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Keywords:

  • Shaker K+ channel;
  • K+ uptake;
  • CIPK–CBL network;
  • grape berry flesh cells;
  • in situ hybridization;
  • expression pattern

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental Procedures
  7. Accession numbers
  8. Acknowledgements
  9. References
  10. Supporting Information

The grape berry provides a model for investigating the physiology of non-climacteric fruits. Increased K+ accumulation in the berry has a strong negative impact on fruit acidity (and quality). In maturing berries, we identified a K+ channel from the Shaker family, VvK1.2, and two CBL-interacting protein kinase (CIPK)/calcineurin B-like calcium sensor (CBL) pairs, VvCIPK04–VvCBL01 and VvCIPK03–VvCBL02, that may control the activity of this channel. VvCBL01 and VvCIPK04 are homologues of Arabidopsis AtCBL1 and AtCIPK23, respectively, which form a complex that controls the activity of the Shaker K+ channel AKT1 in Arabidopsis roots. VvK1.2 remained electrically silent when expressed alone in Xenopus oocytes, but gave rise to K+ currents when co-expressed with the pairs VvCIPK03–VvCBL02 or VvCIPK04–VvCBL01, the second pair inducing much larger currents than the first one. Other tested CIPK–CBL pairs expressed in maturing berries were found to be unable to activate VvK1.2. When activated by its CIPK–CBL partners, VvK1.2 acts as a voltage-gated inwardly rectifying K+ channel that is activated at voltages more negative than –100 mV and is stimulated upon external acidification. This channel is specifically expressed in the berry, where it displays a very strong induction at veraison (the inception of ripening) in flesh cells, phloem tissues and perivascular cells surrounding vascular bundles. Its expression in these tissues is further greatly increased upon mild drought stress. VvK1.2 is thus likely to mediate rapid K+ transport in the berry and to contribute to the extensive re-organization of the translocation pathways and transport mechanisms that occurs at veraison.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental Procedures
  7. Accession numbers
  8. Acknowledgements
  9. References
  10. Supporting Information

Grapevine (Vitis vinifera) is a productive water stress-adapted plant, and one of the most economically important fruit crops (Coombe, 1989; Kanellis and Roubelakis-Angelakis, 1993). Furthermore, the grape berry provides a model for investigating non-climacteric fruit development. Also, because the berry displays a low vacuolar pH (in the 2.2–3.5 range; Hrazdina et al., 1994), and acidity is a major determinant of fruit quality, it is of interest for investigating acid fruit physiology (Ros et al., 1995; Terrier et al., 2001).

Berry acidity is dependent on the level of K+ accumulation in berry cells. As a general rule, the higher the K+ content, the less acidic the fruit. At a qualitative and oenological level, high K+ contents reduce the tartaric to malic acid ratio, resulting in wine of poor quality (Gawel et al., 2000). The roles that K+ plays in the developing berry have not been investigated in detail. However, they are likely to correspond to the basic roles that this cation plays in plant cells, which include control of membrane polarization and electrical neutralization of dissociated organic acids and anionic groups, in addition to regulation of cell turgor.

The developing berry is a strong sink for K+, especially during ripening. The growth pattern of grape berries describes a double sigmoidal curve, with two distinct phases separated by a lag phase (Kanellis and Roubelakis-Angelakis, 1993). After fertilization and berry formation (fruit set), the first phase consists of rapid cell divisions followed by a marked expansion of berry volume as solutes accumulate. This initial growth phase is followed by a lag period of slow or no growth. The entry into the second growth phase begins with veraison, the onset of ripening. This transition results in and is characterized by softening and colouring of the fruit (Coombe and McCarthy, 2000). Although green and colouring berries co-exist on racemes, the transition is very rapid at the single berry level and occurs within 24 h. Growth after veraison is due to cell expansion. The berries undergo major changes in chemical composition, including sugar accumulation and organic acid depletion (Coombe and McCarthy, 2000). A sharp increase in berry K+ content is also observed at veraison and during the whole maturing period (Ollat and Gaudillère, 1996; Mpelasoka et al., 2003). Profound developmental changes occur at the same time. Phloem unloading into the berry shifts from a symplastic mode to an apoplasmic mode. Berry stomata evolve into non-functional lenticels. The xylem conductance between the berry and pedicel is progressively reduced, as in many fleshy fruits, probably because tracheary elements of the peripheral xylem break as a result of fruit growth (Chatelet et al., 2008). Hence, the berry becomes dependent on phloem sap flux not only for sugars and other organic molecules but also for mineral ions and water (Düring et al., 1987; Findlay et al., 1987).

Given the importance of berry K+ accumulation for fruit (and wine) quality, molecular determinants of K+ transport in grapevine have been researched (Pratelli et al., 2002; Davies et al., 2006; Hanana et al., 2007; Cuéllar et al., 2010). Here we report the identification of a plasma membrane K+ channel belonging to the Shaker family. The encoding gene, named VvK1.2, is expressed in the berry, phloem tissues and flesh cells, and is strongly induced at veraison and during ripening. Our results provide evidence that CBL-interacting Ser/Thr protein kinase (CIPK)/calcineurin B-like Ca2+ sensor (CBL) complexes are involved in the control of VvK1.2 activity. In Xenopus oocytes, two CIPK–CBL couples, cloned from maturing berries, specifically activate the VvK1.2 channel (VvCIPK04–VvCBL01 and VvCIPK03–VvCBL02). When activated by these CIPK–CBL partners, VvK1.2 behaves as a voltage-gated inwardly rectifying K+ selective channel. Thus, the data indicate that VvK1.2 is likely to play a major role in K+ transport in phloem tissues and flesh cells of the developing berry.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental Procedures
  7. Accession numbers
  8. Acknowledgements
  9. References
  10. Supporting Information

Molecular cloning of VvK1.2 cDNA

A 2697 bp cDNA was cloned by RT–PCR using degenerate primers and 5′ and 3′ RACE extension experiments while the Vitis vinifera genome-sequencing program was in progress (Methods S1). The deduced polypeptide (898 amino acids, 101.6 kDa) displays a structure typical of plant Shaker channels, including a hydrophobic core with six transmembrane segments, named S1 to S6, a well-defined pore region located between S5 and S6, comprising the potassium-selective hallmark sequence ‘TXXTXGYGD’, a cyclic nucleotide-binding domain, an ankyrin domain and at the extreme C-terminus, the so-called KHA region rich in hydrophobic and acidic amino acids. Phylogenetic analysis (Figure 1) revealed that this new channel subunit belongs to group I of the plant Shaker K+ channel family. As it is the second Shaker channel to be identified in group I of the grapevine Shaker family, it was named VvK1.2, according to the nomenclature proposed by Pilot et al. (2003). The VvK1.2 deduced polypeptide shares 55–68% amino acid sequence identity (ASI) throughout the entire protein length with Shaker group I K+ channels that have been functionally characterized to date. When VvK1.2 is aligned with VvK1.1, the group I member previously identified in grapevine (Cuéllar et al., 2010), the greatest identity is found in the transmembrane domains S1–S6 (77% ASI) and the cyclic nucleotide-binding domain (78% ASI). The greatest sequence divergence is found in the C–terminus, mainly in the ankyrin domain (67% ASI) and the downstream sequence (47% ASI), which includes the KHA domain.

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Figure 1. Phylogenetic relationships of Shaker channels in Arabidopsis and grapevine. Unrooted trees constructed using the complete set of Shaker proteins in Arabidopsis and grapevine. The Shaker family consists of five groups in plants (Pilot et al., 2003), named I–V. The Arabidopsis family has nine members: AKT1 (At2 g26650), AKT5 (At4 g32500) and SPIK (At2 g25600) in group I, KAT1 (At5 g46240) and KAT2 (At4 g18290) in group II, AKT2 (At4 g22200) in group III, AtKC1 (At4 g32650) in group IV, and GORK (At5 g35500) and SKOR (At3 g02850) in group V. The grapevine family also has nine members: VvK1.2 (this paper; bold text), VvK1.1 (Cuéllar et al., 2010; CAZ64538), VvK2.1 (AAL09479, also named VvSIRK; Pratelli et al., 2002), and six other members identified by in silico screening of the grapevine genome sequence: VvK3.1 (CAO24664), VvK4.1 (CAO44136), VvK5.1 (CAD35400, also named VvSOR in GenbankPlease check sense ‘also named VvSOR in Genbank’ and give accession number if relevant) and VvK5.2, VvK5.3 and VvK5.4 (CAO44479, CAO65720 and CAO68241, respectively).

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Spatiotemporal expression pattern of VvK1.2

The expression pattern of VvK1.2 was investigated by real-time PCR analysis in grapevine tissues: roots, leaves, stems, tendrils, petioles and berries. Very low VvK1.2 transcript levels were detected in these tissues except berries, with the highest expression levels being detected in ripe berries (Figure 2a). Further real-time PCR experiments were performed to analyse VvK1.2 transcript accumulation during berry development. The results revealed two very interesting features: VvK1.2 transcript accumulation increased continuously during berry development, and the accumulation kinetics showed a striking acceleration at veraison (Figure 2b).

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Figure 2. VvK1.2 transcript levels in grapevine organs and during berry development. Real-time quantitative PCR was performed on first-strand cDNAs synthesized from total RNAs. A VvK1.2 standard curve was used to determine the expression level of VvK1.2 (number of VvK1.2 mRNA molecules) in each sample. Values are means and standard errors of two biological samples (three technical replicates per sample). (a) VvK1.2 expression levels in leaves or roots from rooted canes, or in leaves, stems, petioles, tendrils or berries from grapevines grown in open-field conditions under standard irrigation (no drought stress). Leaves, stems, petioles and tendrils were collected at fruit set. Berries were collected at various developmental stages (from flower to ripeness). (b) VvK1.2 expression levels in berries during berry development. The values at veraison (berry colour transition)Please check definition of veraison as ‘berry colour transition’, cf ‘the inception of ripening’ in text correspond to red berries.

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In order to investigate the expression pattern of VvK1.2 at the tissue level in developing berries, and to check whether the strong increase in VvK1.2 expression from veraison to ripeness involved a change in the localization of expression, in situ hybridization analyses were performed at various stages of berry development: flowers, fruit set, veraison (green or coloured stages) and ripeness (Figure 3). In flowers, the VvK1.2 signal was mainly located in the nucellus, which is the inner part of the ovule in which the embryo sac develops (Figure 3, panel a3). VvK1.2 expression was also detected in the epidermis of the ovarian locule, stigmas and style (Figure 3, panels a2 and a3). At fruit set, VvK1.2 was essentially expressed in berry phloem tissues, and in the seed endosperm and perisperm (a storage tissue derived from the nucellus) (Figure 3, panels b2 and b3). From this stage onwards, only deseeded berries were analysed for technical reasons. VvK1.2 expression suddenly and strongly emerged in flesh cells (mesocarp parenchyma cells) of coloured berries at the onset of ripening. Intense VvK1.2 signals were detected in flesh cells from coloured berries but not in green berries collected from the same raceme at the same time (Figure 3, panels c2 and c3), suggesting that this induction of VvK1.2 expression occurs at veraison and reflects the extensive change in the gene expression program characterizing this transition phase. Expression of VvK1.2 in berries at veraison was also detectable in phloem tissues (data not shown). Analyses performed at later stages of berry development, in ripe berries, showed that expression of VvK1.2 persisted in flesh cells. Interestingly, the signals appeared to be less intense in the outer than the inner mesocarp (Figure 3, panel d3), suggesting a radial gradient of expression. In addition to flesh cells, expression of VvK1.2 appeared in phloem tissues and perivascular cells surrounding vascular bundles feeding the pips (Figure 3, panel d4), where VvK1.2 may be involved in the control of K+ exchange between vessels, berry flesh and pips.

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Figure 3. Localization of VvK1.2 transcripts by in situ hybridization in flowers and berries at various stages of development. Longitudinal and equatorial sections of flowers (A) and berries at fruit set (B), veraison (C) and ripeness (D) were hybridized with a VvK1.2 RNA sense probe (left column: a1, b1, c1, d1 and d2) or antisense probe (two right columns). Sections hybridized with the sense probe (negative control) did not show any significant signals. The antisense probe detected VvK1.2 transcripts, resulting in a blue colour (signal). (A) Flowers. Intense signals were detected in the ovary and the ovule. In the ovary, the signal is located in the epidermis bordering the ovarian locule, the style and the stigma (a2). In the ovule, the signal is located in the nucellus (a3). Weak signals may be detected in the nectaries at the base of the ovary (a2). (B) Berries at fruit set. Signals were detected in the phloem of the vascular bundles, the endosperm and the endocarp (b2 and b3). A weak signal was observed in the perisperm (b2 and b3). (C) Berries at veraison. A rapid change in berry colour occurs at veraison. Green berries on turning grapes were sampled separately from coloured berries from the same grapesPlease check sense ‘Green berries on turning grapes were sampled separately from coloured berries from the same grapes’. Do you mean bunches/racemes (see comment above)? See also the last sentence of this section. After hybridization, no significant signal was detected in green berries (c2), whereas intense signals, localized in flesh cells (parenchyma cells), were observed for coloured berries (c3). The two analysed berries belong to the same raceme and were harvested on the same day (day 60 after flowering). (D) Berries at ripeness. Signals were detected in the parenchyma cells of the inner mesocarp (d3 and d4) and the perivascular cells surrounding the vascular bundles of pips (d5). CCVB, central capillary vascular bundles; End, endocarp; E, endosperm; Ep, epicarp; EOL, epidermis ovarian locule; II, inner integument; IM, inner mesocarp; Mes, mesocarp; N, nectaries; Nu, nucellus; O, ovule; OI, outer integument; OM, outer mesocarp; P, pericarp; Pe, perisperm; Ph, phloem; PVC, peri-vascular cells; S, style; SC, seed coat; St, stigmas; Xy, xylem.

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Effect of drought stress on VvK1.2 expression

The consequences of drought stress on grape berry composition and development strongly depend on the time at which the stress arises during berry development (Ojeda et al., 2001). A moderate drought stress (Ψ values from –0.7 to –0.6 MPa, versus –0.2 in control plants) was applied to plants grown in open-field conditions at five stages of berry development: fruit set (day 15 after flowering in this experiment), during the rapid cell division phase following fruit set (day 25 after flowering), at veraison (day 60 after flowering) and during ripening (days 67 and 75 after flowering). Real-time PCR experiments were performed in order to investigate the effects of drought stress on VvK1.2 expression (Figure 4). VvK1.2 expression in vegetative organs was low (in agreement with the data shown in Figure 2) and was not significantly affected by drought stress. However, the high increase in VvK1.2 expression in the berry during berry development from veraison (Figure 2) was accompanied by a strong sensitivity to drought stress, which increased VvK1.2 transcript accumulation by up to 20-fold at veraison and fourfold at ripeness.

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Figure 4. Effect of drought stress on VvK1.2 expression. Real-time quantitative PCR was performed on first-strand cDNAs synthesized from total RNAs. A VvK1.2 standard curve was used to determine the expression level of VvK1.2 (number of VvK1.2 mRNA molecules) in each sample. Berries or aerial vegetative organs (leaves, stems, petioles and tendrils) were collected from grapevines grown in open-field conditions under standard irrigation (control) or limited irrigation (drought stress). Berries were collected at various stages of berry development. At veraison (berry colour transition, day 60 in this experiment), green and red berries on turning grapesPlease check sense ‘turning grapes’, see comment above were sampled separately. Leaves, stems, petioles and tendrils were collected at fruit set. Values are means and standard errors of two biological samples (three technical replicates per sample).

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Identification of VvCIPKs and VvCBLs as potential regulators of VvK1.2 in maturing berries

In Arabidopsis, CIPK–CBL complexes have been shown to interact with and activate Shaker channels (Figure S1), thereby playing major roles in regulation of K+ transport (Xu et al., 2006; Held et al., 2011; Lan et al., 2011). A report of functional interactions between the grapevine VvK1.1 Shaker channel (Cuéllar et al., 2010) and the Arabidopsis AtCIPK23–AtCBL1 complex, which is known to interact with and activate the Arabidopsis AKT1 Shaker channel (Xu et al., 2006), provided evidence that CIPK–CBL complexes may also regulate K+ channel activity in grapevine. In a search for such regulatory complexes for VvK1.2, in silico analyses of the grapevine genome were performed. Twenty CIPKs (Figure 5a) and eight CBLs (Figure 5b) were identified. Because VvK1.2 is strongly induced at veraison and during the whole maturing period, nested PCR experiments were performed to identify, among the eight CBL genes, those that are expressed after veraison. Four CBL genes were found to be expressed in ripening berries, where their level of expression was further analysed by quantitative RT–PCR (see below). These CBLs were VvCBL01 (gene ID 100260894), VvCBL02 (gene ID 100266945), VvCBL03 (gene ID 100262221) and VvCBL06 (gene ID 100259377), whose closest homologues in Arabidopsis are AtCBL1/AtCBL9, AtCBL2/AtCBL3, AtCBL6 and AtCBL10, respectively (Figure 5b).

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Figure 5. Phylogenetic relationships of CIPKs and CBLs in Arabidopsis and grapevine. Unrooted trees constructed using complete sets of CIPK and CBL proteins in Arabidopsis and grapevine.(a) Phylogenetic relationships of VvCIPKs. Arabidopsis CIPK family (25 membersPlease check amendment ‘25 members’ (see list and artwork)): CIPK1 (AAG28776); CIPK2 (AAF86506); CIPK3 (AAF86507); CIPK4 (AAG01367); CIPK5 (AAF86504); CIPK6 (AAF86505); CIPK7 (AAK16682); CIPK8 (AAK16683); CIPK9 (AAK16684); CIPK10 (AAK16685); CIPK11 (AAK16686); CIPK12 (AAK16687); CIPK13 (AAK16688); CIPK14 (AAK16689); CIPK15 (AAK16692); CIPK16 (AAF19215); CIPK17 (AAK64513); CIPK18 (AAK59695); CIPK19 (AAK50347); CIPK20 (AAK61493); CIPK21 (AAK59696); CIPK22 (AAL47845); CIPK23 (AAK61494); CIPK24 (AAK72257); CIPK25 (AAL41008). Grapevine CIPK family (20 members): VvCIPK01 (ACQ83517); VvCIPK02 (CAN67244); VvCIPK03 (CAN68670); VvCIPK04 (FR669159); VvCIPK05 (CAN73080); VvCIPK06 (XP_002273178); VvCIPK07 (ACQ83523); VvCIPK08 (ACQ83524); VvCIPK09 (CAN83760); VvCIPK10 (ACQ83526); VvCIPK11 (ACQ83527); VvCIPK12 (ACQ83528); VvCIPK13 (XP_002279331); VvCIPK14 (ACQ83530.1); VvCIPK15 (ACQ83530); VvCIPK16 (ACQ83532); VvCIPK17 (ACQ83533); VvCIPK18 (ACQ83534); VvCIPK19 (ACQ83535); VvCIPK20 (ACQ83536).(b) Phylogenic relationships of VvCBLs. Arabidopsis CBL family (ten members): CBL1 (AAC26008); CBL2 (AAC26009); CBL3 (AAC26010); CBL4 (AAG28402); CBL5 (AAG28401); CBL6 (AAG28400); CBL7 (AAG10059); CBL8 (AAL10300); CBL9 (AAL10301); CBL10 (AAO72364). Grapevine CBL family (eight members): VvCBL01 (FR669117); VvCBL02 (XP_002272971); VvCBL03 (XP_002266438); VvCBL04 (XP_002277839); VvCBL05 (XP_002265356); VvCBL06 (XP_002277878); VvCBL07 (XP_002277917); VvCBL08 (XP_002267431).

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In Arabidopsis, AtCBL1, AtCBL2, AtCBL3 and AtCBL9 are able to interact with AtCIPK6, AtCIPK16 or AtCIPK23 (Figure S1) to form complexes that regulate the activity of the AKT1 K+ channel (Xu et al., 2006; Lee et al., 2007). AtCBL4 has been shown to interact with AtCIPK6 to regulate plasma membrane targeting and activity of the AKT2 K+ channel (Held et al., 2011). The information available from Arabidopsis and data from quantitative RT–PCR experiments to analyse the expression of candidate genes in maturing berries (see below) were used to select grapevine CIPKs that are likely to cooperate with the selected CBLs to regulate VvK1.2 activity in maturing berries (Figure 5). First, we selected three grapevine CIPKs, VvCIPK04 (gene ID 100253180), VvCIPK03 (gene ID 100241657) and VvCIPK02/05 (gene ID 100261839), which are close relatives of Arabidopsis CIPKs involved in K+ channel regulation. VvCIPK04 is the closest relative of Arabidopsis AtCIPK23. VvCIPK03 and VvCIPK02/05 are the closest relatives of Arabidopsis AtCIPK6. Two further grapevine CIPKs were then selected: VvCIPK09 (gene ID 100262837) and VvCIPK07 (gene ID 100243133). VvCIPK09 is the closest grapevine relative of Arabidopsis AtCIPK24 (also named SOS2), which forms complexes with AtCBL1, AtCBL4 (SOS3) or AtCBL10 and regulates ion transport across the cell membrane and tonoplast (Kim et al., 2007; Waadt et al., 2008). VvCIPK07 was selected as a ‘control’ CIPK, as it displayed significant and slightly increased expression during berry ripening (Figure 6) and its closest relatives in Arabidopsis, AtCIPK2 and AtCIPK10, have not been reported so far to play any role in the control of membrane ion transport.

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Figure 6. Expression of selected VvCIPK and VvCBL genes in the developing berry. Real-time quantitative PCR was performed on first-strand cDNAs synthesized from total RNAs prepared from ripening berries. A specific standard curve was used for each gene studied to determine the expression level of each targeted gene. For each experiment, approximately 50 berries were harvested from five vines and pooled. Each pool was used as a biological replicate. Values are means and standard errors of two biological replicates (three technical replicates per sample). (a) Absolute expression level of VvCBL01, VvCBL02, VvCBL03 and VvCBL06. (b) Absolute expression level of VvCIPK04, VvCIPK02/05, VvCIPK07, VvCIPK09 and VvCIPK03.

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Full-length cDNAs were cloned for each of the four grapevine CBLs displaying expression in ripening berries (VvCBL01, VvCBL02, VvCBL03 and VvCBL06) and the five selected CIPKs (VvCIPK02/05, VvCIPK03, VvCIPK04, VvCIPK07 and VvCIPK09), using total RNAs prepared from Cabernet Sauvignon maturing berries (Methods S1 and Table S1).

Expression of the selected CBL and CIPK genes in maturing berries

Expression of the four VvCBL genes and five VvCIPK genes was analysed by real-time PCR using the same preparations of total RNA extracted from berries as those used previously for analysing VvK1.2 expression, at two stages of development: veraison, and during the post-veraison maturing period (Figure 6). Each of the selected genes showed significant expression in ripening berries compared with the data obtained for VvK1.2 (Figure 2). The nine selected genes showed higher or similar levels of expression in the post-veraison maturing berry as in the berry at veraison (Figure 6a,b). The expression levels of VvCBL01 and VvCBL02 in the maturing berry are similar to each other and much higher than those of the other two CBL genes, VvCBL03 and VvCBL06 (Figure 6a). Among the five selected CIPK genes, VvCIPK03 displayed much higher expression levels, by about 20 times, than the other tested VvCIPK genes. It is also worth noting that all tested VvCIPK genes showed increased expression from veraison to ripeness (Figure 6b).

Functional expression of VvK1.2 in oocytes

A preliminary set of experiments revealed that VvK1.2, like AKT1 (Xu et al., 2006) and VvK1.1 (Cuéllar et al., 2010), did not affect the membrane conductance when expressed alone in Xenopus oocytes (Figure 7a, upper panel). Interestingly, when co-expressed with the Arabidopsis protein complex AtCIPK23–AtCBL1, VvK1.2 mediated exogenous inward currents upon membrane hyperpolarization (Figure S2), as previously reported for AKT1 (Xu et al., 2006) and VvK1.1 (Cuéllar et al., 2010). This supports the hypothesis that mechanisms of K+ channel regulation involving CIPK–CBL complexes are conserved between Arabidopsis and grapevine.

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Figure 7. VvK1.2 current activationPlease check sense ‘VvK1.2 current activation’ by the grapevine VvCIPK04–VvCBL01 pair in Xenopus oocytes.(a) Representative current traces upon voltage-clamp pulses from +40 mV to –160 mV in oocytes expressing VvK1.2 alone (upper panel), VvCIPK04 and VvCBL01 (middle panel) or VvK1.2 with VvCIPK04 and VvCBL01 (lower panel), in 100 mm K+ solution at pH 6.5.(b) VvK1.2 inward currents (recorded in the presence of VvCIPK04 and VvCBL01) are inhibited by 10 mm Cs+. A representative inhibition of approximately 90% is shown in comparison to currents in (a).(c,d) The VvK1.2 inward currents (recorded in the presence of VvCIPK04 and VvCBL01) are dependent on external potassium concentration (c) and external pH (d). Steady-state currents at the end of voltage pulses as shown in (a) were normalized by the current recorded in the corresponding oocyte at –150 mV in K100 at pH 6.5, and plotted against corresponding applied membrane potentials. Values are means ± SE (= 5). The external potassium concentration (c) was 100 mm (K100), 10 mm (K10) or 1 mm (K1) at pH 6.5. The external pH (d) was 5, 6.5 or 7.5 in 100 mm K+.

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This hypothesis was further investigated by determining the effects of VvCIPK04 and VvCBL01 (the closest grapevine relatives of Arabidopsis AtCIPK23 and AtCBL1) on VvK1.2 activity. Large slowly activating exogenous K+ currents were recorded in oocytes co-expressing VvK1.2 with VvCIPK04 and VvCBL01, but not in oocytes co-expressing VvCIPK04 and VvCBL01 only (Figure 7a), providing further support to the above hypothesis.

The functional properties of VvK1.2 were then investigated in oocytes in the presence of VvCIPK04 and VvCBL01. Altogether, the data shown in Figure 7 indicate that VvK1.2 is an inwardly rectifying potassium channel that is activated at membrane potentials more negative than approximately –100 mV (Figure 7c), blocked by Cs+ (Figure 7b) and activated by external acidification (Figure 7d).

A second set of experiments was then performed to investigate the effects on VvK1.2 activity of other selected CIPK–CBL pairs from grapevine upon co-expression in oocytes. Four pairs were tested: VvCIPK03–VvCBL02, VvCIPK09–VvCBL01, VvCIPK09–VvCBL06 and VvCIPK07–VvCBL02. The rationale for this selection is described above, and clarified in Figure 5 and Figure S1. Only the first pair, VvCIPK03–VvCBL02, was found to induce VvK1.2 K+ channel activity in oocytes, giving rise to Cs+-sensitive potassium currents that were much smaller (mean value −1.33 ± −0.51 μA, = 6, at −160 mV in 100 mm external K+) than those induced by the VvCIPK04–VvCBL01 pair (−6.81 ± 1.81 μA, = 8) in experiments performed using oocytes from the same batches (Figure 8).

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Figure 8. VvK1.2 current activation by the grapevine VvCIPK03–VvCBL02 pair in Xenopus oocytes. The voltage-clamp protocol involved pulses from +40 mV to −160 mV (as in Figure 7). (a) Representative current traces recorded in an oocyte co-expressing VvK1.2 with VvCIPK03 and VvCBL02, and inhibition of the VvK1.2 currents by Cs+. Currents were recorded in 100 mm K+ external solution, pH 6.5, in the absence (upper panel) or presence of 10 mm Cs+ (lower panel). (b) Representative current traces recorded in an oocyte co-expressing VvK1.2 with VvCIPK04 and VvCBL01. The oocyte was from the same batch as used in (a) for comparisonPlease check amendment ‘The oocyte was from the same batch as used in (a) for comparison’. (c) Steady-state current–voltage (I–V) relationships for VvK1.2 co-expressed with either VvCIPK04 and VvCBL01 (black squares) or VvCIPK03 and VvCBL02 (black circles) in 100 mm K+ external solution (K100). Values are means ± SE (= 6–8). (d) Cs+ inhibition of VvK1.2 currents recorded in the presence of VvCIPK03 and VvCBL02: steady-state I–V plots obtained in 100 mm K+ external solution in the absence (black circles) or presence (open circles) of 10 mm Cs+.

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In a final series of experiments, the three CIPKs VvCIPK02/05, VvCIPK07 and VvCIPK09 were tested one by one via co-injection with VvK1.2 and the four VvCBLs (VvCBL01, VvCBL02, VvCBL03 and VvCBL06 together). The co-injected oocytes did not display any significant typical potassium currents at days 3–5 after injection.

Altogether, the analyses performed in oocytes indicate that VvK1.2 functionally interacts with and is activated by at least two CIPK–CBL pairs, but in a specific way and with distinct levels of activation. Note that the two grapevine CIPK–CBL pairs that were shown to have the capacity to regulate VvK1.2 activity in these experiments, VvCIPK04–VvCBL01 and VvCIPK03–VvCBL02, have close relatives in Arabidopsis, AtCIPK23 and AtCBL1, and AtCIPK6 and AtCBL2, respectively, that have been characterized as regulatory partners of Shaker group I K+ channels in this model plant.

Effect of drought stress on expression of CIPKs and CBLs identified as regulatory partners of VvK1.2

Quantitative RT–PCR data indicated that drought stress results in a strong increase in VvK1.2 expression in maturing berries (Figure 4). Using the same approach and RNA samples, we investigated the effect of drought stress on expression of the CBLs and CIPKs identified as regulatory partners of VvK1.2 by the experiments in oocytes: VvCIPK04–VvCBL01 and VvCIPK03–VvCBL02. For VvCIPK04, VvCIPK03 and VvCBL02, the applied drought stress was found to result in an increase in gene expression of approximately two- to threefold, in both berries at veraison and ripening berries (Figure 9). For VvCBL01, drought stress resulted in an increase in gene expression of approximately twofold at veraison, and a decrease in expression, again of approximately twofold, while the berries were ripening.

image

Figure 9. Effect of drought stress on gene expression of CIPKs and CBLs identified as regulatory partners of VvK1.2.Berries were collected at veraison (day 60 after flowering) or during the post-veraison ripening phase (at day 75 after flowering) from control (grey bars) or drought-stressed (black bars) grapevines. The same RNA samples and protocol for quantitative PCR analysis were used as in Figure 6 experiments. Values are means and standard errors of two biological replicates.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental Procedures
  7. Accession numbers
  8. Acknowledgements
  9. References
  10. Supporting Information

Identification of a K+ channel expressed in ripening berries

K+ is essential for vine growth and yield. The berry is a strong sink for this cation, particularly during ripening. However, high K+ levels in berries lead to decreased vacuolar acidity and changes in relative concentrations of organic acids, affecting fruit quality and resulting in poor quality wine (Hale, 1977; Delas et al., 1989). The present study aims to identify molecular mechanisms responsible for K+ transport and accumulation in grapevine. Here we report the identification and characterization of a K+ channel that is essentially expressed in the ripening berry.

The K+ transport systems characterized at the molecular and functional levels so far in dicotyledonous plants essentially belong to two families, named KUP/KT/HAK and Shaker (Véry and Sentenac, 2003). The KUP/KT/HAK family (13 members in A. thaliana) encodes K+ transporters, including high-affinity H+:K+ symport systems (Gierth and Mäser, 2007). The Shaker family (nine members in grapevine, as in A. thaliana) encodes voltage-gated K+ channels that dominate the cell membrane conductance to K+ in most cell types (Lebaudy et al., 2007). So far, in grapevine, in addition to VvK1.2 two KUP/KT/HAK transporters and two Shaker channels have been cloned and characterized. The transporters, named VvKUP1 and VvKUP2, have been shown to possess K+ transport capacity by functional expression in Escherichia coli. They are expressed in the berry skin during the first phase of berry development (pre-veraison) (Davies et al., 2006). The Shaker channels, named VvSIRK (stomatal inward rectifying K+ channel, also named VvK2.1; Pratelli et al., 2002) and VvK1.1 (Cuéllar et al., 2010), have been shown to mediate voltage-gated inward K+ currents by expression in Xenopus oocytes. The VvSIRK promoter is essentially active in guard cells. In berries, the amounts of SIRK transcript decrease drastically by the time of veraison, possibly because berry stomata evolve into non-functional lenticels after veraison (Pratelli et al., 2002). VvK1.1 is expressed in root peripheral cells and, to a lower level, in young berries, mainly in the seed. It does not appear to be regulated during berry development and ripening. Thus, the Shaker channel we have identified from berry mRNA, VvK1.2, is the only K+ transport system identified so far that is specifically expressed in the berry flesh and is up-regulated during fruit maturation.

Shaker family group I in grapevine

In silico analysis indicates that VvK1.2 belongs to Shaker family group I (Figure 1). This gene encodes a voltage-gated inwardly rectifying K+-selective channel, like all Shaker group I genes characterized so far in plants (Lebaudy et al., 2007). The activation potential of VvK1.2 is rather negative, below –100 mV. Large differences in activation potentials are found amongst Shaker group I channels. It should be also noted that interactions with other proteins, in heterologous contexts as well as in planta in the native cells, strongly affect the channel activation potential, as shown for AKT1 and the regulating Shaker subunit AtKC1 (Reintanz et al., 2002; Duby et al., 2008).

The Shaker group I family has two members in grapevine, VvK1.1 (Cuéllar et al., 2010) and VvK1.2, and three members in Arabidopsis, AKT1 (Sentenac et al., 1992), SPIK (Mouline et al., 2002) and AKT5 (Lacombe et al., 2000; Pilot et al., 2003) (Figure 1). AKT1 shows preferential expression in root peripheral cells (Hirsch et al., 1998), SPIK shows preferential expression in pollen (Mouline et al., 2002) and AKT5 shows preferential expression in flowers and developing siliques (Lacombe et al., 2000). Based on sequence identities, both VvK1.1 and VvK1.2 are more closely related to AKT1 than to SPIK or AKT5. However, sequence identity with AKT1 is greater for VvK1.1 (71% ASI) than for VvK1.2 (62% ASI). Furthermore, the expression pattern of VvK1.1 is more similar to that of AKT1 (both genes being expressed in the root cortex) than expression of VvK1.2 is. VvK1.2 shares with SPIK and AKT5 the fact that it is more specifically expressed in reproductive tissues/organs. Thus, this analysis suggests that the grapevine orthologue of AKT1 is VvK1.1. In other words, in grapevine Shaker group I, VvK1.1 is more specifically dedicated to K+ uptake from the soil solution and VvK1.2 is more specifically dedicated to K+ transport in reproductive organs.

Conservation of K+ channel regulation by CIPK–CBL complexes between Arabidopsis and grapevine

The Arabidopsis channel AKT1 physically and functionally interacts with the complex formed by the kinase AtCIPK23 and its regulating partner, the Ca2+ sensor AtCBL1. Upon co-expression with this complex in Xenopus oocytes, AKT1 becomes functional and electrically detectable at the cell membrane (Xu et al., 2006). Evidence has been obtained, based on genetic analyses, that such interactions occur in planta and play a role in the control of K+ transport and homeostasis (Xu et al., 2006; Cheong et al., 2007; Lee et al., 2007; Batistič and Kudla, 2012).

The grapevine channel VvK1.2 is activated by Arabidopsis AtCIPK23 and AtCBL1 and their grapevine homologues VvCIPK04 and VvCBL01 (Figure 7), supporting the hypothesis of conservation of channel regulation mechanisms. In addition, the specificity of K+ channel regulation mechanisms involving CIPK–CBL partners was investigated by cloning grapevine cDNAs encoding other VvCIPKs and VvCBLs expressed in ripening berries and determining their capacity to activate VvK1.2 when co-expressed in oocytes. These experiments provided evidence that VvK1.2 activation by VvCIPK–VvCBL complexes does not occur with just any VvCIPK and VvCBL partners but involves specific complexes.

In addition to the first VvCIPK04-VvCBL01 pair, a second grapevine VvCIPK–VvCBL pair, VvCIPK03–VvCBL02, has been shown to activate VvK1.2 currents (Figure 8). It is worth noting that VvCIPK03 displays much higher levels of expression in the ripening berry than the other four tested CIPKs. The closest relative of VvCIPK03 in Arabidopsis is AtCIPK6, which is known to be able to activate AKT1 in a CBL-dependent manner, upon interaction with AtCBL1/AtCBL9 or AtCBL2/AtCBL3 (Lee et al., 2007). Interestingly, AKT1 activation by AtCIPK6 in interaction with either AtCBL1 or AtCBL2 has been shown to produce currents of lower amplitudes than activation by AtCIPK23 and AtCBL1. Similarly, in grapevine, the present report shows that the complex VvCIPK03–VvCBL02 is less efficient at activating VvK1.2 (producing currents of lower amplitudes) than the complex VvCIPK04–VvCBL01. Such consistent observations fully agree with the hypothesis that at least part of the regulatory networks associating Shaker channels with CIPK–CBL complexes are strongly conserved between Arabidopsis and fruit crops. This hypothesis deserves to be further investigated in other fruit species, such as tomato, due to the major role that K+ plays in fleshy fruit development and fruit quality. It is also worth noting that the four VvCBLs and five VvCIPKs that we cloned from ripening berries show enhanced (or at least stable) expression during ripening and upon drought stress. Thus, CIPK–CBL complexes are likely to play various roles in berry development and adaptation to drought stress, in addition to regulation of K+ channel activity.

Role of VvK1.2 in the plant

As an inwardly rectifying K+ channel activating at membrane potentials more negative than –100 mV, VvK1.2 is very probably dedicated to K+ uptake. Its stimulation upon acidification of the external medium suggests regulation of K+ influx by the apoplastic pH, which is under the control of H+ secretion by plasma membrane H+–ATPases. Interestingly, the maturing berry is probably the main site at which VvK1.2 plays such a role in K+ uptake, as suggested by analysis of its expression pattern and transcriptional regulation. With regard to fruit development and quality, it should be emphasized that VvK1.2 is the only K+ uptake system identified so far as specifically induced in maturing berry flesh cells.

After veraison, the grape berry becomes a strong sink for K+. Long-distance transport in the xylem and phloem vasculature towards the berry is profoundly altered by the time of veraison due to developmental processes. Discontinuities of the xylem occur in the vascular tissues that connect the berry to the stalk, resulting in hydraulic isolation of the berry xylem (Düring et al., 1987; Findlay et al., 1987). The berry thus becomes dependent on phloem sap flux not only for sugars and other organic molecules but also for mineral nutrient ions and water. Also, by the time of veraison or just before, the mechanisms of phloem unloading in the berries are shifted from symplasmic to apoplasmic mode, probably due to a decrease in the plasmodesmal conductivity between the sieve element/companion cell complexes and the surrounding parenchyma cells (Zhang et al., 2006). It has been hypothesized that destruction of the xylem pathway may be a mechanism to reduce the transport of apoplastic solutes out of the berries (Findlay et al., 1987; Mpelasoka et al., 2003). The shift from the symplastic to the apoplasmic mode of phloem unloading is thought to prevent reduction of the pressure difference between the import phloem in source leaves and the terminal release phloem in the sink organ, and hence to ensure efficient long-distance phloem transport from source leaves to ripening berries (Patrick, 1997; Zhang et al., 2006). Occurring in the context of these profound developmental changes, VvK1.2 expression in the berry at veraison, in flesh cells, phloem tissues and perivascular cells surrounding vascular bundles, probably contributes to re-organization of the translocation pathways and transport mechanisms feeding the berry. For instance, VvK1.2 activity under control of specific VvCIPK–VvCBL pairs may allow rapid K+ retrieval from the apoplast by perivascular and flesh cells, decreasing the apoplastic concentration of this cation and thereby favouring its unloading from sieve element/companion cell complexes. Also, rapid unloading of K+ may stimulate the flux of phloem sap towards the sink and thereby sugar import (Mpelasoka et al., 2003). Finally, the strong sensitivity of VvK1.2 expression to plant water status suggests that control of K+ transport is crucial in the maturing berry under fluctuating water availability and drought conditions. The identification of the grape berry K+ channel VvK1.2 and specific CIPK–CBL partners controlling its activity clearly opens stimulating perspectives for investigating the roles of K+ in fleshy fruit development and fruit quality.

Experimental Procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental Procedures
  7. Accession numbers
  8. Acknowledgements
  9. References
  10. Supporting Information

Plant growth and plant material collection

Four-year-old grapevines (Vitis vinifera L. cv. Cabernet Sauvignon) grafted on Fercal rootstock were grown in field conditions in 70 litre containers under controlled irrigation as described previously (Cuéllar et al., 2010). A drip-irrigation system allowing various irrigation programs was used to control the plant water status, which was assessed by measuring the leaf water potential (ψ) at dawn using the pressure chamber technique (Scholander, 1965). Control plants (not submitted to drought stress) were irrigated throughout the experiment so that ψ at dawn remained close to –0.2 MPa. Drought-stressed plants were obtained by progressively decreasing the water supply over a period of 2 weeks in order to obtain ψ values between –0.7 and –0.6 MPa for 2 days at least, before tissue/organ collection (Cuéllar et al., 2010).

Leaves, stems, petioles and tendrils were collected from control or drought-stressed plants at the initial berry formation period. Berries were sampled at various stages of grape development from control or drought-stressed plants. Roots were collected from 2-month-old rooted canes planted in perlite (Cuéllar et al., 2010). All collected samples were immediately frozen in liquid nitrogen and stored at –80°C until use.

Isolation of cDNAs encoding VvK1.2, VvCIPK and VvCBL proteins

VvK1.2 was cloned while the Vitis vinifera genome-sequencing program was in progress. The cDNA cloning strategy and sequencing are described in Methods S1. The availability of the grapevine reference genome sequence (Jaillon et al., 2007; Weinl and Kudla, 2009) allowed in silico analysis for cloning of the selected VvCIPK and VvCBL genes. Primers were designed from predicted sequences, and corresponding cDNAs were isolated from post-veraison berry RNAs by nested PCR (Methods S1). The primers used in this study are listed in Table S1.

Functional characterization of VvK1.2

Oocyte handling and voltage-clamp experiments were performed as described previously (Cuéllar et al., 2010). VvK1.2, VvCIPK and VvCBL cRNAs were injected using a microinjector (Nanoliter 2000, WPI, http://www.wpi-europe.com/fr/) into oocytes (6 ng VvK1.2 cRNA co-expressed with the same amount of VvCIPK/CBL). Control oocytes were injected with 7 ng VvK1.2 alone, with 7 ng VvCIPK/CBL or with water (23 nl). Membrane currents were measured 3–5 days after injection in solutions containing 100 (K100), 10 (K10) or 1 mm (K1) potassium gluconate, supplemented with 0, 90 or 1 mm sodium gluconate, respectively, in 1 mm CaCl2, 2 mm MgCl2 and 10 mm HEPES, pH 6.5 or 7.5, or 10 mm MES, pH 5.0. Inhibition by Cs+ was tested by addition of 10 mm CsCl to the K100 solution at pH 6.5. Voltage-clamp protocols were applied with 2 sec voltage pulses from +40 to −160 mV (or −170 mV), with –10 mV steps (or –15 mV steps), followed by a 250 msec voltage step to –40 mV at a holding potential of 0 mV. Normalized current–voltage (I–V) 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 −150 mV in K100 at pH 6.5 to −1 for each oocyte.

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

Total RNA extraction, synthesis of first-strand cDNAs and quantitative RT–PCR procedures were performed as described by Cuéllar et al. (2010). The oligonucleotides used are listed in Table S2.

Localization of VvK1.2 expression by mRNA in situ hybridization

In situ hybridization experiments were performed as described by Cuéllar et al. (2010). K1.2–136–F and K1.2–136–R, which are VvK1.2-specific primers (Table S2) that span intron 10 were used to synthesize RNA probes as described by Alemanno et al. (2008).

Accession numbers

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental Procedures
  7. Accession numbers
  8. Acknowledgements
  9. References
  10. Supporting Information

The EMBL accession numbers for the VvK1.2 gene, VvK1.2 cDNA, VvCIPK04 cDNA and VvCBL01 cDNA are FR669115, FR669116, FR669159 and FR669117, respectively.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental Procedures
  7. Accession numbers
  8. Acknowledgements
  9. References
  10. Supporting Information

This work was supported in part by a grant from the Institut National de la Recherche Agronomique to T.C., a grant from Genoplante (ANR-05-GPLA-024) to I.G. and H.S., a scholarship from the Higher Education Commission of Pakistan to F.A., and a scholarship from the Conabex Commission of Madagascar to M.A.

References

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  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental Procedures
  7. Accession numbers
  8. Acknowledgements
  9. References
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental Procedures
  7. Accession numbers
  8. Acknowledgements
  9. References
  10. Supporting Information
FilenameFormatSizeDescription
tpj12092-sup-0001-FigS1.pdfapplication/PDF124KFigure S1. CIPK/CBL Shaker channel partners in Arabidopsis thaliana
tpj12092-sup-0002-FigS2.pdfapplication/PDF352KFigure S2. VvK1.2 current activation by the Arabidopsis AtCIPK23–AtCBL1 pair in Xenopus oocytes.
tpj12092-sup-0003-MethodsS1.pdfapplication/PDF91KMethods S1. Supplementary experimental procedures.
tpj12092-sup-0004-TableS1.pdfapplication/PDF57KTable S1. Primers used for VvK1.2, VvCIPK and VvCBL cloning.
tpj12092-sup-0005-TableS2.pdfapplication/PDF51KTable S2. Primers for quantitative PCR.
tpj12092-sup-0006-legends.docxWord document14K 

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