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(number and positions of introns) as AKT1 (Figure 1b), whereas VvK1.2 comprises one further intron (T.C. and I.G., unpublished data); and (iii) both VvK1.1 and AKT1 are expressed in the root cortex, whereas VvK1.2 does not display any expression in roots (absence of transcripts in real-time PCR experiments; T.C. and I.G., unpublished data).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.