Shaping the calcium signature


Authors for correspondence:
Jon Pittman
Tel: +44 161 275 5235
Fax:+44 161 275 5082
Martin McAinsh
Tel: +44 1524 593 929
Fax: +44 1524 593 192



  • Summary 275

  • I. Introduction 276
  • II. Ca2+ signalling pathways 276
  • III. Shaping Ca2+ signatures 278
  • IV. Ca2+ influx channels 278
  • V. Ca2+ influx channels as modulators of Ca2+ signatures 281
  • VI. Ca2+ efflux transporters 282
  • VII. Ca2+ efflux transporters as modulators of Ca2+ signatures 284
  • VIII. The shaping of noncytosolic Ca2+ signatures 285
  • IX. Future insights into the role of Ca2+ oscillators from modelling studies 287
  • X. Conclusions and perspectives 288
  • Acknowledgements 288

  • References 288


In numerous plant signal transduction pathways, Ca2+ is a versatile second messenger which controls the activation of many downstream actions in response to various stimuli. There is strong evidence to indicate that information encoded within these stimulus-induced Ca2+ oscillations can provide signalling specificity. Such Ca2+ signals, or ‘Ca2+ signatures’, are generated in the cytosol, and in noncytosolic locations including the nucleus and chloroplast, through the coordinated action of Ca2+ influx and efflux pathways. An increased understanding of the functions and regulation of these various Ca2+ transporters has improved our appreciation of the role these transporters play in specifically shaping the Ca2+ signatures. Here we review the evidence which indicates that Ca2+ channel, Ca2+-ATPase and Ca2+ exchanger isoforms can indeed modulate specific Ca2+ signatures in response to an individual signal.

I. Introduction

A change in the cytosolic concentration of the second messenger Ca2+ ([Ca2+]cyt) is an important component of the signalling network by which plant cells respond to environmental and developmental stimuli (reviewed by Sanders et al., 2002; Hetherington & Brownlee, 2004; Lecourieux et al., 2006; McAinsh & Schroeder, in press; Oldroyd & Downie, 2008). Stimulus-induced changes in plant [Ca2+]cyt are observed in many different cell types in response to a diverse range of abiotic and biotic stimuli, examples of which include osmotic, salt and drought signals (Knight et al., 1997; Ranf et al., 2008), oxidative stress (Evans et al., 2005), cold (Knight et al., 1991, 1996), gaseous pollutants (Evans et al., 2005), light (Shacklock et al., 1992), plant hormones (McAinsh et al., 1990; Allen et al., 2001), pathogens (elicitors) (Knight et al., 1991) and bacterial and fungal signals (Ehrhardt et al., 1996; Kosuta et al., 2008). The apparent ubiquity of this simple nonprotein messenger however, raises fundamental questions regarding how stimulus specificity is maintained within the complex network of Ca2+ signalling pathways in plant cells (McAinsh & Hetherington, 1998; Evans et al., 2001; Ng & McAinsh, 2003). Several mechanisms have emerged that may contribute to specificity in Ca2+ signalling (Sanders et al., 2002; McAinsh & Schroeder, in press): (i) the requirement for additional signalling events to occur in parallel with changes in [Ca2+]cyt; (ii) the expression of the appropriate signalling machinery required for the transduction of a given signal (this has been referred to as the ‘physiological address’ of cells, McAinsh & Hetherington, 1998); (iii) Ca2+ sensitivity priming, whereby the Ca2+ sensitivity of specific responses is enhanced, enabling a defined response to occur in cells that express many different Ca2+ sensors (Young et al., 2006); (iv) localized elevations in Ca2+ (e.g. plasma membrane micro-domains or at the nucleus; see Schneggenburger & Neher, 2005; Oldroyd & Downie, 2008) that enable the compartmentalization of signalling pathways and/or the selective activation of discrete response elements; and (v) the encryption of signalling information in the pattern or temporal dynamics of [Ca2+]cyt elevations, that is, oscillations and transients (Fig. 1) (Evans et al., 2001; Berridge et al., 2003). Stimulus-specific Ca2+ signals, in terms of the spatial and temporal dynamics of the stimulus-induced changes in [Ca2+]cyt, have been referred to as a ‘Ca2+ signature’ (McAinsh & Hetherington, 1998; Ng & McAinsh, 2003). In this review we will consider the role that Ca2+ signatures play in maintaining specificity in Ca2+ signalling, focusing on the cellular machinery responsible for the generation and encryption of signalling information in complex Ca2+ signals and the evidence that Ca2+ influx channels and Ca2+ efflux transporters function as modulators of Ca2+ in shaping Ca2+ signatures in plants. We will not discuss the decoding of Ca2+ signals in plant cells, but this subject has been reviewed extensively in a number of articles (Batistic & Kudla, 2004; Harper et al., 2004; Sathyanarayanan & Poovaiah, 2004).

Figure 1.

Encoding specificity in plant Ca2+ signatures. (a) A schematic representation of the encryption of signalling information in the temporal dynamics of Ca2+ oscillations. (b) In Commelina communis guard cells, the strength of the external Ca2+ ([Ca2+]ext) stimulus has been correlated directly with the pattern of Ca2+ oscillations (i.e. the period, frequency and amplitude), which in turn dictates the resultant steady-state stomatal aperture (from McAinsh et al., 1995,, ©American Society of Plant Biologists). (c) Nod factors and the mycorrhizal fungi produce Ca2+ oscillations in Medicago truncatula, which differ in their period and amplitude; this may provide a mechanism for the observed differences in the physiological response to rhizobial bacteria and mycorrhizal fungi (from Kosuta et al., 2008, ©2008, The National Academy of Sciences).

II. Ca2+ signalling pathways

The spatial and temporal dynamics of plant Ca2+ signatures vary markedly and include localized increases in [Ca2+]cyt together with transients, spikes and oscillations in [Ca2+]cyt (reviewed by Evans et al., 2001; Hetherington & Brownlee, 2004; McAinsh & Schroeder, in press). In animal cells, spatial and temporal heterogeneities in Ca2+ are known to play an important role in the encryption of stimulus-specific signalling information (Berridge et al., 2000, 2003). For example, Ca2+-activated gene expression in mouse pituitary cells is differentially regulated through elevations in nuclear Ca2+ ([Ca2+]nuc) and [Ca2+]cyt, whilst differences in the amplitude and duration of Ca2+ signals affect the activation of transcriptional regulators in mammalian cells (Dolmetsch et al., 1998) and regulate distinct cellular functions (Ito et al., 1997). In plants, arguably the most compelling evidence that signalling information can be encoded in the spatiotemporal dynamics of plant Ca2+ signatures comes from studies of Ca2+ signalling in stomatal guard cells and symbiosis signalling in legumes.

1. Stomatal guard cells

Changes in guard cell [Ca2+]cyt are observed in response to a wide range of stimuli that affect stomatal aperture, leading to the suggestion that [Ca2+]cyt constitutes a key hub in the guard cell signalling network (Hetherington & Woodward, 2003; Israelsson et al., 2006; Li et al., 2006b). Changes in guard cell [Ca2+]cyt include localized increases and oscillations (reviewed by Evans et al., 2001; McAinsh, 2007). A direct correlation has been shown between the pattern of external Ca2+ ([Ca2+]ext)- or ABA-induced oscillations in guard cell [Ca2+]cyt, the strength of the [Ca2+]ext or ABA stimulus and the resultant steady-state stomatal aperture (Fig. 1b; see also Fig. 4a,b) (McAinsh et al., 1995; Staxén et al., 1999). This suggests the potential for encoding information about the nature and strength of a stimulus in the temporal dynamics of plant cell Ca2+ signals as in animal cells (Fig. 1) (McAinsh & Hetherington, 1998; Ng & McAinsh, 2003; McAinsh, 2007). The mechanism(s) by which signalling information is encrypted in guard cell Ca2+ signatures have been extensively studied (Allen et al., 2000, 2001; Mori et al., 2006; Young et al., 2006). In Arabidopsis, ABA, cold, [Ca2+]ext and hydrogen peroxide (H2O2) have all been shown to induce oscillations in [Ca2+]cyt in the guard cells of wild-type plants, resulting in steady-state, ‘Ca2+ programmed’ stomatal closure (Allen et al., 2000, 2001). By contrast, in the det3 mutant, which has reduced vacuolar H+-ATPase activity, oscillations in guard cell [Ca2+]cyt and steady-state stomatal closure only occur in response to cold and ABA (Allen et al., 2000). [Ca2+]ext and H2O2 result in prolonged increases in [Ca2+]cyt, which fail to induce stomatal closure. Importantly, in wild-type guard cells, artificially imposed prolonged increases in [Ca2+]cyt fail to elicit steady-state stomatal closure, whereas in det3, artificially imposed oscillations in guard cell [Ca2+]cyt rescue [Ca2+]ext-induced steady-state stomatal closure. These data position oscillations in [Ca2+]cyt as a key component in the guard cell signalling pathway maintaining stomatal closure.

Figure 4.

Modelling of abscisic acid (ABA)-induced guard cell cytosolic Ca2+ ([Ca2+]cyt) oscillations. (a, b) Experimental traces of ABA-induced oscillations in [Ca2+]cyt in guard cells of Commelina communis (from Staxén et al., 1999, ©1999, The National Academy of Sciences) in the presence of 10 nm ABA (a) or 1 µm ABA (b). (c–f) Simulated kinetics of guard cell [Ca2+]cyt oscillations in response to 10 nm ABA (c, e) or 1 µm ABA (d, f) generated from a mathematical model based on the kinetics and parameters of endoplasmic reticulum inositol 1,4,5-trisphosphate-activated Ca2+ channel and Ca2+-ATPase, and tonoplast cyclic ADP-ribose-activated Ca2+ channel and H+/Ca2+ exchanger. Data taken from Veresov et al. (2003). Simulations were generated in a ‘wild-type’ guard cell (c, d) and following removal of the tonoplast H+/Ca2+ exchanger (e, f).

The parameters of the oscillations in guard cell [Ca2+]cyt that encode signalling information have been investigated (reviewed by McAinsh & Schroeder, in press). Only oscillations within a defined window of frequency, transient number, duration and amplitude result in steady-state stomatal closure (Allen et al., 2001). Oscillations outside this window result in immediate short-term, ‘Ca2+-reactive’ stomatal closure. Interestingly, the Arabidopsis Ca2+-dependent protein kinase (CDPK) double mutant, cpk3-1cpk6-1, exhibits a reduction in short-term, but not steady-state, stomatal closure in response to oscillations in guard cell [Ca2+]cyt with a period that falls within this window, demonstrating the functional separation of these two responses (Mori et al., 2006). The physiological significance of these data can be seen in the differential response of stomata of wild-type Arabidopsis and the ABA-insensitive mutant gca2 to [Ca2+]ext, ABA and elevated CO2 (Allen et al., 2001; Young et al., 2006). Wild-type guard cells exhibit [Ca2+]cyt oscillations with kinetics within the window that results in steady-state stomatal closure. By contrast, in guard cells of gca2, the period and duration of oscillations in [Ca2+]cyt are significantly shorter, and steady-state stomatal closure is abolished. Importantly, artificially imposed oscillations in guard cell [Ca2+]cyt with kinetics consistent with steady-state stomatal closure partially rescue steady-state stomatal closure in gca2, confirming that the guard cells of this mutant remain competent to respond to [Ca2+]cyt oscillations.

Taken together, these data strongly support a role for [Ca2+]cyt oscillations in the signalling pathway associated with the maintenance of steady-state stomatal closure. However, observations of stomatal closure in the absence of oscillations in guard cell [Ca2+]cyt (McAinsh et al., 1990, 1992; Schroeder & Hagiwara, 1990; Levchenko et al., 2005) and spontaneous Ca2+ transients that do not always result in stomatal closure (Grabov & Blatt, 1998; Klüsener et al., 2002; Young et al., 2006) highlight additional complexities in the guard cell signalling network.

2. Symbiosis signalling in legumes

A change in intracellular Ca2+ is a key component of the symbiosis signalling pathway by which leguminous plants respond to nitrogen-fixing bacteria, collectively known as rhizobia, and arbuscular mycorrhizal fungi (Kosuta et al., 2008). Rhizobial-derived nodulation (nod) factor-induced oscillations in Ca2+ have been widely reported (Oldroyd & Downie, 2008). Recently, Ca2+ oscillations have also been observed in root hairs of Medicago truncatula in response to the arbuscular mycorrhizal fungi Glomus intraradices (Kosuta et al., 2008), suggesting that they are central to symbiosis signalling. Interestingly, nod factor- and mycorrhizal-induced Ca2+ oscillations are both predominantly localized to the nuclear region (Ehrhardt et al., 1996; Walker et al., 2000; Sun et al., 2007; Kosuta et al., 2008), highlighting the potential contribution of noncytosolic sources of Ca2+ to the generation of Ca2+ signatures and in maintaining specificity in Ca2+ signalling pathways, which we will return to later.

Several studies imply a functional role for Ca2+ oscillations in symbiosis signalling. Nodulation-defective mutants, such as dmi1 and dmi2 of M. truncatula, all fail to exhibit nod-factor-induced oscillations (Wais et al., 2000; Walker et al., 2000; Kanamori et al., 2006; Miwa et al., 2006). Furthermore, dmi1 and dmi2, which are also defective in mycorrhizal infection (Catoira et al., 2000), both abolish mycorrhizal-induced Ca2+ oscillations (Kosuta et al., 2008). This suggests that nod factors and mycorrhizal fungi use common components of a symbiotic signalling pathway to generate Ca2+ oscillations. Interestingly, the heterotrimeric G-protein agonist mastoparan induces expression of early nodulation genes such as ENOD11 and Ca2+ oscillations in M. truncatula root hairs with kinetics similar to those observed in response to nod factors, albeit occurring throughout the cell (Pingret et al., 1998; Sun et al., 2007). Furthermore, mastoparan can also induce ENOD11 expression and Ca2+ oscillations in dmi1 and dmi2 (Charron et al., 2004; Sun et al., 2007), suggesting that artificially imposed Ca2+ oscillations can rescue early nodulation responses in these mutants. Importantly, nod factor- and mastoparan-induced Ca2+ oscillations are both inhibited by Ca2+-ATPase and phospholipase D inhibitors, suggesting similarities in the way in which the Ca2+ oscillations are generated (Engstrom et al., 2002; Sun et al., 2007). Taken together, these data provide strong evidence of the importance of Ca2+ oscillations in symbiosis signalling.

Fourier analysis reveals that nod factor- and mycorrhizal-induced Ca2+ oscillations differ markedly in both their period and amplitude; Ca2+ oscillations observed in response to mycorrhizal fungi have a considerably shorter period and are much lower in amplitude (Fig. 1c) (Kosuta et al., 2008). In addition, in M. truncatula, ENOD11 induction and the frequency of nod factor-induced Ca2+ oscillations vary depending on the position of root hairs along the root; the period between Ca2+ spikes is longer in younger root hairs, producing a frequency gradient (Miwa et al., 2006). ENOD11 expression is only observed in the region of the root where Ca2+ oscillations occur with a period of approximately 100 s between spikes. Interestingly, jasmonic acid treatment results in a reduction in the frequency of Ca2+ oscillations, although it has no effect on the number of Ca2+ oscillations necessary for the induction or pattern of ENOD11 expression (Miwa et al., 2006). Therefore, it is attractive to suggest that signalling information may be encoded in both the frequency and the number of nod factor-induced Ca2+ oscillations and that the kinetics of symbiosis signalling-related Ca2+ signatures provides a mechanism for differentiating between bacterial and fungal signals. However, it is clear from the work of Miwa et al. (2006) that other as yet undefined inputs, such as the developmental state of the cell (i.e. the physiological address of the cell; McAinsh & Hetherington, 1998), may also influence the signalling pathway.

III. Shaping Ca2+ signatures

Multiple pathways have evolved for the regulation of [Ca2+]cyt in plants (Bothwell & Ng, 2005), providing the potential to generate complex Ca2+ signatures that encode information relating to the nature and strength of stimuli in their spatiotemporal dynamics. These include Ca2+ influx channels in the plasma membrane and endomembranes which mediate Ca2+ release into the cytosol, contributing to stimulus-induced increases in [Ca2+]cyt, and Ca2+ efflux transporters which rapidly remove Ca2+ from the cytosol, restoring [Ca2+]cyt to resting values (Table 1, Fig. 2a). The range and properties of Ca2+ influx channels and efflux transporters in plants have been reviewed extensively (Sanders et al., 2002; Hetherington & Brownlee, 2004; Shigaki & Hirschi, 2006; Boursiac & Harper, 2007; Demidchik & Maathuis, 2007; Pottosin & Schönknecht, 2007). However, it is only recently that direct evidence has begun to emerge to support a role for specific components of the Ca2+ regulatory machinery in shaping plant Ca2+ signatures.

Table 1.  Summary of Ca2+ transport pathways which generate Ca2+ fluxes in a typical plant cell
Membrane where Ca2+ flux occursDirection of Ca2+ fluxTransporter typeMain physiological regulatorsa
  • ABA, abscisic acid; ACA, autoinhibited Ca2+-ATPase activated by calmodulin (CaM); [Ca2+]cyt, cytosolic Ca2+; [Ca2+]vac, vacuolar lumen Ca2+; CAX, H+/Ca2+ exchanger; CNGC, cyclic nucleotide-gated channel; DACC, depolarization-activated Ca2+ channel; ΔpH, pH gradient; ECA, ER-type Ca2+-ATPase; GLR, glutamate receptor-like channel; HACC, hyperpolarization-activated Ca2+ channel; InsP3R-like, inositol 1,4,5-trisphosphate receptor-like channel; MCC, mechanosensitive Ca2+ channel; NAADPR, nicotinic acid adenine dinucleotide phosphate receptor-like channel; NSCC, nonselective cation channel; ROS, reactive oxygen species; RyR-like, cyclic ADP-ribose (cADPR)-activated ryanodine receptor-like channel; SV channel, slow-activating vacuolar Ca2+ channel; VVCa channel, vacuolar voltage-gated Ca2+ channel.

  • a

    In addition to the regulators listed, Ca2+-ATPases have an absolute requirement for ATP and Mg2+, and many Ca2+ transporters are regulated by phosphorylation.

  • b

    ?, indicates that the evidence is not clear-cut or is inferred from the animal literature.

Plasma membraneInto the cytosolCNGCcAMP, cGMP, CaM
GLRAmino acids
HACCVoltage, ROS, [Ca2+]cyt, ABA
Out of the cellACA[Ca2+]cyt, CaM
TonoplastInto the cytosolInsP3R-likeInsP3
SV channel (AtTPC1)Voltage, [Ca2+]cyt, pH, CaM
VVCa channelVoltage, [Ca2+]cyt, [Ca2+]vac
Into the vacuoleACACaM
CAXΔpH, [Ca2+]cyt
Endoplasmic reticulum (ER)Into the cytosolInsP3R-likeInsP3
Into the ER lumenACA[Ca2+]cyt, CaM
Mitochondria (inner membrane)Into the cytosolCa2+ exchanger?ΔpH?, Ca2+?
Into the matrixCa2+ uniporter?Ca2+?, voltage?
Chloroplast (inner envelope)Into the cytosolACA?[Ca2+]cyt, CaM
Into the stromaCa2+ uniporterΔpH, voltage
GolgiInto the cytosolunknown 
Into the GolgiECA[Ca2+]cyt?
Nuclear envelopeInto the envelopeECA?[Ca2+]cyt?
Into the nucleusNSCCVoltage
Figure 2.

Ca2+ signature generation by Ca2+ transporters. (a) Schematic representation of the generation of a cytosolic Ca2+ ([Ca2+]cyt) transient and the coordinated action of Ca2+ channels (Ca2+ influx) and Ca2+ pumps/exchangers (Ca2+ efflux). Before a Ca2+ transient, the resting concentration of [Ca2+]cyt is maintained through the action of one or more Ca2+ efflux transporters. Following a stimulus, activation of one or more Ca2+ channels will lead to a rapid elevation in [Ca2+]cyt and the generation of a Ca2+ spike. Further Ca2+ efflux activity will control the upper value of [Ca2+]cyt elevation and lead to a reduction in [Ca2+]cyt and a return to resting concentration. The latter Ca2+ efflux transporters may be different from those that maintained the initial [Ca2+]cyt resting concentration. Ca2+ buffering by binding proteins may also play a role in reducing free [Ca2+]cyt concentrations. (b) Hypothetical [Ca2+]cyt transients that might be observed during different scenarios of Ca2+ influx and efflux transporter knockout (ko). A ‘wild-type’[Ca2+]cyt transient is denoted by a solid line. Knockout of a critical ‘housekeeping’ Ca2+ efflux pathway (Ca2+ efflux ko 1) will lead to an inability to maintain [Ca2+]cyt resting concentration and will be toxic to the cell. Knockout of a Ca2+ efflux pathway that is solely involved in responding to a Ca2+ transient (Ca2+ efflux ko 2) will lead to an attenuation of the [Ca2+]cyt transient and a slow return to resting concentration. Knockout of a Ca2+ efflux pathway that is required to rapidly remove a large, fast pulse of [Ca2+]cyt (Ca2+ efflux ko 3) will control the amplitude of the Ca2+ spike. Knockout of a Ca2+ channel that is critical in response to the hypothetical stimulus (Ca2+ channel ko) will prevent a [Ca2+]cyt transient from occurring.

IV. Ca2+ influx channels

The generation of stimulus-specific Ca2+ signatures in animal cells is associated with Ca2+ influx through specific Ca2+-selective channels (Zou et al., 2002). However, in plants, Ca2+ influx channels are typically permeable to cations, including Ca2+, rather than Ca2+-selective (Schroeder & Hagiwara, 1990; Thuleau et al., 1994; Pei et al., 2000; Very & Davies, 2000). Although many plant Ca2+-permeable channels have been characterized electrophysiologically, there are only a few examples where the molecular identity of the channel has been established. Nevertheless, recent studies provide important insights into the roles of these channel types in the generation of specific Ca2+ signatures.

1. Plasma membrane Ca2+ influx channels

The stimulus-induced influx of Ca2+ into cells is well documented (Schroeder & Hagiwara, 1990; MacRobbie, 2000; Pei et al., 2000). Three main groups of Ca2+-permeable channel have been characterized electrophysiologically in the plasma membrane of plant cells. These are the mechanosensitive Ca2+ channel (MCC), the depolarization-activated Ca2+ channel (DACC) and the hyperpolarization-activated Ca2+ channel (HACC), which are all examples of nonselective cation channel (NSCC) (Table 1) (reviewed by Sanders et al., 2002; Hetherington & Brownlee, 2004; Demidchik & Maathuis, 2007).

Mechanosensitive Ca2+ channels have been recorded in various cell types, including guard cells of Vicia faba (Cosgrove & Hedrich, 1991), Arabidopsis mesophyll cells (Qi et al., 2004), and pollen grain and tube tip protoplasts of Lilium longiflorum (Dutta & Robinson, 2004). They are therefore good candidates for shaping mechanically induced increases in [Ca2+]cyt. Several studies invoke MCCs as key components of the signalling pathway by which cells respond to mechanical stimuli. Pharmacological studies suggest that mechanical stimulation of chloroplast movement in ferns (Sato et al., 2001) and pollen tube germination and elongation (Dutta & Robinson, 2004) are both dependent on Ca2+ influx through MCCs. MCCs may also be responsible for the mechanically induced increase in [Ca2+]cyt recently reported in roots (Monshausen et al., 2007). However, despite the identification of 10 MC-like genes in the Arabidopsis genome based on homology with bacterial MC genes (Haswell & Meyerowitz, 2006), very few data exist about the specific function(s) of this channel type.

Depolarization-activated Ca2+ channels have been reported in a number of tissues, including maize, Arabidopsis and carrot suspension culture cells (Marshall et al., 1994; Thuleau et al., 1994; Thion et al., 1998). Depolarization of the plasma membrane is an early response to many environmental stresses which stimulate increases in [Ca2+]cyt (Lhuissier et al., 2001; Okazaki et al., 2002), suggesting a potential role for DACCs in shaping stress-induced Ca2+ signatures. However, there is, to our knowledge, no direct evidence linking DACC activity to stress-induced changes in [Ca2+]cyt in planta. Furthermore, the pharmacology of DACCs has not been widely studied and their molecular identities remain obscure.

Hyperpolarization-activated Ca2+ channels were first reported in tomato cells activated in response to fungal elicitors (Gelli & Blumwald, 1997) and have subsequently been described in a range of different cell types (Grabov & Blatt, 1999; Pei et al., 2000; Very & Davies, 2000; Coelho et al., 2002; Demidchik et al., 2002; Foreman et al., 2003). Studies in guard cells and root hairs provide important evidence for a role of HACCs in shaping Ca2+ signatures. HACC activity in guard cells is stimulated by ABA (Pei et al., 2000) and reactive oxygen species (ROS) (Pei et al., 2000; Murata et al., 2001), both of which induce increases in guard cell [Ca2+]cyt (McAinsh et al., 1990, 1996). Importantly, ABA also increases ROS concentrations (Pei et al., 2000; Bright et al., 2006) while the activation of HACCs by ABA requires the presence of NADPH in the cytosol (Murata et al., 2001). Furthermore, in the atrbohD atrbohF guard cell NADPH oxidase double mutant, HACC activity is impaired, along with changes in ABA-induced [Ca2+]cyt and stomatal aperture (Kwak et al., 2003). These studies suggest that ROS is essential for the regulation by ABA of HACCs which contribute to the generation of the guard cell ABA Ca2+ signature. Interestingly, phosphorylation events also regulate guard cell HACC activity (Köhler & Blatt, 2002; Mori et al., 2006). The molecular identity of the guard cell HACC is still to be determined, but possible candidates include ABC transporters. The atmrp5 ABC transporter mutant partially inhibits ABA-induced stomatal closure (Gaedeke et al., 2001) and impairs HACC activation by ABA in guard cells (Suh et al., 2007). However, atmrp5 guard cells also exhibit altered anion channel activity, suggesting that AtMRP5 encodes a central regulator of guard cell ion channel activity rather than the HACC itself (Suh et al., 2007).

In roots, HACC activity is localized to the apical region of Arabidopsis root hairs and is down-regulated in subapical regions of growing root hairs and at the tips of mature hairs, suggesting a role in the generation of the root hair apical [Ca2+]cyt gradient (Very & Davies, 2000). As in guard cells, root hair HACCs are also directly regulated by ROS (Foreman et al., 2003), whilst ROS accumulation, the generation of the root hair apical [Ca2+]cyt gradient and root hair formation are all inhibited in the Arabidopsis rhd2 NADPH oxidase mutant (Foreman et al., 2003). These data provide additional evidence for the regulation of HACCs by ROS and confirm the role of these channels in the generation of plant Ca2+ signatures. Furthermore, guard cell and root hair HACCs exhibit differential responses to [Ca2+]cyt (Very & Davies, 2000), suggesting that more than one class of HACC may exist in plants. However, the molecular characterization of these different channels is required before this can be confirmed.

2. Endomembrane Ca2+-permeable channels

In contrast to plasma membrane channels, the electrophysiological characterization of Ca2+ channels in the endomembrane is not possible in intact cells, imposing additional challenges when assigning channel activities to the generation of specific Ca2+ signatures. There are at least four Ca2+-permeable channels that have been identified in the vacuolar membrane and that may contribute to stimulus-induced increases in [Ca2+]cyt. These include the inositol 1,4,5-trisphosphate (InsP3)- and cyclic ADP-ribose (cADPR)-gated channels, and the vacuolar voltage-gated Ca2+ (VVCa) and slow-activating vacuolar (SV) channels (Table 1) (reviewed by Sanders et al., 2002; Hetherington & Brownlee, 2004; Pottosin & Schönknecht, 2007). Despite the absence of homologues for animal InsP3 and ryanodine receptor channels in either the Arabidopsis or rice genomes, InsP3 and cADPR have both been shown to cause the release of Ca2+from plant vacuoles and to cause increases in [Ca2+]cyt (Schumaker & Sze, 1987; Gilroy et al., 1990; Wu et al., 1997; Leckie et al., 1998; MacRobbie, 2000). Furthermore, InsP3 and/or cADPR have been implicated in multiple Ca2+-mediated stress signalling pathways in plants (Knight et al., 1996; Wu et al., 1997; Drobak & Watkins, 2000; Lecourieux et al., 2006). Whether InsP3- and cADPR-gated Ca2+-permeable channels reside solely in the vacuolar membrane (Allen et al., 1995; Lommel & Felle, 1997) or are more widely distributed, such as at the endoplasmic reticulum (ER) (Muir & Sanders, 1997; Navazio et al., 2001), remains to be established. In addition, Ca2+ release from the vacuole and ER in response to inositol hexakisphosphate (InsP6) (Lemtiri-Chlieh et al., 2003) and nicotinic acid adenine dinucleotide phosphate (NAADP) (Navazio et al., 2000), respectively, has been reported, highlighting further the potential for ligand-gated endomembrane Ca2+-permeable channels to shape Ca2+ signatures.

Vacuolar voltage-gated Ca2+ and SV channels are both voltage-dependent channels with a high affinity for Ca2+ (Pottosin & Schönknecht, 2007). The VVCa channel is gated by membrane hyperpolarization and activated by Ca2+ from the vacuolar side (Johannes et al., 1992), whereas the SV channel is gated by membrane depolarization and activated by [Ca2+]cyt (Hedrich & Neher, 1987). This has led to the suggestion that they are the same channel but in opposite orientation (Pottosin & Schönknecht, 2007). The SV channel is also regulated by calmodulin (CaM) (Bethke & Jones, 1994), phosphorylation (Allen & Sanders, 1995), and 14-3-3-proteins (van den Wijngaard et al., 2001). Interestingly, the SV channel is the most abundant channel in the vacuolar membrane (Hedrich & Neher, 1987) and is widely distributed among terrestrial plants (Hedrich et al., 1988). This apparent ubiquity, together with the Ca2+-permeability and [Ca2+]cyt-dependent activation of the channel, has led to the suggestion that SV channels contribute to increases in [Ca2+]cyt through the process of Ca2+-induced Ca2+ release (CICR) (Ward & Schroeder, 1994; Bewell et al., 1999). Importantly, there is strong evidence that in Arabidopsis, the SV channel is encoded by the AtTPC1 (two-pore channel 1) gene (Peiter et al., 2005), although it is interesting to note that studies in rice, tobacco and wheat suggest that the AtTPC1 gene homologues NtTPC1, OsTPC1 and TaTPC1 may encode putative Ca2+-permeable channels in the plasma membrane, implying the species-dependent targeting of TPC1 channel proteins to different membranes (reviewed by Demidchik & Maathuis, 2007; Pottosin & Schönknecht, 2007). The demonstration that AtTPC1 encodes the Arabidopsis SV channel has permitted the first functional analysis of endomembrane Ca2+-permeable channel involvement in the generation of plant Ca2+ signatures at the molecular level (Peiter et al., 2005; Ranf et al., 2008).

V. Ca2+ influx channels as modulators of Ca2+ signatures

Although a number of genes have been identified that are thought to encode plant Ca2+-permeable channels (reviewed by Hetherington & Brownlee, 2004; Demidchik & Maathuis, 2007; Pottosin & Schönknecht, 2007), there is little direct evidence linking the gene products to the channel activity observed in vivo or for their role in shaping Ca2+ signatures. To date, there are only two classes of ion channel homologous genes which are good candidates for plasma membrane Ca2+-permeable channels in plants, both of which are NSCCs. These are the cyclic nucleotide-gated channel (CNGC; Mäser et al., 2001) and the glutamate receptor-like (GLR; Lacombe et al., 2001) genes. In addition, the SV channel has recently been shown to be encoded by the AtTPC1 gene (Peiter et al., 2005), providing a candidate for an endomembrane Ca2+-permeable channel that may contribute to the shaping of plant Ca2+ signatures.

In animals, cation channels activated by the cyclic nucleotides cAMP and cGMP are involved in the transduction of sensory stimuli and in Ca2+ signalling (Kaupp & Seifert, 2002). Plant CNGCs were first identified in barley (Schuurink et al., 1998), and the Arabidopsis genome includes 20 full-length CNGC genes (Mäser et al., 2001). Initial indications of a role of CNGCs in plant Ca2+ signalling derive from reports that cAMP is able to stimulate Ca2+ influx in cultured carrot cells (Kurosaki et al., 1994) and that cAMP and cGMP both induce increases in [Ca2+]cyt in aequorin-expressing tobacco protoplasts (Volotovski et al., 1998). Furthermore, cAMP stimulates HACC activity in guard cells (Lemtiri-Chlieh & Berkowitz, 2004). Studies of established Ca2+-dependent processes also provide evidence for a physiological role of specific CNGCs in plants. Mutants of the Arabidopsis CNGC2 and CNGC4 genes exhibit altered patterns of pathogen-induced cell death during attack by Pseudomonas syringae, suggesting that these ion channels function in the pathway(s) by which pathogen-mediated responses are modulated (Clough et al., 2000; Balagué et al., 2003). Similarly, a mutation that generates a chimeric CNGC-encoding gene, CNGC11/12, constitutively activates Arabidopsis defence responses and produces stunted plants which exhibit enhanced resistance to the virulent pathogen Hyaloperonospora parasitica Emco5 (Yoshioka et al., 2006). Recently, the Arabidopsis CNGC18 gene, which encodes a Ca2+-permeable channel in pollen tubes, has been shown to localize preferentially to the growing tip (Frietsch et al., 2007), and therefore may be important for regulating the tip-focused Ca2+ gradient during pollen tube growth. The cngc18 knockout mutants produce short, thin pollen tubes which exhibit nondirectional growth before prematurely bursting (Frietsch et al., 2007). By contrast, overexpression of CNGC18 results in the formation of short, wide pollen tubes that exhibit depolarized growth which is enhanced at high [Ca2+]ext and suppressed at low [Ca2+]ext (Chang et al., 2007). These results strongly support a role for CNGC18 in the regulation of pollen tube growth. However, the mechanisms by which the putative CNGC Ca2+-permeable channels are activated by their respective signalling pathways remain unknown.

Animal ionotropic glutamate receptors are glutamate- and glycine-activated cation channels that mediate synaptic transmission and generate Ca2+ signals in the mammalian central nervous system (Madden, 2002). In the Arabidopsis genome, 20 GLR genes have been identified with similarities to those for the animal ionotropic glutamate receptors (Lacombe et al., 2001; Davenport, 2002). Glutamate and glycine both stimulate rapid, transient depolarization of the plasma membrane and increases in [Ca2+]cyt in aequorin-expressing Arabidopsis seedlings (Dennison & Spalding, 2000; Dubos et al., 2003; Demidchik et al., 2004; Meyerhoff et al., 2005). Glutamate-activated cation currents have also been detected in the plasma membrane of Arabidopsis root protoplasts (Demidchik et al., 2004). These may contribute to the observed glutamate-induced membrane depolarization and increases in [Ca2+]cyt by allowing Ca2+ influx into cells. Importantly, glutamate-induced increases in [Ca2+]cyt are inhibited by antagonists of animal ionotropic glutamate receptors, suggesting a functional role for GLRs in the generation of these [Ca2+]cyt increases (Dubos et al., 2003; Meyerhoff et al., 2005). This suggestion is reinforced by the observation that glutamate affects a range of Ca2+-dependent processes, including microtubule depolymerization and root elongation (Sivaguru et al., 2003) and root branching (Walch-Liu et al., 2006). Furthermore, disruption of GLR3.1 in rice has been shown to disrupt Ca2+-dependent processes, such as cell division, differentiation and programmed cell death in roots (Li et al., 2006a), while overexpression of a radish GLR in Arabidopsis results in increased glutamate-induced Ca2+ influx and enhanced resistance to pathogen attack (Kang et al., 2006). However, perhaps the most compelling evidence of a role for GLRs in stimulating increases in [Ca2+]cyt comes from a recent study of the Arabidopsis glr3.3 knockout mutant expressing aequorin, in which glutamate-induced membrane depolarization is attenuated and the associated increases in [Ca2+]cyt completely blocked (Qi et al., 2006). Taken together, these data demonstrate unequivocally the potential for GLRs to contribute to the shaping plant Ca2+ signatures.

As discussed previously, the SV channel has been implicated in the process of CICR in plants (Ward & Schroeder, 1994; Bewell et al., 1999). Importantly, the Arabidopsis tpc1-2 knockout mutant appears to lack SV channel activity, whereas AtTPC1 overexpressing lines exhibit increased SV channel activity (Peiter et al., 2005; Ranf et al., 2008), demonstrating that the AtTPC1 gene encodes the SV channel. Furthermore, AtTPC1 is the only member of the two-pore channel family of voltage-gated cation channels present in Arabidopsis (Furuichi et al., 2001). Importantly, tpc1-2 fails to show ABA inhibition of germination or [Ca2+]ext-induced stomatal closure, while both of these responses are unaffected in AtTPC1 overexpressing lines (Peiter et al., 2005). This makes AtTPC1 an excellent candidate for an endomembrane Ca2+ influx channel involved in shaping Ca2+ signatures. However, a recent study was unable to detect any effect of AtTPC1 knockout or overexpression on abiotic or biotic stress-induced changes in [Ca2+]cyt (Ranf et al., 2008). Although, it is not possible to exclude a role for the AtTPC1/SV channel in mediating highly localized Ca2+ release from the vacuole, which might not be detected by cytosolic-targeted aequorin (as used by Ranf et al., 2008), it is clear that the precise role played by the channel in plant Ca2+ signalling requires further analysis.

VI. Ca2+ efflux transporters

Calcium is an essential nutrient, yet in all organisms Ca2+ is extremely toxic when present at high concentrations in the cytosol. Thus transport mechanisms to rapidly remove Ca2+ from the cytosol developed early in evolution (Bothwell & Ng, 2005; Case et al., 2007). In addition to providing [Ca2+]cyt tolerance by reducing [Ca2+]cyt and for refilling Ca2+ stores, the presence of multiple Ca2+ efflux transporters at various membrane locations provides the potential to generate Ca2+ signatures in a sophisticated manner. Plants, like virtually all eukaryotes, have two main pathways for [Ca2+]cyt removal: high-affinity Ca2+-ATPases and lower-affinity Ca2+ exchangers. Unlike the Ca2+ influx pathways, the genetic basis of Ca2+ efflux transporters has been known for many years, principally because of the significant sequence conservation of these transporters throughout all branches of life. Therefore we have significant knowledge of the kinetic characteristics, regulation, membrane localization, expression pattern, and physiological function of these transporters, most notably in Arabidopsis (Table 1) (reviewed by Sze et al., 2000; Pittman & Hirschi, 2003; Shigaki & Hirschi, 2006; Boursiac & Harper, 2007). Moreover, evidence is beginning to accumulate that demonstrates a role for these Ca2+ transporters in particular Ca2+ signalling pathways and in shaping specific Ca2+ signatures.

1. High-affinity Ca2+ efflux: Ca2+-ATPases

A subgroup of the P-type ATPases (the P2-ATPases) encompass the Ca2+pumps (Baxter et al., 2003). These are further divided into P2A-ATPases, which include the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) in animals, and the ER-type Ca2+-ATPase (ECA) in plants, and P2B-ATPases, including the animal CaM-regulated plasma membrane Ca2+-ATPase (PMCA) and the autoinhibited Ca2+-ATPase (ACA) in plants. While the animal pumps are clearly delineated on the basis of their membrane location and CaM regulation, plant Ca2+ pumps have proliferated in numbers (14 in Arabidopsis and rice) and gained more flexibility. Isoforms of both ECAs and ACAs have been observed at the ER, plasma membrane and tonoplast (Ferrol & Bennett, 1996; Liang et al., 1997; Hong et al., 1999; Bonza et al., 2000; Lee et al., 2007). There is also some evidence that regulation by CaM is not solely a characteristic of ACAs, as CaM binding by ECAs has been observed (Subbaiah & Sachs, 2000; Navarro-Aviñó & Bennett, 2003). For example, CAP1 from maize has been shown to bind and be stimulated by CaM following heterologous expression in yeast (Subbaiah & Sachs, 2000). This variation in Ca2+-ATPase characteristics in higher plants suggests the potential for a wider range of roles for these pumps and an increased flexibility in responding to [Ca2+]cyt.

While CAP1 may be CaM-regulated, most plant ECAs, such as AtECA1, are not regulated by CaM or possess CaM-binding domains (Sze et al., 2000). The membrane proteins phospholamban and sarcolipin are primary regulators of SERCAs, but there are no known plant homologues of these proteins. Thus potential mechanisms of post-translational regulation of ECAs are unknown, although there is circumstantial evidence that they are regulated by interacting proteins (Hwang et al., 2000a). By contrast, ECAs are clearly regulated transcriptionally and so can be implicated in stress-dependent signalling pathways (Maathuis et al., 2003). For example, tomato LCA1 is regulated by salt stress and phosphate starvation (Wimmers et al., 1992; Muchhal et al., 1997), while a role for rice OsECA1 in gibberellin-dependent signalling in aleurone cells has been demonstrated (Chen et al., 1997). Further studies are needed, however, to unequivocally demonstrate whether ECAs do function in the shaping of Ca2+ signatures during signalling.

A number of studies have begun to provide information regarding the regulatory and functional properties of plant ACAs. These pumps are of particular relevance to Ca2+ signalling, because of their ability to be rapidly switched on in a Ca2+-dependent manner via CaM. Following an elevation in [Ca2+]cyt, CaM will interact with an N-terminal autoinhibitory domain and cause its release, leading to activation of Ca2+ transport activity (Bækgaard et al., 2005). These CaM-binding domains appear to be ubiquitous on all ACAs but are highly divergent with little consensus sequence (Harper et al., 1998; Chung et al., 2000; Malmström et al., 2000; Bækgaard et al., 2006). In addition, plants have multiple isoforms of CaM, with some being more divergent than others (Yang & Poovaiah, 2003). This suggests the potential for specificity in the regulation of Ca2+-ATPases, with CaM isoforms regulating pumps differentially. Whether this does occur is unclear, particularly as the CaM-binding domain of cauliflower BCA1 can bind both conserved and divergent isoforms of CaM (Yamniuk & Vogel, 2004). ACAs are also regulated by additional means, such as phospholipid stimulation or phosphorylation (Bonza et al., 2001; Nühse et al., 2004), thus providing a degree of specificity of activation and deactivation. For example, AtACA2 is phosphorylated by the CDPK AtCPK1, which inhibits basal and CaM-stimulated activity (Hwang et al., 2000b). The ability to switch AtACA2 on and off by two independent Ca2+ signalling components indicates the great flexibility that ACAs can provide in modulating Ca2+ oscillations.

Unlike with mammalian Ca2+-ATPases, knockout studies suggest that none of the Arabidopsis Ca2+-ATPases are essential as individual pumps (Boursiac & Harper, 2007). There is good evidence, however, for the involvement of ACAs in specific Ca2+ signalling pathways. AtACA8 and AtACA10 are transcriptionally regulated by cold stress (Schiøtt & Palmgren, 2005), while knockout studies of AtACA10 indicate a role in vegetative development (George et al., 2008). The plasma membrane pump AtACA9 is important for pollen tube growth and fertilization (Schiøtt et al., 2004). Pollen tubes from an aca9 knockout are almost completely unable to reach the end of the pistil and if an aca9 pollen tube does reach an ovule, fertilization is usually aborted. There is a considerable body of evidence describing the importance of Ca2+ signalling pathways in pollen tube growth (Holdaway-Clarke & Hepler, 2003). The AtACA9 study indicates an important role for plasma membrane Ca2+ efflux in this signalling pathway, although it is as yet unclear whether the function of AtACA9 is solely in preventing [Ca2+]cyt over-accumulation or in directly modulating pollen tube tip [Ca2+]cyt oscillations such as by priming Ca2+ influx (Schiøtt et al., 2004).

2. Low-affinity, high-capacity Ca2+ efflux: Ca2+ exchangers

Most cells possess Ca2+ exchangers that are energized by the counter exchange of another ion. These transporters are usually of lower Ca2+ affinity than the Ca2+ pumps but transport Ca2+ from the cytosol rapidly at high capacity. In animal cells, Ca2+ efflux by Na+/Ca2+ exchangers is coupled to Na+ flux, while plants possess a structurally related family of cation exchanger (CAX) genes that encode H+/Ca2+ exchangers (Cai & Lytton, 2004; Shigaki & Hirschi, 2006). Arabidopsis has six CAX genes (AtCAX1-AtCAX6) plus five related genes, designated cation/Ca2+ exchanger (CCX) (originally named AtCAX7-AtCAX11) that are more similar to an animal Na+/Ca2+ exchanger isoform (Cai & Lytton, 2004; Shigaki et al., 2006). Whether any of the plant CCX transporters function in Ca2+ transport is unclear but is a distinct possibility, although recent studies show that AtCCX3 can transport Na+ and K+ but not Ca2+(Morris et al., in press).

H+/Ca2+ exchange activity has long been known to be a major route for Ca2+ removal from the cytosol into the vacuole (Schumaker & Sze, 1985; Blumwald & Poole, 1986), although exchange activity has also been detected at the plasma membrane (Kasai & Muto, 1990). CAX genes encoding tonoplast H+/Ca2+ exchangers have been subsequently identified from various plant species (Hirschi et al., 1996; Ueoka-Nakanishi et al., 2000; Kamiya et al., 2006). H+/Ca2+ exchangers have predominant roles in Ca2+ homeostasis, with maintenance of low [Ca2+]cyt being a significant function. For example, knockout or overexpression of AtCAX1 or AtCAX3 leads to significant perturbations in Ca2+ homeostasis, Ca2+ tolerance and impaired plant development (Hirschi, 1999; Cheng et al., 2003, 2005; Mei et al., 2007). H+/Ca2+ exchange activity of AtCAX1 can be regulated via an N-terminal autoinhibitory domain, analogous to that of the ACAs, although it does not bind CaM (Pittman & Hirschi, 2001; Pittman et al., 2002a; Mei et al., 2007). Other plant CAXs appear to share this mode of regulation (Pittman et al., 2002b, 2004; Kamiya et al., 2005). AtCAX1 activity may be regulated by phosphorylation (Pittman et al., 2002a) or via various CAX interacting proteins (CXIP), including CXIP4, a plant-specific protein of unknown function, and the Ser/Thr kinase SOS2 (Cheng & Hirschi, 2003; Cheng et al., 2004a,b). Regulation by SOS2 is notable as it is a component of a Ca2+-regulated salt stress tolerance pathway (Zhu, 2003). Interestingly, AtCAX3 has been identified as another putative regulator of AtCAX1 (Cheng et al., 2005). This suggests that under some developmental conditions, AtCAX1 and AtCAX3 may interact to form a complex potentially with altered kinetics of transport activity.

The potential for differential regulation of H+/Ca2+ exchangers suggests that they may be activated during specific signalling pathways. Impaired hormone and developmental responses following AtCAX1 and AtCAX3 deletion further suggest signalling roles for these Ca2+ transporters (Cheng et al., 2003, 2005; Zhao et al., 2008). Moreover, abiotic stress phenotypes of cax1 and cax3 knockout plants indicate a function for these transporters in responses to these stresses. For example, cax1 has increased tolerance to freezing following cold acclimation, suggesting that AtCAX1 is a negative regulator of the cold-acclimation response (Catalá et al., 2003). By contrast, cax3 has increased sensitivity to salt stress (Zhao et al., 2008). Both cold and salt stress elicit [Ca2+]cyt oscillations in Arabidopsis, each with specific characteristics and resulting, in part, from Ca2+ release from the vacuole (Knight et al., 1996, 1997; Evans et al., 2001). H+/Ca2+ exchangers may be involved in resetting the [Ca2+]cyt elevation following stress induction. The differential stress sensitivities of the cax mutants may be a consequence of specific responses by AtCAX1 and AtCAX3 to the individual stresses.

VII. Ca2+ efflux transporters as modulators of Ca2+ signatures

Most Ca2+ efflux transporters clearly have a critical housekeeping function for providing tolerance to excess concentrations of Ca2+ and maintaining optimal Ca2+ concentration in certain organelles. It is unclear, however, in both plant and nonplant cells, whether all transporters play major roles in modulating [Ca2+]cyt during signalling events. For example, AtECA1 and AtECA3 can transport Ca2+ and Mn2+ equally and are important in manganese homeostasis (Wu et al., 2002; Mills et al., 2008), yet it is unknown whether they have a role in Ca2+ signalling. The ability of many Ca2+ transporters to be regulated, such as the dynamic modes of regulation of the ACAs, is certainly consistent with a role as Ca2+ modulator. We can also hypothesize as to how loss of individual Ca2+ efflux transporters may alter a Ca2+ transient (Fig. 2b). Several studies in animals support such models and demonstrate that manipulation of Ca2+ efflux transporters can significantly perturb stimulus-induced [Ca2+]cyt changes and, in some cases, alter downstream responses. For example, manipulation of SERCAs can give rise to frequency and amplitude alteration of agonist-induced [Ca2+]cyt oscillations or waves (Lechleiter et al., 1998; Zhao et al., 2001). Similarly, overexpression of PMCA2 reduces glucose-induced [Ca2+]cyt oscillations and changes the downstream response by altering insulin secretion patterns (Kamagate et al., 2002). However, for many Ca2+-ATPase manipulations, changes in [Ca2+]cyt are not observed. What is not always clear is whether this indicates a nonsignalling function for the transporter or whether any changes are being masked by redundancy or compensation. In the case of SERCA knockout animals, upregulation of Na+/Ca2+ exchange activity and/or plasma membrane Ca2+-ATPase is frequently encountered (Prasad et al., 2004).

Despite such evidence from nonplant systems, there is not as yet any published data confirming that the in vivo plant Ca2+-ATPases play a direct role in shaping Ca2+ signatures, as opposed to merely preventing high Ca2+ elevation. A recent study utilizing yeast heterologous expression has begun to give some indication that generation of a Ca2+ signature by a plant Ca2+ pump can mediate a specific stress response (Anil et al., 2008). A Saccharomyces cerevisiae mutant lacking two Ca2+-ATPases, has increased sensitivity to NaCl when grown under low-calcium conditions. Expression of AtACA4 or AtACA2 in this yeast mutant can provide tolerance to salt stress (Geisler et al., 2000; Anil et al., 2008). The mechanism of this tolerance might result from the action of the Ca2+-ATPase generating an internal Ca2+ signal that mediates a downstream stress tolerance response. This is supported by observations using the [Ca2+]cyt reporter aequorin that mutant yeast expressing AtACA2 produces a rapid [Ca2+]cyt transient following salt treatment, which decays quickly (Anil et al., 2008). By contrast, untransformed yeast shows a prolonged [Ca2+]cyt elevation and slow decline following salt stress, which precedes the inability of the yeast to survive. AtACA2-mediated salt tolerance of yeast appears to be the result of Na+ sequestration via the H+/Na+ exchanger NHX1. Furthermore, treatment with a Ca2+/CaM-dependent protein kinase inhibitor abolished the salt tolerance provided by AtACA2 and blocked AtACA2-mediated transcription of NHX1 (Anil et al., 2008). This study demonstrates some of the advantages of assessing Ca2+ transporter signalling function in a simple cell system without the complication of many other compensatory mechanisms. However, yeast is certainly not a plant cell and many of the regulators of the transporter will be lacking.

As with some of the mammalian Ca2+-ATPases, there has been conflicting evidence as to whether Ca2+ exchangers can directly modulate [Ca2+]cyt elevations. These high-capacity transporters clearly have a predominant role in Ca2+ homeostasis and will efflux excess concentrations of [Ca2+]cyt much more efficiently than the Ca2+-ATPase. However, deletion of the yeast vacuolar H+/Ca2+ exchanger VCX1 can perturb changes in [Ca2+]cyt observed in response to hypertonic shock (Denis & Cyert, 2002). By contrast overexpression of the human plasma membrane Na+/Ca2+ exchanger NCX1 alters the depolarization-induced [Ca2+]cyt elevation pattern (Van Eylen et al., 2002). There is indirect yet convincing evidence that supports a role for plant H+/Ca2+ exchangers in modulating Ca2+ signatures. As previously discussed, the Arabidopsis det3 mutant, which has a 60% reduction in vacuolar H+-ATPase activity, exhibits oscillations in guard cell [Ca2+]cyt and steady-state stomatal closure in response to cold and ABA, but not high [Ca2+]ext and H2O2 (Allen et al., 2000). One explanation for this disruption of stress-induced [Ca2+]cyt oscillations is indirect inhibition of H+/Ca2+ exchange activity, as the vacuolar H+-ATPase is the primary energizer of H+-coupled antiport activity. Studies assessing stimulus-induced [Ca2+]cyt in direct H+/Ca2+ exchange knockout lines may provide confirmation of this. However, compensatory mechanisms will make such studies challenging; indeed, it has already been shown that deletion of AtCAX1 leads to up-regulation of various CAX genes and tonoplast Ca2+-ATPase activity (Cheng et al., 2003).

VIII. The shaping of noncytosolic Ca2+ signatures

Spatial localization of a Ca2+ signal is an obvious means by which Ca2+ can provide specificity in signalling. In addition to spatial variation in cytosolic location, spatial separation of Ca2+ signals can occur within organelles (Fig. 3). The vacuole, ER and apoplast are important pools for Ca2+ release in plant cells but it is becoming clear that other organelles, such as chloroplasts, can also function as sites for Ca2+ release (Weinl et al., 2008). However, it is also apparent that noncytosolic Ca2+ oscillations can be generated in some of these organelles and may have specific Ca2+ signalling roles or act as an internal switch to regulate organellar processes.

Figure 3.

Stimulus-induced Ca2+ oscillations in the cytosol and in noncytosolic locations, including the nucleus, chloroplast stroma and mitochondria. Ca2+ oscillations are generated by an integrated Ca2+ oscillator that comprises a Ca2+ influx pathway, such as a Ca2+ channel (shown as cylinders), which mediates the flow of Ca2+ down its concentration gradient, and an active Ca2+ efflux pathway, including Ca2+-ATPases and H+/Ca2+ exchangers (shown as ovals), which transport Ca2+ against its concentration gradient. Cytosolic Ca2+ signatures, such as external Ca2+-induced Ca2+ oscillations in Commelina guard cells (from McAinsh et al., 1995,, ©American Society of Plant Biologists), may be generated by Ca2+ influx from outside the cell or release from an internal store, and through the action of Ca2+ efflux transporters at the plasma membrane or internal membranes. Nuclear Ca2+ signatures, such as nod factor-induced Ca2+ oscillations observed in the nuclear region of Medicago root hair cells (reprinted from Ehrhardt et al., 1996, with permission from Elsevier), are likely to be dependent on the release and accumulation of Ca2+ from nearby stores, such as the nuclear envelope. Chloroplastic Ca2+ oscillations have been observed in the stroma of tobacco seedlings following dark stimulation (from Johnson et al., 1995,, reprinted with permission from AAAS). Ca2+ transporters identified at the inner envelope membrane and the thylakoid membrane may be required for the generation of these oscillations. Mitochondrial Ca2+ oscillations, such as touch-induced Ca2+ elevation in Arabidopsis mitochondria (from Logan & Knight, 2003,, ©American Society of Plant Biologists, reproduced with permission American Society of Plant Biologists), will be dependent on Ca2+ influx and efflux into and from the mitochondrial lumen, although the identity of the transporters that mediate this are unknown. The cytosolic and nuclear Ca2+ oscillations shown are from single-cell imaging using Fura-2 (McAinsh et al., 1995; Ehrhardt et al., 1996), while the chloroplast and mitochondrial Ca2+ oscillations shown are derived from luminescence measurements of whole seedlings using stromal-localized and mitochondrial-localized aequorin, respectively (Johnson et al., 1995; Logan & Knight, 2003).

1. Nuclear Ca2+

The insights from the studies of nodulation signalling in legumes (Oldroyd & Downie, 2008) have clearly reminded us that Ca2+ signalling does not only take place in the cytosol. As described earlier, nod factor-induced changes in intracellular Ca2+ are frequently observed as Ca2+ oscillations that are restricted to the nuclear region (Fig. 3) (Ehrhardt et al., 1996; Walker et al., 2000; Sun et al., 2007). This pathway and the advancing genetic and genomic tools of model legumes such as M. truncatula should be able to enhance our knowledge of how [Ca2+]nuc homeostasis is controlled. Various candidate genes have been identified from the detailed genetic dissection of this pathway. One of the M. truncatula genes downstream of the Ca2+ response and required for [Ca2+]nuc elevation is DMI1. DMI1 encodes a putative cation channel which is localized to the nuclear periphery, possibly the nuclear envelope (Ané et al., 2004; Riely et al., 2007). However, recent functional analysis of DMI1 demonstrates that it does not directly generate [Ca2+]nuc oscillations, as it does not appear to be a Ca2+ channel, but DMI1 is able to regulate Ca2+ release, possibly via membrane potential modulation (Peiter et al., 2007). Other proteins required for induction of [Ca2+]nuc oscillations are nucleoporins, which are components of the nuclear pore complex (Kanamori et al., 2006; Saito et al., 2007). It can be hypothesized that these proteins may regulate the transport of either Ca2+ from the cytosol into the nucleus or another signal that is required to activate Ca2+ release into the nuclear interior.

It is not fully clear what directly generates the [Ca2+]nuc signal. Ca2+ may permeate from the cytosol into the nucleus, although various stimulus-induced [Ca2+]nuc elevations have been shown to be independent of [Ca2+]cyt (Pauly et al., 2000; Xiong et al., 2004). The nuclear envelope is an obvious Ca2+ store, with the envelope membrane possessing voltage-dependent and Ca2+-activated channels, Ca2+-ATPase activity and possible localization of an ECA (Downie et al., 1998; Grygorczyk & Grygorczyk, 1998; Bunney et al., 2000). Pharmacological studies provide the most convincing evidence to date for the role of such transport pathways in shaping [Ca2+]nuc signatures. An assessment of known inhibitors of Ca2+ transport and homeostasis found that a Ca2+-ATPase, phospholipase C and possibly a InsP3-regulated Ca2+ channel are required for M. truncatula [Ca2+]nuc oscillations in response to nod factor (Engstrom et al., 2002).

2. Mitochondrial Ca2+

Mitochondria can accumulate extremely high concentrations of Ca2+ (Putney & Thomas, 2006). Mitochondrial Ca2+ ([Ca2+]mit) and mechanisms of Ca2+ transport at this organelle are areas of intense study in the animal field, particularly because of the role of [Ca2+]mit in modulating [Ca2+]cyt and regulating apoptotic cell death (Giacomello et al., 2007). In plants, relatively little is known of the dynamics of [Ca2+]mit, although Ca2+ concentrations and Ca2+ oscillations have been determined using [Ca2+]mit indicators (Logan & Knight, 2003) (Fig. 3). A variety of stimuli cause rapid increases in [Ca2+]mit in Arabidopsis, with touch stimulation in particular inducing a [Ca2+]mit signature that is distinct from the [Ca2+]cyt signature (Logan & Knight, 2003). The principal pathway for [Ca2+]mit accumulation is the mitochondrial uniporter, a Ca2+-selective channel at the inner mitochondrial membrane that is neither ATP-dependent nor coupled to ion exchange (Kirichok et al., 2004). Conversely, release of Ca2+ from the mitochondria is mediated by ion-coupled Ca2+ exchangers (Putney & Thomas, 2006). The molecular identity of the uniporter (and the Ca2+ exchangers) in any organism is unknown, although human mitochondrial uncoupling proteins have recently been shown to be essential for Ca2+ uniport either as a regulator or as a component of the uniporter itself (Trenker et al., 2007). It will be interesting to see if orthologous proteins in plants are essential for [Ca2+]mit accumulation.

3. Chloroplast Ca2+

Chloroplasts can also generate independent Ca2+ oscillations ([Ca2+]chl) (Fig. 3). The chloroplast has as essential requirement for Ca2+, and, as in the cytosol, excess concentrations are toxic. Studies using Ca2+ reporters targeted to the tobacco stroma have shown the occurrence of circadian oscillations of [Ca2+]chl. Interestingly, these stromal Ca2+ oscillations are significantly enhanced following darkness, with the magnitude of Ca2+ flux proportional to the duration of light exposure before the onset of darkness (Johnson et al., 1995; Sai & Johnson, 2002). These circadian [Ca2+]chl oscillations may have a regulatory role to make sure that photosynthetic processes are switched off at night (Sai & Johnson, 2002). It is not fully clear what generates [Ca2+]chl fluxes. Ca2+ is transported across the inner envelope membrane by ATP- and pH-dependent means during light conditions. A pH gradient- or membrane potential gradient-dependent Ca2+ uniport mechanism is present at the inner envelope membrane of pea (Roh et al., 1998). In addition, the putative CaM-regulated Ca2+-ATPase AtACA1 may be located at the inner envelope (Huang et al., 1993), although there is no detectable Ca2+-ATPase activity at this membrane (Roh et al., 1998), and AtACA1 has been assigned as ER-localized by proteomic analysis (Dunkley et al., 2006). Nevertheless, a CaM-binding protein of the expected size for an ACA has been detected in isolated chloroplast membranes (Johnson et al., 2006). Another ATPase present at the envelope membrane is AtHMA1, a Cu+-ATPase that is essential for plant survival under high-light conditions (Seigneurin-Berny et al., 2006). AtHMA1 also appears to have high-affinity Ca2+ transport activity and is sensitive to the SERCA inhibitor thapsigargin (Moreno et al., 2008). Whether this pump has in vivo Ca2+-ATPase activity in chloroplasts will be of interest. In addition to flux across the inner envelope, Ca2+ may be released into the stroma from the thylakoid by an as yet unknown pathway, while Ca2+ can be loaded into the thylakoid by a H+/Ca2+ exchanger (Ettinger et al., 1999).

The potential of the chloroplast as an alternative source for Ca2+ release for [Ca2+]cyt signalling events must also be considered. Arabidopsis guard cells that overexpress a pea chloroplast protein, PPF1, a putative Ca2+ channel, have reduced [Ca2+]cyt elevations, as more Ca2+ is retained in the chloroplast (Wang et al., 2003; Li et al., 2004). The Arabidopsis membrane-associated Ca2+-binding protein CAS also appears to be specifically required for regulating stomatal closure in response to [Ca2+]ext. Deletion of CAS causes a loss of stomatal closure in response to [Ca2+]ext but not in response to other signals such as ABA, and this is coincident with a loss of [Ca2+]ext-induced guard cell [Ca2+]cyt oscillations (Han et al., 2003; Nomura et al., 2008; Weinl et al., 2008). Intriguingly, although CAS was originally thought to be plasma membrane-localized (Han et al., 2003), it is clearly present specifically at the thylakoid membrane (Nomura et al., 2008; Vainonen et al., 2008; Weinl et al., 2008). CAS therefore appears to regulate [Ca2+]ext-induced [Ca2+]cyt changes by controlling Ca2+ storage and release from the chloroplast, although the mechanisms for this are still unclear.

IX. Future insights into the role of Ca2+ oscillators from modelling studies

Genetic analysis of individual Ca2+ transporters through knockout or overexpression has begun to provide direct evidence that they play a role in the generation of Ca2+ oscillations. It is more difficult, however, certainly from genetic studies alone, to determine how these influx and efflux transporters integrate to form a Ca2+ oscillator which can generate Ca2+ spikes and oscillations (Fig. 3). As we have discussed, plant cells possess a variety of mechanisms and pathways which may affect Ca2+ (Table 1). The potential complexity of interplay between these pathways is difficult to understand intuitively. Mathematical modelling provides a powerful tool to understand this complexity and the mechanisms underlying the Ca2+ oscillation. Ca2+ oscillations in animal cells have been modelled extensively over a number of years (reviewed by Schuster et al., 2002; Falcke, 2004; Sneyd, 2005). These models provide reasonably accurate descriptions of [Ca2+]cyt oscillations in various cell types and provide some useful insights. Many of these models have highlighted a central role for agonist-facilitated CICR from internal stores in the generation of such oscillations, most notably mediated by the ER InsP3R, the regulatory properties of which appear to be largely responsible for the oscillatory nature of many signals.

While models describing some Ca2+-mediated signalling pathways in plants now exist (Li et al., 2006b), there has as yet been little attempt to model plant cell Ca2+ oscillations. Clearly a good model is based on experimental data, and unlike in the animal field, our understanding of some Ca2+ homeostatic mechanisms is lacking, particularly for Ca2+ release from internal stores. One of the few models produced describes Ca2+ oscillations in guard cells in response to high (1 µm) and low (10 nm) concentrations of ABA (Veresov et al., 2003). This is a minimal mathematical model based on the kinetics of four Ca2+ transport pathways – ER InsP3-activated Ca2+ release, ER Ca2+-ATPase, tonoplast cADPR-activated Ca2+ release, and tonoplast H+/Ca2+ exchange – plus the parameters of ABA stimulation on these pathways in guard cells. Simulated [Ca2+]cyt oscillations by this model (Fig. 4) show good similarity to oscillations observed in Commelina communis guard cells (Staxén et al., 1999). Removal of either Ca2+ channel leads to a loss of oscillations, while removal of H+/Ca2+ exchange activity causes a loss of the simulated [Ca2+]cyt oscillations but only in response to high ABA (Veresov et al., 2003) (Fig. 4). Interestingly, in the det3 mutant in which H+/Ca2+ exchange activity is expected to be reduced, guard cell [Ca2+]cyt oscillations in response to 10 µm ABA were unaltered (Allen et al., 2000).

In many ways, plant cellular Ca2+ dynamics are more complex than a typical animal cell. In addition to the ER and mitochondria, the vacuole is a major pool for Ca2+ buffering and release. Furthermore, we are beginning to appreciate the impact that the chloroplast can play on [Ca2+]cyt (Weinl et al., 2008). To understand the roles of these components, further modelling of plant Ca2+ signalling systems is required. In addition to the guard cell system, circadian regulation of Ca2+ modulation (Dodd et al., 2006), and the legume rhizobial symbiosis system are obvious systems for which modelling should be achievable and rewarding. For example, [Ca2+]nuc signatures generated in M. truncatula in response to mycorrhizal fungi and rhizobial bacteria differ in characteristics, despite being dependent on the same proteins for their activation (Kosuta et al., 2008) (Fig. 1c). Modelling of these pathways may provide insights into the unknown factors that determine these differential Ca2+ signatures.

X. Conclusions and perspectives

Plants clearly have the potential to generate complex Ca2+ signatures. However, the number of components of the Ca2+ signalling network that have been functionally assigned remains limited and there are many questions that need answering before we understand the mechanisms by which plant Ca2+ signatures are shaped. The technology now exists to reliably and sensitively measure stimulus-induced changes in both cytosolic and noncytosolic Ca2+ (Allen et al., 2000; Kosuta et al., 2008) and to study the contribution of individual transporters by manipulating the expression of putative Ca2+ transporter genes, where known (e.g. GLR3.3 knockout, Qi et al., 2006). Such analyses are hampered, however, by genetic redundancy in pathways and the lack of candidate Ca2+-permeable channel genes. Approaches such as heterologous expression (Anil et al., 2008), together with in silico gene knockout or overexpression studies using models derived from existing physiological and biochemical data for pathways (Fig. 4), provide powerful tools for addressing outstanding questions. It is also likely that additional and potentially as yet unknown components will play a role in regulating and shaping Ca2+ signatures. For example, the role of novel proteins, such as thylakoid-localized CAS on [Ca2+]cyt modulation (Weinl et al., 2008), are only just being appreciated. In addition, although we do not consider the role of Ca2+-binding proteins and buffers in this review, it is probable that soluble Ca2+-buffering proteins will further fine-tune and shape a Ca2+ transient. Cytosolic Ca2+ binding proteins such as calretinin and paravalbumin are known in animal cells (Berridge et al., 2003), and whilst progress is being made in plants (Ide et al., 2007), our knowledge of plant Ca2+ buffers, particularly in the cytosol, is limited. Finally, the presence of multiple Ca2+ signals within plants and different cell types highlights the dynamic and flexible nature of the Ca2+ signalling network and the many points where crosstalk may occur (McAinsh & Schroeder, in press). Consequently, analysis of individual genes in isolation will only take us so far and a key challenge in the future will therefore be to understand how these signalling components interact to shape Ca2+ signatures.


The authors acknowledge the financial support of the BBSRC, NERC and Royal Society. JKP is grateful for the award of a BBSRC David Phillips Fellowship.