1. The functional subclasses of NSCCs
Even though no definitive molecular candidates have thus far emerged, a strong consensus has developed in recent years, largely based on electrophysiological studies, that various classes of NSCCs catalyse primary influx of Na+ under saline conditions. NSCCs are thoroughly characterized in animals, and their functions are well understood in their cellular signaling, vascular endothelial function, Ca2+ influx in response to store depletion, and renal ion homeostasis (Kaupp & Seifert, 2002; Clapham, 2003; Firth et al., 2007; Venkatachalam & Montell, 2007; Kauer & Gibson, 2009). In plants, several categories of NSCCs have also been identified, and these have been subdivided (Demidchik & Tester, 2002; Demidchik & Maathuis, 2007), according to their response to changes in membrane electrical potential, into the following major classes: (1) depolarization-activated NSCCs (DA-NSCCs), (2) hyperpolarization-activated NSCCS (HA-NSCCs), and (3) voltage-insensitive NSCCs (VI-NSCCs). Additional classification systems distinguish NSCCs by their reponsiveness to certain ligands and physical stimuli and include cyclic-nucleotide-gated NSCCs (CNGCs), amino-acid-gated NSCCs (AAG-NSCCs), and reactive-oxygen-species-activated NSCCs (ROS-NSCCs). These may well constitute representatives of subclasses (1) through (3), as may other minor types of NSCCs not discussed here (see Demidchik & Maathuis, 2007).
The first definitive demonstration, using patch-clamp approaches, of NSCC-type conductances in plants dates to 1989, when Stoeckel and Takeda reported constitutive cation fluxes across the plasma membranes of triploid endosperm cells in species from the genera Haemanthus and Clivia that displayed minimal selectivity for various alkali, and some earth alkali, ions, and could be activated following depolarizations of the membrane potential (Stoeckel & Takeda, 1989). Despite some constitutive activity, these types of NSCCs have thus been classified in category 1 above. DA-NSCC operation has since been confirmed in a large number of experimental systems, including leaf and root cell preparations from Arabidopsis thaliana, Thlaspi arvense and T. caerulescens, Hordeum vulgare and Phaseolus vulgaris (Cerana & Colombo, 1992; Spalding et al., 1992; de Boer & Wegner, 1997; Pei et al., 1998; Piñeros & Kochian, 2003; Zhang et al., 2004). Their main function appears to be in conducting Ca2+ (White & Ridout, 1999; White et al., 2000), although a role in catalyzing K+ release from root cells under sudden imposition of saline conditions has also been proposed (Shabala et al., 2006). By contrast, the role of DA-NSCCs in catalyzing primary Na+ fluxes under salt stress conditions has been much less conclusively demonstrated. Nevertheless, in a major review on the topic (Demidchik & Maathuis, 2007), it was suggested that members of this depolarization-activated class of NSCCs may well be involved in this function. The proposal was based upon reference to a series of comparative electrophysiological studies conducted in Arabidopsis thaliana and its natural halophyte relative Thellungiella halophila (Volkov et al., 2004; Volkov & Amtmann, 2006; Wang et al., 2006); studies that, however, concluded that the predominant Na+ conductances observed were voltage-insensitive, not depolarization-activated. A role for the subclass of depolarization-activated NSCCs in catalyzing significant Na+ fluxes under saline conditions therefore remains purely speculative at this point.
NSCC category 2 (HA-NSCCs) can be excluded from further in-depth discussion in the context of primary Na+ fluxes under salinity, as hyperpolarization of the plasma membrane, inherent to the gating properties of these channels (see e.g. Gelli & Blumwald, 1997; Hamilton et al., 2000; Véry & Davies, 2000; Demidchik et al., 2007), does not typically accompany the imposition of salinity, neither in short-term nor in long-term applications of Na+ (Laurie et al., 2002; Carden et al., 2003; Shabala et al., 2006; Volkov & Amtmann, 2006; Malagoli et al., 2008).
2. VI-NSCCs: the current consensus
In contrast to the above categories, a substantial number of studies support a role for VI-NSCCs (category 3) in catalyzing Na+ fluxes across the plasma membrane, in particular in roots (some reports have also focused on shoots: see e.g. Elzenga & van Volkenburgh, 1994; Véry et al., 1998), and it is here where more extensive discussion is warranted. CNGCs, AAG-NSCCs and ROS-NSCCs may well represent subclasses of this type of NSCC (Demidchik & Maathuis, 2007). The earliest demonstration of VI-NSCCs was in wheat (Triticum aestivum; Moran et al., 1984; see also: Tyerman et al., 1997; Buschmann et al., 2000; Davenport & Tester, 2000), followed by extensive work in rye (Secale cereale; White & Tester, 1992; White & Lemtiri-Chlieh, 1995; White & Ridout, 1995; White, 1996), maize (Zea mays; Roberts & Tester, 1997), barley (Hordeum vulgare; Amtmann et al., 1997), A. thaliana (Maathuis & Sanders, 2001; Demidchik & Tester, 2002; Shabala et al., 2006; Volkov & Amtmann, 2006), Thellungiella halophila (Volkov et al., 2004; Volkov & Amtmann, 2006; Wang et al., 2006), and Capsicum annuum (Murthy & Tester, 2006). Common features unite the observations in this large body of studies: VI-NSCCs are so named because their open probability is not significantly, or at best weakly, modulated by membrane potential, in contrast to the categories of NSCCs discussed above. Currents are constitutive and instantaneous (i.e. permanently present when ensemble averages, not individual channel traces, are examined), and they lack time-dependent activation (Tyerman et al., 1997; Amtmann & Sanders, 1999; White, 1999b). VI-NSCCs have been shown, in classic current–voltage relationships, to conduct both inward and outward currents, and thus may constitute both influx and efflux pathways in planta (see e.g. Shabala et al., 2006; Volkov & Amtmann, 2006). VI-NSCCs also exhibit several pharmacological characteristics that separate them from other classes of ion channels (see Demidchik et al., 2002; Demidchik & Maathuis, 2007): they are not sensitive to the potassium channel inhibitors Cs+ and tetra-ethyl-ammonium (TEA+), are not affected by the alkali cations Li+ and Na+, the sodium channel inhibitor tetrodotoxin (cf. Allen et al., 1995) or the calcium channel inhibitors verapamil and nifedipine, but are greatly inhibited by the trivalent cations lanthanum (La3+) and gadolinium (Gd3+; it should be noted, however, that these two cations are very broad-spectrum; see e.g. Qu et al., 2007). One class of VI-NSCCs can also be partially blocked by divalent cations, including Ba2+ and Zn2+, as well as, especially importantly, Ca2+ and Mg2+, while another class is not inhibited by these ions, but instead transports them (Demidchik & Maathuis, 2007). Some VI-NSCCs are also inhibited by the organic compound quinine (Demidchik & Tester, 2002), but this feature is not universal (White & Lemtiri-Chlieh, 1995; White & Broadley, 2000). Other treatments, including pH changes (stimulation by alkaline pH and inhibition by acidic pH) and application of the histidine modifier diethylpyrocarbonate (DEPC; strong inhibition), have also been shown to be effective in selected experimental systems, such as A. thaliana (Demidchik & Tester, 2002) and rye (White, 1999a), but have as yet not been tested widely. Within a given experimental system (e.g. A. thaliana), such responses, in addition to the more universally exhibited ones, provide valuable gauges for a critical comparative evaluation of physiological results obtained by different methods (for further discussion, see Section II.3 below).
In most studies on VI-NSCCs, clear demonstration of Na+ conductance was provided. As suggested by their name, VI-NSCCs are, to a high degree, nonselective for cations, that is, similar permeation of a variety of cations can be observed when such tests are conducted. Nevertheless, ion preferences are still encountered, resulting in selectivity series. Many such series have been published, and, while generally similar, they vary in their detail. In a seminal study on A. thaliana (Demidchik & Tester, 2002), the series observed (cation permeabilities are listed relative to Na+) was: K+ (1.49) > NH4+ (1.24) > Rb+ (1.15) > Cs+ (1.10) > Na+ (1.00) > Li+ (0.73) > TEA+ (0.47). In rye roots (White & Tester, 1992), the series was: K+ (1.36) = Rb+ (1.36) > Cs+ (1.17) > Na+ (1.00) > Li+ (0.97) > TEA+ (0.41). In wheat, NH4+ (2.06) > Rb+ (1.38) > K+ (1.23) > Cs+ (1.18) > Na+ (1.00) > Li+ (0.83) > TEA+ (0.20) was reported (Davenport & Tester, 2000). In other words, in these three benchmark studies (see also Tyerman et al., 1997 and Volkov & Amtmann, 2006), the macronutrient potassium (and, where tested, also ammonium) was transported to a significantly greater extent than sodium, from equimolar concentrations (see also Zhang et al., 2010). Thus, for this category of NSCCs, the cation selectivity series appear to follow a more consistent pattern than the frequently cited range of K+ : Na+ selectivity ratios for NSCCs of 0.3 to 3 (Demidchik et al., 2002; Demidchik & Maathuis, 2007). The published selectivity series should provide an important gauge for determining the contribution of NSCCs to Na+ conductance in planta.
Additional subclasses of NSCCs that have been the subject of some discussion in the context of Na+ fluxes are cyclic nucleotide-gated and amino-acid- (in particular, glutamate-) gated NSCCs (CNGCs and AAG-NSCCs; see also Demidchik & Maathuis, 2007). Among these, CNGCs are perhaps the best studied. They are characterized by gating mediation involving the second messengers cAMP and cGMP, and their role in animal physiology is diverse and has been extensively investigated, in particular within the context of transduction of visual and olfactory stimuli and Ca2+ signalling (Kaupp & Seifert, 2002; Talke et al., 2003; Gobert et al., 2006; Takeuchi & Kurahashi, 2008). However, functional expression of plant CNGCs has proved difficult, and thus little functional consolidation has occurred to date, even though some 20 CNGCs have been found in the A. thaliana genome (Gobert et al., 2006; Demidchik & Maathuis, 2007). However, in a few cases, expression in heterologous systems, including Xenopus laevis oocytes and yeast, has been successful (Leng et al., 2002; Balaguéet al., 2003; Gobert et al., 2006), and sensitivity to cAMP and cGMP has been observed, as well as sensitivity to Cs+ (Balaguéet al., 2003) and Mg2+ (Leng et al., 1999). Interestingly, in planta Na+ fluxes, in glycophytes under toxic conditions, are typically reported to be insensitive to Cs+ (see later discussion on fluxes; also see, however, Kader & Lindberg (2005) for work examining the protoplasts of rice; Wang et al. (2007) for work on the halophyte Sueda maritima, and Voigt et al. (2009) for Na+ tissue content data in cowpea (Vigna unguiculata) – these studies present evidence of Cs+ sensitivity of Na+ uptake). Cesium sensitivity, and the voltage sensitivity seen in many CNGCs, reduce the likelihood of their significant involvement in catalyzing Na+ fluxes in whole plants for extended periods of time (the roles of AtCNGC2, 4, 11 and 12 in response to pathogen attack, and the flow of Ca2+ under such conditions, are, by contrast, well documented; see e.g. Balaguéet al., 2003; Demidchik & Maathuis, 2007; Guo et al., 2010). Two CNGCs from the A. thaliana genome, AtCNGC3 and AtCNGC10, have nevertheless been linked to primary K+ and Na+ fluxes in roots. In the case of the former (AtCNGC3), tissue expression analysis has localized the transporter to root epidermal and cortical cells, and a null mutation in the gene has been shown to reduce the net uptake rate of Na+ during the initial (although not the later) stages of NaCl exposure, resulting in slightly enhanced growth on intermediate (40–80 mM) NaCl concentrations; the Na+ content of mutant seedlings, however, was not different from that of the wild type following longer term treatments at high (80–120 mM) NaCl concentrations (Gobert et al., 2006). The work may indicate a role for AtCNGC3 in Na+ uptake in the early phases (the initial few hours) of salt stress. In the case of AtCNGC10, tissue expression studies have also localized the transporter to root tissues, and the gene was able to complement the reduced K+ uptake phenotype of the A. thaliana akt1;1 mutant (see Hirsch et al., 1998; Spalding et al., 1999), establishing a possible role for the transporter in alkali ion fluxes in roots (Li et al., 2005). Other more recent studies, however, have shown a greater role of AtCNGC3 in the transport of the earth alkali ions Ca2+ and Mg2+ (Guo et al., 2010), although Na+ transport may be involved indirectly (Guo et al., 2008), while other CNGCs, such as AtCNGC2, are strongly selective for K+ over Na+ (Leng et al., 2002). In support of an in planta involvement of CNGCs in Na+ transport under toxic conditions, some studies have indeed reported a sensitivity of unidirectional or net fluxes of Na+ to cyclic nucleotides (Maathuis & Sanders, 2001; Essah et al., 2003; Rubio et al., 2003; Maathuis, 2006; see, however, Section II.3). Additionally, the observation that salt-tolerant varieties of rice down-regulate OsCNGC1 to a greater extent than salt-sensitive varieties under saline conditions (Senadheera et al., 2009) may also be taken as circumstantial evidence for an involvement of CNGCs in Na+ influx. Based on these findings, therefore, the role of CNGCs in primary Na+ fluxes cannot be dismissed at this point, and deserves careful further investigation, but the balance of the evidence does not currently favour a significant involvement (see also Zhang et al. (2010), who review conflicting information regarding whether CNGCs are blocked, or activated, by cyclic nucleotides).
Another subgrouping of ligand-sensitive NSCCs that may be involved in Na+ transport is that of AAG-NSCCs, and, in particular, those gated by glutamate. Precedents for glutamate-activated NSCCs abound in the animal literature (Dingledine et al., 1999; Traynelis et al., 2010), but their role in plant physiology, and under conditions of sodium toxicity, is more obscure (Lam et al., 1998; Davenport, 2002; Demidchik & Maathuis, 2007). At this time, convincing functional analyses of these channels are lacking, despite the fact that, as with CNGCs, some 20 AAG-NSCCs have been identified in the A. thaliana genome. The voltage insensitivity and instantaneous activation of currents, along with sensitivity to quinine and lanthanides in one study (Demidchik et al., 2004), suggest that AAG-NSCCs may represent subclasses of VI-NSCCs. While some evidence from Xenopus oocytes indicates the possibility of Na+ transport in at least some members of this family (AtGLR1;1, AtGLR 1;4 and AtGLR3;7; Roy et al., 2008; Tapken & Hollmann, 2008), the preponderance of evidence currently supports a role for AAG-NSCCs in Ca2+ transport (Dennison & Spalding, 2000; Dubos et al., 2003; Demidchik et al., 2004) and signalling during development (Kim et al., 2001; Turano et al., 2002; Li et al., 2006; Qi et al., 2006; Walch-Liu et al., 2006), rather than a role in primary Na+ fluxes under saline conditions (cf. Essah et al., 2003). Similarly, ROS-NSCCs (perhaps most NSCCs?) appear to be predominantly involved in Ca2+ transport (Demidchik & Maathuis, 2007).
3. Linking electrophysiological readings from protoplasts to fluxes in the whole plant: the challenge
It has to be strongly emphasized, and we will return to this critical point later, that essentially all demonstrations of the role of NSCCs, and in particular of VI-NSCCs, in catalyzing Na+ fluxes have been achieved by patch-clamp analysis with isolated protoplasts or artificial lipid bilayers. By contrast, the connection between such measurements and Na+ fluxes at the level of whole tissues and the whole plant is, in fact, much less secure (Malagoli et al., 2008; Britto & Kronzucker, 2009; Zhang et al., 2010), although the opposite conclusion is often stated (see e.g. Davenport, 2002; Munns & Tester, 2008). Several key studies have attempted to relate Na+ currents measured by electrophysiology in protoplasts and artificial lipid bilayer systems to Na+ fluxes and accumulation in intact plants and/or plant tissues. Once such set of comparative experiments was carried out in wheat (Davenport & Tester, 2000), and another in A. thaliana (Demidchik & Tester, 2002; Essah et al., 2003). Both sets of studies employed 22Na+-labelling of excised plants roots alongside electrophysiological examinations of protoplast and lipid bilayer preparations within a genotype. In the first of these studies, the authors showed that ‘Na+ influx through the NSC channel resembled 22Na+ influx’ (Davenport & Tester, 2000), and, indeed, concluded, even within the paper’s title, that a ‘nonselective cation channel mediates toxic sodium influx in wheat’.
This attribution was supported in large part by the partial sensitivity of both radiolabelled Na+ fluxes and Na+ currents to Ca2+, Mg2+ and Gd3+, and their insensitivity to other inhibitors, including those specific to potassium channels (TEA+ and Cs+; cf. Kader & Lindberg, 2005; Wang et al., 2007; Zhang et al., 2010). While Ca2+ sensitivity may indeed link NSCC operation well to the frequently (albeit not universally: see Yeo & Flowers, 1985; Schmidt et al., 1993; Malagoli et al., 2008) observed amelioration of Na+ toxicity by Ca2+ in whole plants (LaHaye & Epstein, 1969; Greenway & Munns, 1980; Rengel, 1992; Epstein, 1998), it should be kept in mind that Ca2+ has a myriad of other effects on plants (Britto et al., 2010; Zhang et al., 2010) and thus can hardly be seen as specific, and that the similarly strong Mg2+ sensitivity documented for NSCC operation (Davenport & Tester, 2000; their Fig. 4) is not typically reflected in the Na+ toxicity rescue of plants (LaHaye & Epstein, 1969). In addition, however, other issues deserve discussion. First, Ca2+ sensitivity, while exhibiting similar Ki values for electrical currents in bilayer preparations and tracer fluxes in roots (in the range of 610–650 μM; Davenport & Tester, 2000; see also White, 1999b; cf.Wang et al., 2007; Malagoli et al., 2008), was much more pronounced in single-channel preparations (> 50%) than it was in roots, where, at Ca2+ concentrations above 3 mM, c. 75% of the influx seen at the lowest [Ca2+] was still observed, measuring in excess of 70 μmol g−1 FW h−1, a very high cationic flux indeed (see Britto & Kronzucker, 2009; and discussion of data in Table 1). Ascribing Ca2+ sensitivity of Na+ influx in cereals exclusively to NSCCs (see also Davenport et al., 1997) is further complicated by the recent demonstration of Ca2+ suppression of OsHKT2;1-mediated Na+ transport in rice (Yao et al., 2010), contrary to the earlier claim of Ca2+ insensitivity of HKT-mediated transport (Davenport & Tester, 2000; citing Schachtman et al., 1997; see also other demonstrations of HKT-mediated Na+ influx under toxicity in wheat, e.g. Laurie et al., 2002– to be discussed in Section V). Similarly, the Ca2+ sensitivity of other potential transport candidates, such as LCT1 (see Section III below), undermines the clear attribution of Ca2+-sensitive fluxes to NSCCs. Moreover, it may be of paramount importance in this context that, as has been argued before (Schachtman & Liu, 1999; Amtmann et al., 2001; Britto & Kronzucker, 2009), Ca2+ concentrations in saline soils are typically high (10 mM or more is not unusual: Schachtman & Liu, 1999; Garciadeblás et al., 2003; Hirschi, 2004; Kronzucker et al., 2008), and thus a Ca2+-insensitive component(s) of Na+ influx should, in fact, be of greater interest as a target for engineering salt tolerance. The at times nearly exclusive focus on NSCCs in the context of Na+ acquisition under toxic, saline conditions is thus puzzling.
Interestingly, in wheat, sensitivity to low concentrations of Gd3+, a hallmark of many VI-NSCCs, was not observed (Demidchik & Tester, 2002; Demidchik et al., 2002; Demidchik & Maathuis, 2007), and only at 1 mM Gd3+ were significant reductions in Na+ flux evident (Davenport & Tester, 2000; unfortunately, only one flux value was provided in that study, in low-salt plants; sensitivity to La3+, another key uniting feature of VI-NSCCs, was not tested). By contrast, in A. thaliana, strong Gd3+ sensitivity (complete inhibition could be achieved at 0.1 mM; this was similar for La3+) was seen in electrophysiological characterizations of NSCC conductances (Demidchik & Tester, 2002), but none at all in corresponding tracer studies on plant roots of the same ecotypes (Essah et al., 2003; in this study, La3+ actually produced a 34%increase in roots from the same genotype; their Table 4). Thus, the pharmacological agreement is actually far less compelling than is frequently stated.
More importantly, however, as illustrated in Fig. 1, a fundamental characteristic evident in electrophysiological trials, but not in root influx measurements, is the saturability of the Na+ flux. In electrophysiological characterization, an NSCC proclaimed to mediate toxic Na+ influx in wheat (Davenport & Tester, 2000) failed to produce any flux enhancement at Na+ concentrations beyond 7–10 mM, that is, far below the toxicity threshold for the ion, with complete saturation being observed between 10 and 80 mM (using a simple Michaelis–Menten model, a Km value of 1.2 mM was reported for this saturable pattern; see also the discussion by Amtmann et al. (2001) and that of White & Davenport (2002), who developed a permeation model for this NSCC). A similar, if slightly less pronounced, saturable pattern was reported in a follow-up study in A. thaliana (Demidchik & Tester, 2002; cf. White & Ridout, 1995, who did see a nonsaturating increase in current with increasing external [Na+], in an NSCC from rye root plasma membranes; however, parallel tracer flux studies have not been conducted in this system). By stark contrast, Na+ influx into roots produced a linearly increasing flux in both wheat and A. thaliana that showed no signs of abating even at 200 mM Na+ (Fig 1; Davenport & Tester, 2000; Essah et al., 2003). Interestingly, the influx measured at 200 mM external [Na+] in Essah et al. (2003) was c. 300 μmol g−1 (FW) h−1, one of the highest purported trans-plasma-membrane cation fluxes ever reported in glycophytes (Table 1, and Britto & Kronzucker, 2009). Indeed, in the extensive study by Essah et al. (2003), all Na+ fluxes, even some at rather low external Na+ (see their Fig. 4, for values at 1 mM Na+) were very high (e.g. a flux of over 210 μmol g−1 FW h−1 was reported at 1 mM). It is unclear whether translations of the magnitudes of currents in patch-clamp experiments (reported in pS or pA) into tissue fluxes (typically reported in μmol g−1 FW h−1) can be achieved in principle, but what is clear, at this time, is that such correspondence has not yet been achieved in the case of NSCCs and their corresponding Na+ fluxes at the tissue level.
Figure 1. Idealized comparison of Na+ current measured electrophysiologically through nonselective cation channels (left; G, conductance through channel; redrawn from Davenport & Tester, 2000), and Na+ influx into plant roots measured using radiotracing with 22Na+ (redrawn from Essah et al., 2003). Note the early saturability of the current (cf. White & Ridout, 1995), as compared with the continued linearity of the tracer flux.
Download figure to PowerPoint
We have previously shown (Malagoli et al., 2008; Britto & Kronzucker, 2009), using established energetic models of transport (Poorter et al., 1991; Scheurwater et al., 1999; Kurimoto et al., 2004; Britto & Kronzucker, 2006), that fluxes of the magnitudes reported in the above studies, and indeed many others (Table 1), are not explicable energetically, if they are to follow currently proposed mechanisms of Na+ transport (Tester & Davenport, 2003; Apse & Blumwald, 2007; Malagoli et al., 2008; Munns & Tester, 2008; Teakle & Tyerman, 2010). In Table 1, we summarize, using the currently established model of cation transport and its energization (Britto & Kronzucker, 2009), minimal respiratory oxygen fluxes required to energize the reported Na+ fluxes. In halophytes, at 100 mM external Na+ supply, unidirectional Na+ fluxes as high as 600 μmol g−1 h−1 have been reported (Jefferies, 1973; Lazof & Cheeseman, 1986), corresponding to a respiratory O2 flux of 120 μmol g−1 h−1, with 50% of these values being attained in the glycophyte A. thaliana at 200 mM Na+ (Essah et al., 2003). No precedents for respiratory values of this magnitude can be found in the literature, and we previously showed (Malagoli et al., 2008), in the IR29 variety of Indica rice, that the respiratory requirement for the measured Na+ fluxes in that variety (as high as 225 μmol g−1 h−1 at 25 mM Na+) exceeded measured total respiratory values by 100%. Thus, a critical look at many, although not necessarily all (see e.g. Laurie et al., 2002), of the Na+ fluxes summarized in Table 1 is essential, if one is to successfully interpret measured Na+ fluxes and link them to plant performance. In addition, it would behoove experimenters to conduct respiratory analyses in their systems when exceptionally large fluxes are observed, as a partial test of the correct assignment of the measurements to a genuine plasma membrane flux. In this context, the need to distinguish between apoplastic and symplastic phases of uptake may be critically important (Yeo et al., 1987; Kronzucker et al., 1995, 1998; Britto & Kronzucker, 2001; see Section VIII). As we have also argued previously (Malagoli et al., 2008; Britto & Kronzucker, 2009), alternative explanations for such fluxes, or a revision of the accepted transport energization model, must be considered. These alternative possibilities may include tracer absorption by the plant root apoplast (see Yadav et al., 1996; Gong et al., 2006; Krishnamurthy et al., 2009), or an as yet unsatisfactorily characterized means of flux coupling (Colmenero-Flores et al., 2007), or vesicular transport (Peiter et al., 2007).
Additional evidence, independent of tracer analysis, for the participation of NSCCs in Na+ influx in planta comes from tissue analysis (Volkov & Amtmann, 2006), and use of Na+-sensitive fluorescent dyes (Kader & Lindberg, 2005; Anil et al., 2007). On the basis of insensitivity to the potassium-channel blockers Cs+ and TEA+ of both instantaneous Na+ currents and tissue Na+ accumulation, Volkov & Amtmann (2006) (see their Fig. 8) came to the conclusion that NSCCs are responsible for Na+ fluxes in T. halophila. However, it should be pointed out that data sets for results obtained using fluorescing dyes are scant at this time, and relationships with tissue accumulation may be problematic in cases of long-term treatment with pharmacological inhibitors, as illustrated by the increase in Na+ accumulation in plants treated with Cs+ for 2 d in the aforementioned study, in disagreement with the premise that accumulation can directly reflect the pharmacological profile of the channels carrying instantaneous currents in patch-clamp experiments. In studies on protoplasts and suspension-culture cells using the sodium-sensitive dye SBFI, the appearance of Na+ in the cytosol, upon sudden Na+ exposure, was reduced by Ca2+ (Anil et al., 2007) and some additional channel inhibitors (Zn2+ and La3+; Kader & Lindberg, 2005) known to target NSCCs. However, it should be kept in mind that such pharmacological agents can give conflicting results (Balkos et al., 2010), and assignment to specific mechanisms can be difficult.
We argue that, for a match-up between electrophysiology readings and excised tissue or whole- plant tracer studies to be achieved, several criteria must be met. First, responses to pharmacological treatments must match not just for a few, but for the majority of agents applied within a given genotype. They must produce changes in the same direction (inhibition vs enhancement) in both experimental approaches and, in particular, sensitivity to La3+ and Gd3+ at low concentrations should be observed as a gauge of NSCC involvement. Secondly, the kinetic response of currents and in planta fluxes must assume comparable shapes (saturable vs linear). Thirdly, fluxes measured in planta must be subjected to an energetic analysis, and, where excessive fluxes are seen, respiration data must be provided to test the proposed interpretation that fluxes in fact proceed across the plasma membrane. As such criteria are currently not met, assignments of electrophysiological Na+ currents of the NSCC type to in planta Na+ fluxes and vice versa must be viewed as preliminary.