the plasma membrane inward conductance to K+ in root periphery cells, and to play a major role in K+ uptake from the soil solution. VvK1.1 shares common functional properties with AKT1, such as inward rectification (resulting from voltage sensitivity) or regulation by calcineurin B-like (CBL)-interacting protein kinase (CIPK) and Ca2+-sensing CBL partners (shown upon heterologous expression in Xenopus oocytes). It also displays distinctive features such as activation at much more negative membrane voltages or expression strongly sensitive to drought stress and ABA (upregulation in aerial parts, downregulation in roots). In roots, VvK1.1 is mainly expressed in cortical cells, like AKT1. In aerial parts, VvK1.1 transcripts were detected in most organs, with expression levels being the highest in the berries. VvK1.1 expression in the berry is localized in the phloem vasculature and pip teguments, and displays strong upregulation upon drought stress, by about 10-fold.VvK1.1 could thus play a major role in K+ loading into berry tissues, especially upon drought stress.

Introduction

Grapevine is a highly productive water stress-adapted plant and a major crop, based on cultivated hectares and economical value. The recently reported genome sequence of this species (Jaillon et al., 2007; Velasco et al., 2007) is the only one available so far for a fruit crop. The grapevine berry is considered as a model for investigating (non-climacteric) fleshy fruit development and acid fruit physiology (Combe, 1989; Terrier et al., 2005).

Grape berry exhibits a double sigmoid pattern of development, with two distinct phases of growth separated by a lag phase (Kanellis and Roubelakis-Angelakis, 1993). Phase I (which begins by the nouaison step) consists in rapid cell divisions followed by a marked expansion of berry volume as solutes accumulate. The beginning of the second phase (véraison) is characterized by the softening and colouring of the fruit (Coombe, 1992). Sugars and amino acids are rapidly accumulated. The grape berry is also a strong sink for K+ during this period, and its content in this cation increases in a biphasic manner, with a sharp rise when ripening starts (Mpelasoka et al., 2003; Davies et al., 2006).

In grapevine as in other plants, K+ is an essential macronutrient and a major osmoticum. The cells use this cation in functions such as electrical neutralization of dissociated organic acids and anionic groups or cell turgor regulation (Véry and Sentenac, 2003; Lebaudy et al., 2007). On the other hand, increased K+ accumulation in the fruit has a negative impact on fruit acidity (as the exchange of organic acid hydrogen ions with cations reduces the free acid concentration), a phenomenon that has deleterious effects on must and wine quality. Information on major molecular determinants of K+ accumulation in grapes is thus strongly required (Mpelasoka et al., 2003). Efforts in this field have led to the identification of several K+ transport systems, among which are: VvSIRK, a Shaker K+ channel expressed in guard cells (Pratelli et al., 2002); two KUP/KT/HAK-type potassium transporters expressed in the berry skin (Davies et al., 2006); and a cation/proton antiporter associated with berry ripening (Hanana et al., 2007).

Here we report the characterization of a new grapevine Shaker K+ channel named VvK1.1. In Arabidopsis, Shaker genes have been shown to code for voltage-gated highly K+-selective channels active at the plasma membrane, which provide major pathways for wholesale K+ uptake or secretion in most tissues and cell types (Véry and Sentenac, 2003; Gambale and Uozumi, 2006). VvK1.1 is mainly expressed in the root cortex like its Arabidopsis AKT1 counterpart, which has been shown to be involved in K+ uptake from the soil (Hirsch et al., 1998). VvK1.1 displays common features with AKT1, such as voltage sensitivity resulting in inwardly rectifying channel activity and regulation by calcineurin B-like (CBL)-interacting protein kinase (CIPK) activity under the control of CBL calcium sensors. It also displays distinctive features, such as activation at much more negative membrane voltages, suggesting that VvK1.1 transport activity is especially dedicated to K+ uptake from external media (soil or apoplast) containing low K+ concentrations. Furthermore, besides being expressed in root cortex like AKT1, VvK1.1 displays expression in the berry phloem vasculature and in seed teguments.

Results

Cloning of VvK1.1 cDNA

Aiming at identifying grapevine homologues of the Arabidopsis Shaker K+ channel AKT1, a cDNA (2619 bp) was obtained from total RNAs of Vitis vinifera root (cv. Cabernet Sauvignon) by a combination of reverse transcription (RT)-PCR amplifications with degenerated primers and 5′ rapid amplification of cDNA ends (RACE) extension (Appendix S1). The deduced amino acid polypeptide (873 amino acids) shares sequence homology with AKT1 and other members of the plant Shaker family (Figure 1; Table 1), with the levels of amino-acid sequence identity (ASI) ranging from 65 to 77% or from 60 to 64% with K+ channels from Shaker group I (Pilot et al., 2003a), identified in dicots or monocots, respectively. The corresponding cDNA was therefore named VvK1.1, being the first member from Shaker group I identified in grapevine, according to the nomenclature proposed by Pilot et al. (2003a). The hydrophobicity profile of VvK1.1 predicts six membrane-spanning domains (S1–S6) and a hydrophilic C terminus (not shown), in agreement with the typical structure of Shaker channels (Véry and Sentenac, 2003). The C-terminus region comprises a cyclic nucleotide binding domain, an ankyrin domain and a KHA domain (not shown). Like its counterparts, VvK1.1 possesses a highly conserved domain named P (pore), located between S5 and S6, comprising a TxxTxGYGD motif, which is the hallmark of highly K+-selective channels. Phylogenetic analysis reveals that VvK1.1 is more related to AKT1 (Sentenac et al., 1992) than to the other two Arabidopsis Shaker channels from group I, namely SPIK (Mouline et al., 2002) and AKT5 (AT4g32500; Lacombe et al., 2000) (Figure 1a).

Figure 1.

 Phylogenetic relationships of VvK1.1 and chromosomal localization on the grapevine genome.
(a) Phylogenetic analysis of polypeptide Shaker K+ channel sequences. An unrooted consensus phylogenetic tree was constructed with a set of 25 protein sequences comprising the whole family (nine members) of Shaker channels in Arabidopsis, all group-I Shaker channels (Pilot et al., 2003a) functionally characterized in other plant species to date (seven proteins) and the nine proteins from grapevine, namely VvK1.1 (this report), VvK1.2 (CAO44181), VvK2.1 (AAL09479, also named VvSIRK; Pratelli et al., 2002), VvK3.1 (CAO24664), VvK4.1 (CAO44136), VvK5.1 (CAD35400, also named VvSOR in Genbank) and VvK5.2–VvK5.4 (CAO44479, CAO65720, CAO68241, respectively). Arabidopsis sequences: group I, AKT1 (At2g26650), AKT5 (At4g32500) and SPIK (At2g25600); group II, KAT1 (At5g46240) and KAT2 (At4g18290); group III, AKT2 (At4g22200); group IV, AtKC1 (At4g32650); and group V, GORK (At5g35500) and SKOR (At3g02850). Group-I channels functionally characterized in other plants: LKT1 from tomato (Solanum lycopersicum-CAA65254), SKT1 from potato (Solanum tuberosum-CAA60016), DKT1 from carrot (Daucus carota CAG27094), MKT1 from ice plant (Mesembryanthemum crystallinum AAF81249), TaAKT1 from wheat (Triticum aestivum AAF36832), OsAKT1 from rice (Oryza sativa POC550) and ZMK1 from maize (Zea mays CAA68912). Bootstrap values of >50 of 100 replicates are reported near the nodes of the protein phylogram. The branch length is proportional to the evolutionary distance between the channels. Each of the five Shaker groups comprises at least one grapevine member.
(b) Genetic localization and gene structure of the VvK1.1 gene. The linkage group is drawn according to Doligez et al. (2006). The location of the VvK1.1 gene is indicated by an arrow, and the sequence corresponds to bases 4 006 642–4 012 667 on chromosome 11 of the grapevine reference genome sequence (Jaillon et al., 2007). Bottom panel: diagrammatic representation of the intron–exon structure of both VvK1.1 and the Arabidopsis AKT1 gene, the exons being indicated by black boxes. The last VvK1.1 intron (represented by a jagged line) is much longer than the corresponding AKT1 intron.

Table 1.   Amino-acid sequence identity percentages shared by potassium AKT1-like channels
 VvKT1AKT1DKT1SKT1LKT1ZMK1TaAKT1OsAKT1MKT1CAO44181
VvKT110070.775.276.076.663.564.561.365.668.3
AKT1 10068.870.170.760.361.458.761.362.0
DKT1  10075.175.462.562.760.163.066.3
SKT1   10096.963.563.560.564.567.0
LKT1    10063.263.560.564.367.0
ZMK1     10075.674.759.160.0
TaAKT1      10076.159.961.2
OsAKT1       1005863.8
MKT1        10055.2
CAO44181         100

Identification of the VvK1.1 gene and localization in the grapevine genome

Identification of VvK1.1-positive bacterial artificial chromosomes (BACs) and genome walking allowed us to identify the VvK1.1 genomic sequence. Like AKT1 (Basset et al., 1995), VvK1.1 comprises 10 introns. The introns are located exactly at the same positions in the two genes (Figure 1b). Each intron of VvK1.1 displays a size very similar to that of the corresponding intron in AKT1, except the last one, which is much longer in VvK1.1 than in AKT1 (1485 and 94 bp, respectively).

Before the availability of the grapevine reference genome sequence, we were able to assign the VvK1.1 gene to the grapevine genome using the grapevine physical map (Moroldo et al., 2008) and non-ordered genome sequence contigs of an intermediate assembly (6X) of the genome sequence project. The sequence of one end of a VvK1.1-positive BAC (access number CT527471) could be found in the contig_1428, which also contained the SSR marker VVS2. This led to map VvK1.1 on linkage group 11 (Figure 1b).

At the end of 2007 the V. vinifera reference genome sequence was made available (Jaillon et al., 2007), which allowed the location of VvK1.1 as a single gene on chromosome 11 to be confirmed in the region from base 4 006 642 to 4 012 667 (gb:CAO65612 for the protein). Sequence alignment of the predicted protein CAO65612 with VvK1.1 revealed six mismatchs, which could be ascribed to a single nucleotide polymorphism (SNP), as SNP frequency has an average value of 4.0 per Kb across the grapevine genome (Velasco et al., 2007).

Functional expression of the VvK1.1 channel in oocytes

Injection of VvK1.1 cRNA in Xenopus oocytes was found to be without any significant effect on the membrane conductance for K+ (Figure 2a,b). Similar results have been reported for the Arabidopsis AKT1 channel, which remains electrically silent when expressed alone in Xenopus oocytes (Gaymard et al., 1996).

Figure 2.

 Inwardly rectifying voltage-gated K+ channel activity of VvK1.1 in the presence of AtCIPK23 and AtCBL1.
Currents were recorded (two-electrode voltage clamp) 3 days after oocyte injection with cRNAs. Voltage-clamp protocol: 1.6 sec voltage-clamp pulses from +25 mV to −185 mV, with −15-mV steps, and a holding potential of −40 mV. Currents were recorded in 100 mm K+ (K100), 55 mm K+ (K55) or 10 mm K+ (K10) bath solutions at pH 7.5, unless otherwise indicated.
(a) Diagrammatic representation of the voltage-clamp protocol (upper panel) and typical current traces in oocytes injected with VvK1.1 cRNA, recorded in K100 bath medium (middle panel), or in oocytes injected with a mix of VvK1.1, AtCIPK23 and AtCBL1 cRNAs, recorded in K10.
(b) Effect of external K+ concentration on the channel current–voltage relationship. Steady-state currents at the end of the activation step [see the symbol at the end of the current traces shown in panel (a)] were plotted against the membrane potential (I–V curve) for oocytes expressing VvK1.1 + AtCIPK23 + AtCBL1. I–V curves are shown for three external K+ concentrations: 100, 55 or 10 mm. Current values from recordings like those in (a) were normalized, i.e. data obtained in a given oocyte were expressed (Inorm) as a percentage of the current value recorded in the same oocyte at −185 mV in 100 mm K+. Means ± SE (n = 12).
(c) Dependence on the external K+ concentration ([K+]ext) of the measured reversal potential (Erev) of VvK1.1 currents. The dashed line shows the calculated K+ equilibrium potentials (oocyte internal K+ concentration estimated to be close to 130 mm from Erev values obtained in 100 mm K+). Means ± SE (n = 5).
(d) Voltage dependence of the VvK1.1 activation level at steady state in K100 (white symbols) or K10 (black symbols). Relative conductance data (G/Gmax, means ± SE, n = 11) were derived from the steady-state current level and fitted with a two-state Boltzmann relation (dashed line), irrespective of [K+]ext, according to the method described by Lacombe and Thibaud (1998). The best Boltzmann parameter values were zg = 2.09 and Ea50 = −180 mV. Average parameters for individual I–V data sets were zg = 2.11 ± 0.13 and Ea50 = −180 ± 3 mV (n = 22).
(e) Stimulation of the VvK1.1 current by external acidification in K100. I–V curves are shown for four external pHs: 7.5, 6.5, 5.5 or 4.8. Current values were normalized as in (b) (the 100% value for each oocyte is the current at pH 7.5, at −170 mV). Means ± SE (n = 5).

In Arabidopsis, a Ser/Thr protein kinase of the CIPK family, named CIPK23, and Ca2+ sensors of the CBL family, CBL1 and CBL9, which act as positive upstream regulators of CIPK23, have been shown to be part of a regulatory network involved in the control of AKT1 activity and root K+ uptake from the soil solution (Xu et al., 2006). It has also been shown that co-expression in Xenopus oocytes of AKT1 with the regulating kinase CIPK23 and one of the upstream partners CBL1 or CBL9 renders AKT1 active at the oocyte membrane, giving rise to large exogenous currents that can be straightforwardly recorded. As VvK1.1 appeared to be closely related to AKT1, we assumed that a similar regulatory network could control its activity at the cell membrane, and checked its activity in Xenopus oocytes upon co-expression of the Arabidopsis CIPK/CBL regulators. As shown in Figure 2a, this co-expression context enabled large, time-dependent, inwardly-rectifying currents to be recorded upon hyperpolarization, which were never observed in control oocytes injected with water.

These AtCIPK23/AtCBL1-stimulated VvK1.1 currents increased with increasing external K+ concentration (Figure 2a,b), were blocked by external Cs+ (not shown) and were reversed at voltage values that displayed an essentially Nernstian variation in the 10–100 mm external K+ concentration range (Figure 2c), indicating that the inward current mediated by VvK1.1 was mainly carried by K+ ions.

Voltage dependence of VvK1.1 gating was analysed by deriving the relative conductance values (G/Gmax) from the steady-state current–voltage relationships obtained for different external K+ concentrations (in the range 10–100 mm; Figure 2b) as previously described (Lacombe and Thibaud, 1998). The results (Figure 2d) indicated that the gating of VvK1.1, like that of other Shaker channels, was voltage dependent. The voltage dependence was independent of the external K+ concentration, a feature classically reported in inwardly rectifying plant Shaker channels (Véry and Sentenac, 2003). The equivalent gating charge zg was close to 2.1 (2.11 ± 0.13; n = 22), and the half-activation potential Ea50 was close to -180 mV (180 ±3 mV; n = 22). Comparison with the corresponding values in AKT1 (zg = 1.5, Ea50 = −123 mV; Gaymard et al., 1996) indicates that a distinctive feature of VvK1.1 is its activation at much more negative membrane voltages.

Lowering extracellular pH from 7.5 to 6.5 had little effect on the VvK1.1 current, but further lowering to 5.5 and 4.8 increased VvK1.1 currents by 46% (±19%) and 104% (±17%) at −170 mV, respectively (n = 5, Figure 2e). This VvK1.1 stimulation at external pH 5.5 and 4.8 occurred without significant changes in the voltage-dependence of the current (similar zg and Ea50 at pH 7.5, 5.5 and 4.8; data not shown).

VvK1.1 expression pattern

The expression pattern of the VvK1.1 gene was investigated by real-time PCR on total RNAs extracted from roots, leaves, stems, petioles, tendrils and berries at different stages of development (Figure 3), and by in situ hybridization (Figures 4 and 5). Real-time PCR data indicated that VvK1.1 transcripts were preferentially accumulated in root tissues (Figure 3), and, to a lower level, in young berries. In roots, in situ hybridization experiments detected VvK1.1 transcripts in the cortex (in agreement with data on AKT1 in Arabidopsis; Lagarde et al., 1996) and, at much lower levels, in phloem tissues (Figure 4). In berries, in situ hybridization signals were mainly found in the seed (endosperm, outer and medium integuments), raphe and, to a lower extent, in the phloem vasculature present in the pericarp (Figure 5).

Figure 3.

VvK1.1 transcript levels in grapevine organs.
Real-time quantitative PCR was performed on total RNAs isolated from leaves or roots from rooted canes, or from leaves, stems, petioles, tendrils, flowers or berries from open field grapevines grown under standard irrigation.
(a) Leaves, stems, petioles, tendrils and berries were collected at nouaison.
(b) Berries were collected at different developmental stages. At veraison (the phase of berry colour transition, on day 60 in this experiment), green and red berries on turning grapes were sampled separately (arrows 1 and 2, respectively). A VvK1.1 standard curve was used to calculate the number of VvK1.1 mRNA molecules in each sample. The mean values (and standard errors) of two biological replicates are presented.

Figure 4.

 Localization of VvK1.1 transcripts in roots by in situ hybridization.
Root cross sections (a, d) or longitudinal sections (b, e and c, f) were probed using a VvK1.1 RNA antisense probe (a–c) or a control sense probe (d–f). The antisense probe detected (blue colour) VvK1.1 transcripts in root cortex parenchyma cells, and in root phloem. No significant signal was observed upon hybridization with the control sense probe.
Abbreviations: C, cortex; E, epidermis; lrp, lateral root primordium; PC, parenchyma cells; VC, vascular cylinder.

Figure 5.

 Localization of VvK1.1 transcripts in berries at the nouaison stage by in situ hybridization.
(a) Hybridization with VvK1.1 RNA antisense probe. A cross section of a berry at nouaison and enlargements of the dotted line-enclosed regions are shown. VvK1.1 transcripts were detected in the pericarp and in seeds. In the pericarp, the signal (blue colour) is located in the phloem of the peripheric and the central capillary vascular bundles. In seeds, the signal is located in the endosperm, the seed coat and the raphe.
(b) Corresponding control berry sections hybridized with a VvK1.1 RNA sense probe displayed no significant signal.
Abbreviations: CCVB, central capillary vascular bundles; EN, endosperm; END, endocarp; EP, epicarp; IM, inner mesocarp; OM outer mesocarp; P, pericarp; PE, perisperm; PH, phloem; R, raphe; SC, seed coat; VB, vascular bundles; XY, xylem.

Effect of drought stress and ABA on VvK1.1 expression

A first set of real-time PCR experiments was aimed at investigating the effects of drought stress on VvK1.1 expression in berries. Grapevine is a highly productive drought stress-adapted plant, and regulated-deficit irrigation is used to manipulate fruit quality (Coombe, 1989; Kanellis and Roubelakis-Angelakis, 1993). The impact of water stress on berry development and composition depends on its intensity, duration and position within the cycle of development (Ojeda et al., 2002). Our experiments reproduced field conditions of moderate drought stress, the corresponding plants being maintained at a water potential ranging between −0.6 and −0.7 MPa, against −0.2 MPa in control, unstressed plants. Berries were sampled at four stages of development: during the rapid cell division phase following nouaison (day 25 after flowering), at the onset of ripening named the véraison stage (day 60 after flowering) and during ripening (days 67 and 75 after flowering). Drought stress was found to result in strong increases in VvK1.1 mRNA accumulation in the four berry samples, by 6–25-fold (Figure 6a).

Figure 6.

 Regulation of VvK1.1 expression in response to drought stress, ABA and ionic stress.
VvK1.1 transcript accumulation was analysed by real-time quantitative PCR on total RNAs. Leaves and roots were collected from rooted canes. The plants were first grown in perlite in standard watered conditions for 2 months. Then (t = 0) plants were subjected to drought stress by stopping irrigation (a), watered with 100 μm ABA (b), or were transferred (after the root system had been washed) onto water, 50 mm NaCl or 50 mm KCl, and hydroponically grown for five further days (control, NaCl and KCl treatments, respectively) (c). Data are expressed relative to VvK1.1 transcript accumulation in plant material collected at t = 0 [panels (a) and (b)] or in control plants on water (c). Means (±SE) of two biological replicates (with three technical replicates). Berries (a) were collected from field-grown 4-year-old plants under control irrigation. Drought stress was applied by decreasing the level of irrigation for 15 days before berries were collected (the leaf water potential was then in the range from −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 control (grey bars) or water-stressed berries (black bars) were normalized using EF1-alpha transcript signals. The data presented are means (±SE) of two biological replicates.

A second set of experiments aimed to investigate the effects of drought stress on VvK1.1 transcript accumulation in roots and leaves. This was achieved using rooted canes grown in perlite. Drought stress (induced by stopping irrigation) was found to have strong opposite effects on VvK1.1 transcript levels in roots and leaves: transcript accumulation was increased in leaves and decreased in roots, with the relative variation being of more than fivefold in both cases (Figure 6a). In parallel experiments, treatments with the stress hormone ABA (introduced in plant watering solution at 100 μm concentration) were found to rapidly induce (within <4 h) a strong (fivefold) upregulation of VvK1.1 transcript accumulation in leaves, but to be without any significant effect (for at least 8 h) in roots (Figure 6b).

Effects of saline stress on VvK1.1 expression

Integrated control at the whole-plant level of K+ membrane transport system activities is crucial for plant growth in the presence of high Na+ concentrations. This is thought to involve a large diversity of regulation mechanisms, one of which is the transcriptional control of gene expression, which can vary between plant species. For example, accumulation of AKT1 transcripts in Arabidopsis roots is not sensitive to salt stress (Pilot et al., 2003b), whereas that of OsAKT1 in rice (Oryza sativa) is inhibited, a response that leads to a strong decrease in the voltage-gated inwardly rectifying membrane conductance of K+ (Fuchs et al., 2005). Checking the sensitivity of VvK1.1 expression to grapevine watering with 50 mm NaCl or KCl revealed no significant change in transcript accumulation, either in roots or in leaves (Figure 6c).

Discussion

Comparison of VvK1.1 with AKT1 and other group-I Shaker channels

In silico analysis of the grapevine genome reveals that the Shaker K+ channel family comprises nine members in this species, as in Arabidopsis. The phylogenetic tree shown in Figure 1a indicates that two of them, VvK1.1 and another one, CAO44181, thereafter named VvK1.2, form the Shaker channel group I (Pilot et al., 2003a) in grapevine. The Arabidopsis Shaker group I comprises three members, AKT1, SPIK and AKT5 (AT4g32500). Thus, although the total number of Shaker genes is the same in grapevine as in Arabidopsis, the numbers of members within each group are not strictly conserved between the two species (Figure 1a). In Arabidopsis, AKT1 is mainly expressed in roots, where it gives rise to inward K+ channel activity involved in K+ uptake from the soil (Lagarde et al., 1996; Hirsch et al., 1998; Xu et al., 2006). SPIK is specifically expressed in pollen and in the growing pollen tube, and mediates inward K+ channel activity essential for efficient tube growth and pollen competitive ability (Mouline et al., 2002). AKT5 is specifically expressed in flowers and seeds (Lacombe et al., 2000; Botany Array Resource database, http://bbc.botany.utoronto.ca; Arabidopsis thaliana Geneinvestigator, https://iii.genevestigator.ethz.ch/). VvK1.1 can be considered as the grapevine ortholog of Arabidopis AKT1 because: (i) the level of sequence identity is higher between AKT1 and VvK1.1 (71% ASI) than between AKT1 and VvK1.2 (the other group-I Shaker gene in grapevine; 62% ASI) (Table 1); (ii) the VvK1.1 gene displays strictly the same structure (number and positions of introns) as AKT1 (Figure 1b), whereas VvK1.2 comprises one further intron (T.C. and I.G., unpublished data); and (iii) both VvK1.1 and AKT1 are expressed in the root cortex, whereas VvK1.2 does not display any expression in roots (absence of transcripts in real-time PCR experiments; T.C. and I.G., unpublished data).

VvK1.1 displays distinctive features, however, when compared with AKT1, in terms of functional properties on the one hand, and localization and regulation of expression on the other hand. At the functional level, it activates at more negative membrane potentials than AKT1, e.g. at about −120 mV (Figure 2) against −50 mV for AKT1 (in oocytes in the presence of CBL1 and CIPK23, and in Sf9 insect cells; Lee et al., 2007; Gaymard et al., 1996). Interestingly, the Shaker channels SKT1 from potato (Solanum tuberosum) and LKT1 from tomato (Solanum lycopersicum), which are close relatives of VvK1.1 in the phylogenetic tree shown in Figure 1, and which have both been shown to be expressed in roots, also activate at rather depolarized membrane potentials, at around −70 mV and −50 mV, respectively (Zimmermann et al., 1998; Hartje et al., 2000). Major determinants of voltage sensitivity in animal Shaker channels are electrically charged residues (R or K, D or E) present in particular in the S4 segment (the so-called ‘voltage-sensor’), but also in other domains such as the cytoplasmic S4–S5 linker. For example, in animal Shaker channels, salt bridges between residues present in the cytoplasmic S4–S5 linker and the COOH-terminal end of S6 can stabilize the channel in an inactive state, and their breaking upon changes in membrane polarization is required for channel activation (Lu et al., 2002; Barghaan and Bahring, 2009). Based on such information in animal Shaker channels, sequence alignment of VvK1.1, AKT1, SKT1 and LKT1 might provide clues for identifying molecular determinants of voltage sensitivity in plant group-I Shaker channels. Interestingly, the S4 segments and the S4–S5 linkers are highly conserved between the four channels, suggesting that the determinants of voltage sensitivity might be located in other domains. For example, N128 and R155 located respectively in the N- and the C-terminal regions of the S3 segment of VvK1.1 might be considered in such analyses because the corresponding residues in AKT1, LKT1 or SKT1 are different in terms of electric charge.

Regarding the localization and regulation features specific to VvK1.1, in situ hybridization experiments indicate that VvK1.1 is also expressed in root phloem (Figure 4), a localization that has not been reported for AKT1 (Pilot et al., 2003b). In berries, VvK1.1 is expressed at rather high levels in pips, whereas expression of AKT1 in seeds or in siliques has not been reported (Lagarde et al., 1996; Pilot et al., 2003b). Lastly, VvK1.1 expression is strongly upregulated in leaves (and berries) by ABA (and water stress), whereas AKT1 expression in Arabidopsis leaves has been reported to be downregulated by this hormone (Pilot et al., 2003b).

Role and regulation

In roots, expression of VvK1.1 in cortical cells suggests a role in K+ uptake from the soil solution, as shown for the Arabidopsis AKT1 channel by using a mutant line disrupted in the encoding gene (Hirsch et al., 1998). Expression of VvK1.1 can also be detected in phloem tissues, both in roots and in berries (Figures 3 and 4), pointing to a contribution to K+ transport in the phloem vasculature, as already proposed for another group-I Shaker gene, OsAKT1 from rice (Golldack et al., 2003). A striking feature of VvK1.1 is that it also displays high expression levels in pips (Figure 4). Accumulation of mineral nutrients into the developing seeds is thought to be a prerequisite for efficient germination and seedling establishment. It has been shown that the germination capacity can depend on the control of seed K+ content (Zerche and Ewald, 2005). It is likely that VvK1.1 is involved in this control by contributing to the K+ uptake of the developing pips. Identification of molecular determinants of K+ accumulation in berries and pips is of great importance at the biotechnological level, as high K+ contents in berries have strong detrimental effects on fruit acidity, and thus also on wine quality (Mpelasoka et al., 2003).

Plant voltage-gated inwardly-rectifying K+ channels have the ability to allow K+ efflux when the membrane potential is between the channel activation threshold and the K+ equilibrium potential (EK), in other words when the channel is gated open by a membrane potential more negative than the channel activation threshold, and when the transmembrane electrochemical gradient of K+ is outwardly directed because of a low concentration of this cation in the external solution. In these conditions, such channels can behave as leak-like channels at membrane potentials around EK, a behaviour that might allow them to play a role in the control of cell membrane polarization close to EK. This phenomenon has not yet been demonstrated to occur in planta, but outwardly-directed K+ currents have been observed, for instance, in oocytes expressing AKT1 (Duby et al., 2008) and in Arabidopsis guard cell protoplasts (Bruggemann et al., 1999). Regulation of channel activity by external K+, preventing both K+ influx and K+ efflux when the external concentration of K+ is decreased down to values in the submillimolar range, when the channel then enters a non-conducting state, has been observed in a plant group-II Shaker channel, ZmK2.1, from maize (Zea mays) (Su et al., 2005). This type of regulation mechanism has, however, not been observed in any group-I Shaker channel so far. Another type of regulation involves the formation of heteromeric channels. AKT1, for instance, can form heteromeric channels with the regulatory Shaker subunit AtKC1 (Duby et al., 2008). This interaction affects the channel sensitivity to voltage, and shifts the voltage threshold for activation from about −50 mV in homomeric AKT1 channels to values more negative than −100 mV in heteromeric AKT1/AtKC1 channels (Duby et al., 2008). Such a shift in channel activation threshold results in the downregulation of channel activity, affecting the inward K+ currents, but also strongly reducing the ability of the channels to mediate outward K+ currents. As VvK1.1 displays an activation threshold (close to −120 mV) that is much more negative than that of AKT1, its ability to mediate outward currents is therefore further reduced. Furthermore, in silico analysis reveals that an ortholog of AtKC1 is present in the grapevine genome (CAO44136), suggesting that regulation mechanisms involving the formation of heteromeric channels are also likely to occur in this species, giving rise to channels activating at very hyperpolarized membrane potentials. Such channel activity might be dedicated to K+ uptake from external media (soil or apoplast) containing very low K+ concentrations.

VvK1.1 transcript accumulation is strongly sensitive to drought stress, with the leaves and berries displaying an increase in transcript accumulation, by up to sixfold in the berries, whereas the roots display a decrease of up to fivefold. Thus, drought stress in grapevine is likely to result in deep changes in the cell membrane equipment for K+ transport, dependent on the organ or tissue, and probably affecting the membrane conductance for K+, and the balance between transporter and channel activities. Such responses to drought stress are still poorly documented, even in Arabidopsis. ABA resulted in increased levels of VvK1.1 accumulation in leaves, but did not affect the accumulation level in roots. This suggests that VvK1.1 expression regulation upon drought stress is under ABA control in leaves and independent from this hormone in roots. In Arabidopsis, AKT1 transcript accumulation has been reported to be insensitive to ABA in roots, like VvK1.1, and to be decreased by ABA in leaves, in contrast to VvK1.1. The opposite regulation of VvK1.1 and AKT1 in leaves in response to ABA reveals distinctive features that could be related to specificities in water stress adaptation between Arabidopsis and grapevine.

Finally, our results provide evidence that CIPK–CBL complexes are involved in the control of VvK1.1 activity, and thus reveal that this type of regulation of K+ channel activity is strongly conserved in higher plants. In Arabidopsis, a pathway associating CIPK23 with either CBL1 or CBL9 has been shown to play an important role in plant adaptation to low K+ availability (Xu et al., 2006; Lee et al., 2007). The Arabidopsis CIPK and CBL families comprise 25 and 10 members, respectively. In silico analysis indicates that at least 19 CIPK and eight CBL genes are present in the grapevine genome. The grapevine gene CAO40354.1 is the closest relative of Arabidopsis CIPK23, and CAO17301.1 is the closest relative of CBL1 and CBL9. As the relationships within the CBL and CIPK phylogenetic trees seem similar in Arabidopsis and grapevine (Figures S1, S2), it is tempting to speculate that the functions and regulatory networks are highly conserved, and that the aforementioned grapevine CBL and CIPK genes contribute to the regulation of VvK1.1 activity. In Arabidopsis, CIPK23 and its interacting partners CBL1 and CBL9 have been shown to also play a role in the control of ABA sensitivity, stomatal aperture and leaf transpirational water loss, besides regulating root K+ uptake (Cheong et al., 2007). Such a background of knowledge in Arabidopsis, along with conservation of regulatory pathways controlling K+ channel activity, is likely to stimulate advances in our understanding of grapevine drought stress adaptation, berry development, and determinants of fruit acidity and quality.

Experimental procedures

Plant material

Grapevines (V. vinifera L. cv. Cabernet Sauvignon) grown in field conditions were placed in 70-L containers containing a mixture of perlite and sand (90/10 v/v) (Agro-M/INRA collections, Montpellier, France). They were grafted on Fercal rootstock, trained on a lyre system and pruned as a cordon. Water was applied using a drip-irrigation system allowing different irrigation programs. The plant water status was characterized by the measurement of the leaf water potential at dawn (Ψ) using the pressure chamber technique (Scholander, 1965). Control plants (absence of drought stress) displayed leaf water potential close to −0.2 MPa throughout the experimental period. Drought stress was applied by progressively reducing irrigation over a period of 2 weeks, in order to obtain Ψ values at dawn ranging between −0.7 and −0.6 MPa for 2 days, at least, before tissue collection.

Rooted canes grown on ‘perlite’ were 2-months old when they were used for water and saline stress experiments and ABA treatments. For each experiment, five plants were used as biological replicates. Each experiment was repeated twice.

All collected samples were immediately frozen in liquid nitrogen and stored at −80°C until use. Total RNA extraction was performed using the Plant RNeasy Extraction kit (Qiagen, http://www.qiagen.com) according to the manufacturer’s instructions, except for lysis buffer, which was replaced by 200 mm Tris–HCl, pH 8.5, 1.5% (w/v) sodium dodecylsulfate, 300 mm LiCl, 10 mm sodium EDTA, 1% (w/v) sodium deoxycholate, 1% (v/v) Nonidet P-40, 1 mm aurintricarboxylic acid, 10 mm dithiothreitol and 5 mm thiourea.

Isolation of cDNA and gene-encoding VvK1.1 protein

VvK1.1 was cloned while the V. vinifera genome-sequencing program was in progress. cDNA and gene cloning strategies, sequencing and mapping on the grapevine genome are described in Appendix S1.

Bioinformatic analysis

Multiple alignments were performed using ClustalW 1.8 (Thompson et al., 1994). Phylogenic trees were constructed using PhyML 3.0, and were visualized using TreeDyn 198.3 (http://www.phylogeny.fr/version2_cgi/index.cgi). Pairwise sequence comparisons were performed with Align at the genestream server (http://xylian.igh.cnrs.fr/).

Real-time PCR analysis

Total RNAs were quantified with Ribogreen reagent (Molecular Probes, now part of Invitrogen, http://www.invitrogen.com/site/us/en/home/brands/Molecular-Probes.html) after DNase I treatment (Invitrogen, http://www.invitrogen.com), and were used as the template to synthesize first-strand cDNA with SuperScript III reverse transcriptase (Invitrogen), according to the manufacturer’s instructions.

In order to compare data from different PCR runs or cDNA samples, the cycle threshold (CT) values obtained for VvK1.1 were normalized to the corresponding CT values of Ef1-alpha (elongation factor 1α gene; GT181C12 or BQ799343). This isogene, which has been shown to be constitutively expressed during berry development, and to give rise to stable transcript levels, can be taken as housekeeping control in grape berry development studies, including during drought stress (Terrier et al., 2005; Reid et al., 2006; Abbal et al., 2008; T.C. and I.G., unpublished data). The average CT value of EF1-alpha for all templates was 20.1 (±0.46).

Oligonucleotides were designed using Primer3 (http://frodo.wi.mit.edu). The primer pair KT1-121-F (5′-TTGTTGAAACGTGGTCTGGA-3′) and KT1-121-R (5′-GCCCTGCCCCATAATCTAGT-3′) spans intron IX, according to VvK1.1 gene structure (Figure 1). The Ef1-alpha primer pair, EF1-F (5′-TCTGCCTTCTTCCTTGGGTA-3′) and EF1-R (5′-GCACCTCGATCAAAAGAGGA-3′), was designed in the 3′ untranslated region of VvEF1-alpha.

PCR reactions were performed on a 7300 Real Time PCR system (Applied Biosystems, http://www.appliedbiosystems.com) using Power SYBR Green PCR Master Mix (Applied Biosystems) and gene-specific primers according to the manufacturer’s specifications, with the cDNA equivalent of 2 ng of RNA in 25 μl. Reactions were performed in triplicate with two independent biological samples. The following standard thermal profile was used in all PCR: 50°C for 2 min; 95°C for 10 min; 40 cycles of 95°C for 15 sec; and 60°C for 1 min, followed by a melt cycle from 60°C to 95°C. Absence of genomic DNA and primer dimers was confirmed by analysis of minus-RT and water control samples, and by examination of melting curves. Baseline data were collected between cycles 3 and 15. All amplification plots were analysed with an Rn (normalised reporter) threshold of 0.2 to obtain CT values.

Standard curves for VvK1.1 and Ef1-alpha were obtained from dilution series of known quantities of VvK1.1 and Ef1-alpha DNA fragments used as templates. Standard curves were used to calculate the absolute numbers of VvK1.1 cDNA molecules in each cDNA sample, and these values were then normalized against the corresponding Ef1-alpha signals. The normalization factor was calculated assuming that CT is inversely proportional to the logarithm of the quantity of target DNA present at the PCR start. To derive relative expression levels, the comparative CT method (ΔΔCT) was used (Livak and Schmittgen, 2001).

Localization of VvK1.1 expression by mRNA in situ hybridization

VvK1.1-specific primers KT1-121-F and KT1-121-R (see above), which span intron IX, were used to synthesize RNA probes, as described by Alemanno et al. (2008). As a control, an 18S ribosome probe was used. The length of the probe was 121 bp for VvK1.1, and 151 bp for the ribosome control. Sense and antisense probes were labelled with UTP-digoxigenin during the transcription step.

Explants from roots and nouaison berries were fixed in 4% paraformaldehyde. Samples were cut into 8-μm sections and hybridization was performed overnight. Slides were then incubated 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 with VectorBlue KIT III (Vector Laboratories, http://www.vectorlabs.com). Slides were observed with a DM600 microscope (Qimaging, http://www.qimaging.com) and pictures were taken with a Qimaging Retiga2000R camera.

Functional characterization of VvK1.1

The VvK1.1 cDNA open reading frame was cloned into pCI vector (Promega, http://www.promega.com) for expression in oocytes. In vitro transcriptions were performed using the mMESSAGE mMACHINE kit (Ambion, http://www.ambion.com) following the manufacturer’s instructions. Xenopus oocytes were purchased from the Centre de Recherche en Biochimie Macromoléculaire (CNRS, Montpellier, France). Stage V–VI oocytes were injected with 20 nl of deionized water containing either 6 ng of VvK1.1 cRNA alone or 6 ng of VvK1.1 cRNA, mixed with both 3 ng of AtCIPK23 and 3 ng of AtCBL1 (1 : 1) in vitro transcribed RNA (Offenborn, 2006; Xu et al., 2006). Control oocytes were injected with 20 nl of deionized water.

Injected oocytes were maintained at 19°C for 3–4 days in a solution containing 2 mm KCl, 96 mm NaCl, 5 mm HEPES, 2.5 mm sodium pyruvate, pH 7.5, supplemented with gentamycin sulfate (50 μg ml−1). All experiments were performed at room temperature (20–22°C). Macroscopic currents were recorded 3–4 days after oocyte injection using the two-electrode voltage clamp technique, and then analysed as described previously (Lacombe and Thibaud, 1998). The bath solution contained 100, 55 or 10 mm KCl, supplemented with 0, 45 or 90 mm NaCl, respectively, in 1 mm CaCl2, 1.5 mm MgCl2 and 10 mm HEPES-2-amino-2-(hydroxymethyl)-1,3-propanediol (TRIS), pH 6.5 or 7.5, or 10 mm 2-(N-morpholine)-ethanesulphonic acid (MES)-TRIS, pH 5.5 or 4.8. Linear leak currents were digitally subtracted from recorded whole oocyte currents. pClamp 9.0 (Axon Instruments, now part of Molecular Devices, http://www.moleculardevices.com) and Sigmaplot (Jandel Scientific, now part of SPSS, http://www.spss.com) were used to perform voltage pulse protocol application, data acquisition and data analysis.

Acknowledgements

We are grateful to Guy Albagnac (Montpellier, France) for helpful discussions. We thank Pascale Brial for her expert technical assistance. TC was supported by a grant from INRA. FP was supported by a grant from CNRS. The work was partly supported by a grant (ANR-05-GPLA-024) from Genoplante.

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