T G Ca S P: HE Eneration of 2+ Ignals in Lants

Annu. Rev. Plant Biol. 2004. 55:40127 doi: 10.1146/annurev.arplant.55.031903.141624 Copyright c 2004 by Annual Reviews.
All rights reserved First published online as a Review in Advance on January 12, 2004
THE GENERATION OF Ca2+ SIGNALS IN PLANTS

Alistair M. Hetherington and Colin Brownlee
Department of Biological Sciences, Lancaster Environment Center, University of Lancaster, Lancaster LA1 4YQ, United Kingdom; email: A.Hetherington@lancaster.ac.uk Marine Biological Association of the United Kingdom, The Laboratory, Citadel Hill, Plymouth PL1 2PB, United Kingdom; email: cbr@mba.ac.uk
Annu. Rev. Plant Biol. 2004.55:401-427. Downloaded from www.annualreviews.org by University of Uppsala on 12/13/12. For personal use only.
Key Words signal transduction, stimulus-response coupling, nodulation factor, guard cells, calcium channels s Abstract The calcium ion is rmly established as a ubiquitous intracellular second messenger in plants. At their simplest, Ca2+-based signaling systems are composed of a receptor, a system for generating the increase in [Ca2+]cyt, downstream components that are capable of reacting to the increase in [Ca2+]cyt, and other cellular systems responsible for returning [Ca2+]cyt to its prestimulus level. Here we review the various mechanisms responsible for generating the stimulus-induced increases in [Ca2+]cyt known as Ca2+ signals. We focus particularly on the mechanisms responsible for generating [Ca2+]cyt oscillations and transients and use Nod Factor signaling in legume root hairs and stimulus-response coupling in guard cells to assess the physiological signicance of these classes of Ca2+ signals.
CONTENTS
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DISSECTING THE COMPONENTS OF Ca2+ SIGNALS . . . . . . . . . . . . . . . . . . . . . Entry Points: Ca2+ -Permeable Channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Plasma Membrane Depolarization-Activated Ca2+ Channels (DACCs) . . . . . . . . . . Plasma Membrane Hyperpolarization-Activated Ca2+ Channels (HACCs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Plasma Membrane NSCCs Indicate Diverse Functions Including Ca2+ Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Endomembrane Ca2+ Channels: Unidentied Players with Important Roles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Shaping the Ca2+ Signal: Pumping and Binding! . . . . . . . . . . . . . . . . . . . . . . . . . . . Functional Roles: Evidence from Disruption Studies . . . . . . . . . . . . . . . . . . . . . . . . ER Ca2+ Binding and Homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mitochondria: Energetic Players in a Dynamic Ca2+ Network . . . . . . . . . . . . . . . . Mitochondria as Ca2+ Buffers: Marks and Sparks . . . . . . . . . . . . . . . . . . . . . . . . PROPAGATION OF PLANT Ca2+ SIGNALS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [CA2+ ]cyt OSCILLATIONS AND TRANSIENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . Calcium Oscillations in Legume Root Hair NF Signaling . . . . . . . . . . . . . . . . . . . . 1543-5008/04/0602-0401$14.00 402 403 403 403 404 405 406 407 408 408 409 409 410 411 411
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Generation of NF-Induced [Ca2+ ]cyt Oscillations in Root Hair Cells . . . . . . . . . . . . Stimulus-Induced Oscillations and Transients of Guard Cell [Ca2+ ]cyt . . . . . . . . . . Generating Stimulus-Induced Oscillations and Transients in Guard Cell [Ca2+ ]cyt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Signicance of [Ca2+ ]cyt Oscillations and Transients in Plant Cell Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CONCLUSIONS AND FUTURE PERSPECTIVES . . . . . . . . . . . . . . . . . . . . . . . . . .
INTRODUCTION
The calcium ion (Ca2+) is rmly established as a ubiquitous intracellular second messenger in plants (114, 116). An increase in the cytosolic concentration of Ca2+ ([Ca2+]cyt) couples a diverse array of signals and responses. At their simplest, Ca2+based signaling systems are composed of a receptor, a system for generating the increase in [Ca2+]cyt, downstream components that are capable of reacting to the increase in [Ca2+]cyt, and other cellular systems responsible for returning [Ca2+]cyt to its prestimulus level. This apparently simple scheme hides considerable complexity. Although it has been argued recently that plant Ca2+ elevations may more often act as switches in the signaling process (118) rather than encoding specic information, it is clear that the cell has multiple mechanisms for generating increases in [Ca2+]cyt (116, 136), which suggests the capacity to produce highly complex spatio-temporal patterns of [Ca2+]cyt elevation. Downstream of the stimulusinduced [Ca2+]cyt increase (or Ca2+ signal), the cell possesses an array of proteins that can respond to changes in [Ca2+]cyt such as calmodulin (CaM) (85, 120), Ca2+-dependent protein kinases (19, 54), and CaM-binding proteins (113, 143). Further complexity arises with the involvement of other intracellular messengers and signaling proteins that can modulate Ca2+ signaling. In recognition of this increasing complexity, it is perhaps more appropriate to represent intracellular signaling as a network. Within a network-based organization a stimulus-induced increase in [Ca2+]cyt can be regarded as one of a small number of nodes or hubs responsible for orchestrating the events that comprise the full cellular response. The possibility that guard cell signaling might be organized as and exhibit properties reminiscent of a specic type of network known as a scale-free network has been discussed (57, 58). In this review we only consider the processes involved in generating Ca2+ signals. Currently, our understanding of how complex spatio-temporal patterns of [Ca2+]cyt elevation contribute to controlling responses in stimulated cells is fragmentary. Nevertheless, recent work has provided strong evidence that information is encoded in [Ca2+]cyt oscillations and transients, at least in guard cells (2, 3). To assess the physiological importance of [Ca2+]cyt oscillations and transients we review how they are generated. Although we restrict our discussion to the roles of mitochondria and plasma membrane and endomembrane Ca2+-permeable channels in this process, it should not be forgotten that nuclei (106, 107) and chloroplasts
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(65, 115) also contribute to Ca2+ signal generation. The physiological context of our discussions is provided by focusing on the roles of [Ca2+]cyt transients and oscillations in Nod Factor (NF) signaling and stimulus-response coupling in guard cells.
DISSECTING THE COMPONENTS OF Ca2+ SIGNALS Entry Points: Ca2+-Permeable Channels

Ca2+-permeable channels are the key entry points for Ca2+ into the cytosol (116). Elevation of [Ca2+]cyt can arise via increased inux and/or decreased efux (Figure 1), although generally most Ca2+ signals initiate from increased Ca2+ channel activity. In animal cells, specic [Ca2+]cyt elevations have been ascribed to Ca2+ inux through specic Ca2+-selective channels, at both the whole-cell (50) and single-channel level (145). The pharmacological proles and selectivities of plant plasma membrane Ca2+-permeable channels often differ signicantly from their animal counterparts (136). In plants Ca2+ permeable, rather than Ca2+-selective, channels predominant and there is a general lack of specicity of pharmacological agents. These differences may reect signaling strategies of plants that rely largely on adaptive rather than behavioral responses as well as the different modes of energization and more hyperpolarized nature of most plant plasma membranes. The known properties of the major groups of plant Ca2+-permeable channels have been surveyed in several recent comprehensive reviews (26, 130, 136). At the molecular level these have been broadly classed as nonselective cation channels (NSCCs). They are encoded by 41 genes of the cyclic nucleotide gated channel (CNGC) or glutamate receptor channel (GLR) families (see below) and the two pore calcium channel (TPC) gene. However, in only a few cases have physiologically characterized channels been mapped to their molecular counterparts. Here we focus on those channels that have been implicated in bringing about specic Ca2+ elevations.
Plasma Membrane Depolarization-Activated Ca2+ Channels (DACCs)

Plasma membrane depolarization is a frequently observed response to various biotic (32, 81) and abiotic (104) stimuli that also give rise to [Ca2+]cyt elevations, which suggests the involvement of DACCs (135). Nonselective, cation-permeable DACCs have been described in a number of cell types such as Arabidopsis root cells (128). Their activity appears to be downregulated by the microtubule cytoskeleton. However, their pharmacology is relatively unexplored and there are no reports directly relating DACC activity in situ with elevations in [Ca2+]cyt. These are difcult channels to characterize due to small currents and activity rundown, although channel recruitment in whole-cell recordings is possible with strong
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depolarization or microtubule disrupters (128). DACCs from cereal root plasma membrane have also been characterized in lipid bilayers (136). The molecular identities of DACCs have not been veried. AtTPC1, which has homology with the animal 1 subunit of voltage-dependent Ca2+ channels, and the ScCCH/MID1 gene in yeast (41) has been proposed as a candidate gene (137). However, evidence that AtTPC1 encodes a depolarization-activated channel is indirect so far. Expression of AtTPC1 in ScCCH1-decient yeast enhances Ca2+ uptake and growth rate, and in Arabidospis, AtTPC1 is required for the [Ca2+]cyt elevation in response to sucrose supply (41). The sucrose-induced Ca2+ rise depends on the expression of sucrose symporter genes [AtSUC1 and AtSUC2], and it is plausible that depolarization associated with sucrose/H+ cotransport underlies channel activation.
Plasma Membrane Hyperpolarization-Activated Ca2+ Channels (HACCs)

Ca2+ channels that underlie hyperpolarization-activated Ca2+ currents across the plasma membrane have been relatively well characterized. They were rst described in tomato cells (43) and were activated in response to fungal elicitors (44). Signicantly, aequorin-transformed parsley cells show a biphasic elevation of [Ca2+]cyt in response to a Phytophora oligopeptide elicitor (13). HACCs have also been described in root hair apices, and root epidermal, endodermal, and cortical cells from the elongation zone and in guard cells (116). Their activation at hyperpolarized membrane potentials (more negative than 100 mV) leads to elevation of Ca2+ in guard cells (49, 109), and they play a critical role in the response of stomata to abscisic acid (ABA). In root hairs their preferential activity at the growing apex (129) suggests a role in the sustained tip-localized elevation of [Ca2+]cyt, and their presence in elongating root cells (68) suggests a role in sustained Ca2+ inux. Spontaneous or evoked hyperpolarization leads to inward Ca2+ currents and corresponding elevation of [Ca2+]cyt (49) in Vicia guard cells. The observed [Ca2+]cyt elevation is relatively slow, occurring over tens of seconds. Grabov & Blatt (49) suggested that ABA-induced [Ca2+]cyt elevation could result from hyperpolarization-induced HACC activity, although it is also likely that Ca2+ release from intracellular stores plays a signicant role in the ABA response. + In addition to regulation by membrane voltage, Ca2 cyt (129) and reactive oxygen species (ROS), specically OH , increase HACC activity in Arabidopsis root hairs (40). Molecular evidence for upstream activation of HACC activity by ROS, and an essential role in cell growth comes from work with the Arabidopsis rhd2 [AtrbohC] nicotinamide adenine dinucleotide phosphate (NADPH) oxidase mutant. These mutants fail to elongate root hairs and are defective in ROS production and the generation of the root hair apical [Ca2+]cyt gradient (40, 140). Both mutant and wild-type cells have similar OH -activated HACC activity. This study suggests that ROS is an essential upstream signal regulating cell growth via HACC regulation. In guard cells, ROS (specically H2O2) (76, 108) and phosphorylation (73) increase + HACC activity (see below), but in contrast to root hairs, elevated Ca2 cyt decreases it
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(53). This apparent differential regulation by Ca2+ and ROS raises the possibility that different isoforms occur in these two cell types. The molecular identity of HACC is not known. However, two possibilities have been proposed. First, mutation in the AtCNGC2 gene underlies the defense no death (dnd) mutation in Arabidopsis. AtCNGC2 has inward-rectifying properties when expressed in Xenopus and is nonselective for cations. This led Demidchik and colleagues (25) to suggest that it may be an HACC. However, the apparent insensitivity to cyclic nucleotides of the Arabidopsis root hair HACC (V. Demidchik & J.M. Davies, unpublished data) argues against the identity of the HACC as a CNGC. Second, White et al. (137) proposed that an annexin may underlie HACC activity based on their Ca2+ permeability, localization, and known channel-forming properties of annexins in animal cells (45). Annexins comprise a small family in Arabidopsis (21) that have been linked with Ca2+ signaling and uxes (20, 137) and the Ca2+-dependent regulation of membrane cycling (17). Analysis of annexin mutants should shed more light on their roles as Ca2+-permeable channels.
Plasma Membrane NSCCs Indicate Diverse Functions Including Ca2+ Signaling

NSCCs have been characterized in Arabidopsis root epidermal cells and mediate steady-state Ca2+ inux at more depolarized membrane potentials than HACCs (25). These rapidly activating channels show little voltage dependence of activation and are active over a wide range of membrane potentials. They also co-occur with HACCs in the same cell type. Such channels may provide a crucial Ca2+ inux pathway at moderate membrane potentials as found, for example, in root epidermal cells (86), increasing the range of potentials over which plasma membrane channels may play a role in Ca2+ signaling. CNGCs are represented by 20 members in Arabidopsis. In animals their activity is increased by cAMP or cGMP. Members of the Arabidopsis CNGC family possess overlapping nucleotide-binding and CaM-binding regions at their C terminal (116, 126, 137). Binding CaM and cyclic nucleotides provides the potential for dual regulation by distinct signaling pathways and possible mechanisms for integrating Ca2+ and cyclic nucleotide signals (126). However, direct regulation of Ca2+ ux or [Ca2+]cyt dynamics by cyclic nucleotides for plant CNGCs remains to be shown. Cyclic nucleotide modulation of a Na+-permeable channel has been reported in Arabidopsis root protoplasts, but cGMP or cAMP inhibited rather than enhanced activity (87). CNGCs probably have a wide range of functions not all related to Ca2+ signaling. However, human kidney (HEK) cells transformed with AtCNGC2 showed that [Ca2+]cyt increases after treatment with membrane-permeant cAMP and cGMP (80). The demonstration of a role for cAMP in pollen tube growth regulation raises the possibility that CNGC regulation could be involved in the formation of the apical [Ca2+]cyt gradient that directs pollen tube growth (97). The 20 members of the GLR family are also good candidates for nonselective Ca2+-permeable channels (24, 26). GLRs have fundamental roles in animal cells
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in mediating fast chemical transmission and long-term synaptic potentiation (29). Plant GLR ligand binding is suggested by the similarities between the animal and plant genes in the ligand-binding domains (24, 26). Glutamate can induce relatively fast (within a few seconds) increases in [Ca2+]cyt in aequorin-transformed whole Arabidopsis seedlings (27). Ca2+ inux is implicated in this response by the inhibitory effects of La3+.
Endomembrane Ca2+ Channels: Unidentied Players with Important Roles

Because many Ca2+ responses in plant cells involve Ca2+ release from internal stores it is important to understand the nature and regulation of the endomembrane Ca2+ release pathways. However, linking the activity of specic endomembrane Ca2+ channels to [Ca2+]cyt elevations is difcult because they cannot be monitored electrophysiologically in intact cells. None have yet been characterized at the molecular level in plants. Obvious homologues of animal inositol (1, 4, 5) trisphosphate (InsP3) or ryanodine receptors that mediate InsP3 or cyclic ADP ribose (cADPR)-induced Ca2+ release, respectively, have not been identied in the Arabidopsis genome. Nevertheless, there is compelling evidence to suggest fundamental roles for endomembrane channels based on direct patch clamp recordings, caged release, pharmacology, imaging, and more recently, genetic studies. A variety of endomembrane channels that could potentially give rise to cytosolic Ca2+ elevations have been characterized at the physiological level. These include voltage-dependent calcium channels (VDCCs), slow vacuolar (SV) channels, and InsP3-, and cADPRsensitive Ca2+ release channels in the vacuolar membrane (116). Ca2+ release from endoplasmic reticulum (ER)enriched fractions has also been demonstrated in response to InsP3 (98), cADPR (101), and nicotinic acid adenine dinucleotide phosphate (NAADP) (102), although Ca2+-permeable ER channels have been monitored directly in only two plant systems (Bryonia tendris and Lepidium root tips) by incorporation into lipid bilayers (70, 71). The SV channel has been well studied with respect to its potential role in bringing about Ca2+ release from the vacuole during guard cell signaling. There has been considerable discussion about whether this channel can indeed give rise to Ca2+ ux into the cytosol under normal physiological conditions (137); at cytosolic Mg2+ concentrations the activation threshold for this channel is in the physiological range (3 to 50 mV, cytosol negative). This channel can potentially give rise to Ca2+-induced Ca2+ release (CICR) from the vacuole because its activity also increases with elevated cytosolic Ca2+. The SV channel is also activated by CaM and the type IIB phosphoprotein phosphatase, calcineurin, which activates at low concentrations but inhibits at high concentrations. The SV channel is also activated signicantly by increased cytosolic pH in the physiological range (7.3 to 8.0), potentially providing a link between observed increases in pH in response to ABA (11).
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Shaping the Ca2+ Signal: Pumping and Binding!

To act as an effective signal [Ca2+]cyt needs to be maintained at sufciently low levels to allow small inuxes through channels to bring about signicant changes in [Ca2+]cyt. Adenosine triphosphate (ATP)-fueled pumps (Ca2+-ATPases) and transporters driven by the electrochemical gradients of other ions, particularly H+ in higher plants (H+/Ca2+ exchangers), play critical housekeeping roles in maintaining low resting [Ca2+]cyt in the unstimulated cell. However, they may also be important in determining the peak amplitudes and duration of Ca2+ transients (Figure 1). Evidence that they play active roles in shaping Ca2+ signals comes from the knowledge that certain classes of pumps and transporters can be regulated by Ca2+/CaM, among other factors (see below). However, although the properties of transporters and pumps have been elucidated in some detail in various plant cell types, we await concrete evidence to link a specic pump or transporter directly with a specic Ca2+ signaling event. It is also becoming clear that disrupting the expression or activity of one type of pump or transporter can lead to compensatory changes in the activity or expression of other pumps or transporters. However, despite these problems, functions for pumps and transporters in stress signaling and Ca2+ homeostasis are beginning to emerge. In general Ca2+-ATPases belong to a group of high-afnity pumps whereas + H /Ca2+ exchangers have lower afnity (42, 112, 124). The Ca2+-ATPases can be grouped as type IIA or IIB based on their homologies with animal counterparts. Type IIA ATPases include the ER-type Ca2+ ATPases (ECA) whereas type IIB contain the autoinhibited Ca2+-ATPases (ACA) (31). Each group contains multiple members (4 ECA and 10 ACA genes in Arabidopsis). Although there is evidence for an ER location for type IIA Ca2+-ATPases in plants (82, 83), plasma membrane, vacuole, and Golgi locations are also evident (38, 42, 105). Type IIB Ca2+-ATPases in animal cells are targeted to the plasma membrane whereas plant IIB members are also targeted to endomembranes (42, 60). For example, ACA8 occurs on the plasma membrane (14) but ACA2 has been identied with an ER location (82) and ACA4 occurs on small vacuolar membranes but not the large central vacuole, which suggests functional diversity within the vacuolar membrane system in relation to Ca2+ homeostasis or signaling (42). Type IIA and IIB Ca2+-ATPases show distinctly different modes of regulation. Type IIB (ACA) members contain an N-terminal autoinhibitory domain (in contrast to animal counterparts, which contain a C-terminal autoinhibitory domain) and can be activated directly through binding Ca2+/CaM (56). ACA activity can be inhibited by calcium-dependent protein kinase (CDPK) binding and phosphorylation (63). In plants no direct regulation of a type IIA ATPase has been demonstrated, which may suggest a constitutive role in maintaining resting cytosolic Ca2+ levels. However, animal type IIB pumps are highly regulated by an inhibitory subunit phospholamban and possibly also ER lumen Ca2+ content (31). Physiological and molecular data indicate vacuolar localization of H+/Ca2+ exchangers such as the Arabidopsis cation exchanger CAX1, although there is also
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physiological evidence for H+/Ca2+ exchange at other locations such as the plasma membrane (67), the chloroplast thylakoid membrane (35), and the nuclear envelope (30). CAX transporter activity can be regulated via an N-terminal autoinhibitory domain. Pittman & Hirschi (111, 112) postulated that this is effected via interaction with a regulatory protein. However, the N terminal does not interact with CaM (112), unlike the ACA Ca2+-ATPases, and the nature of the regulatory protein remains unknown.
Functional Roles: Evidence from Disruption Studies

Recent manipulation of expression of both Ca-ATPases and H+/Ca2+ exchangers has begun to shed light on their roles in Ca2+ homeostasis. T-DNA insertion mutants of ECA1 (139) showed reduced growth on Ca2+-depleted media. This was the rst knockout of a Ca2+-ATPase gene and provided evidence for a role for ECA1 in ER Ca2+ homeostasis. Overexpression of CAX1 in tobacco plants gave rise to increased vacuolar H+/Ca2+ exchange activity and increased plant Ca2+ levels. CAX1 overexpressing plants showed severe Ca2+ deciency symptoms, probably due to reduced [Ca2+]cyt levels. These plants also showed increased vacuolar H+ pumping. Knockout of CAX1 (18) led to increased tolerance to both Ca2+ deciency and abiotic stress and also resulted in decreased vacuolar V-ATPase H+ pumping activity. Signicantly, these knockouts led to increased tonoplast Ca2+-ATPase activity and increased expression of other H+/Ca2+ exchangers CAX3 and CAX4. Although evidence for a direct involvement in Ca2+ signaling was not provided in these studies, a possible signaling role was suggested by the impaired hormone responses and auxin-regulated gene expression of cax1 mutants. These results from overexpression and knockout experiments suggest complex interactions between the activities and expression levels of different pumps and transporters, and the operation of a highly integrated compensatory network that may also include primary H+ pumps. Direct monitoring of Ca2+ transients in plants where specic pump or transporter activity has been manipulated is eagerly awaited, and their precise cellular localizations need to be cataloged. In frog oocytes, overexpression of a type IIA Ca2+-ATPase led to increased frequency of Ca2+ spiking (77). Although there is currently no direct evidence for a role for Ca2+ pumps or transporters in shaping Ca2+ signals, there is evidence for a role of a V-type H+-ATPase in determining the patterns of Ca2+ signals. Arabidopsis det3 mutants that are defective in a V-type H+ ATPase show disrupted Ca2+ signaling in stomatal guard cells in response to elevated [Ca2+]ext and oxidative stress but not to ABA or cold (3). The simplest mechanistic interpretation of this result may relate to energization of a CAX that is involved in generating stimulus-specic [Ca2+]cyt oscillations.
ER Ca2+ Binding and Homeostasis

Ca2+-binding proteins also play important roles in Ca2+ homeostasis in plants, although a direct signaling role is not yet evident. Overexpression of maize
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calreticulin (CRT) in tobacco leads to increased Ca2+ storage in the ER (110). Arabidopsis plants overexpressing CRT show increased resistance to Ca2+ depletion, and expression of CRT antisense leads to increased sensitivity to low Ca2+. This suggests that the ER can provide an exchangeable store of Ca2+ for [Ca2+]cyt homeostasis. Vacuolar Ca2+-binding proteins have also been identied (141), although their role in Ca2+ signaling or homeostasis has not been determined.
Mitochondria: Energetic Players in a Dynamic Ca2+ Network

The role(s) of mitochondria in signaling in plants cells are relatively unexplored. There is evidence that they may function to provide a source of Ca2+ in maize suspension cells in response to anoxia (122), or respond to elevated [Ca2+]cyt by increasing their Ca2+ content in Fucus rhizoid cells (22). Recently, Logan & Knight (84) targeted aequorin to the mitochondrial matrix and recorded average cellular resting [Ca2+]mit around 200 nM, approximately double that of [Ca2+]cyt. Treatments that caused elevation of [Ca2+]cyt also gave rise to elevations of [Ca2+]mit. However, although [Ca2+]mit elevations were comparable in kinetics (though lower in amplitude) to those in the cytosol in response to cold and osmotic treatments, signicantly different kinetics were observed in response to touch stimuli, and [Ca2+]cyt recovered to resting levels much more rapidly than [Ca2+]mit. In response to H2O2, [Ca2+]mit elevated to a similar amplitude as [Ca2+]cyt. These results indicate separate mechanisms for regulating [Ca2+]mit and [Ca2+]cyt, and existence of mitochondria-specic Ca2+ signals. Logan & Knight (84) proposed that the elevations in [Ca2+]mit might be involved in stimulating dehydrogenases of the TCA cycle, which in turn would stimulate respiration and increased ATP production. Similar mechanisms have been elucidated in animal cells (10, 51).
Mitochondria as Ca2+ Buffers: Marks and Sparks

In animal cells, evidence is emerging for a very tight coordination of ER/SR Ca2+ release and uptake by individual mitochondria (51). It is likely that very highly localized [Ca2+]cyt sparks, resulting from Ca2+ ux although endomembrane InsP3 or ryanodine receptors, permit targeted delivery of Ca2+ to neighboring mitochondria, resulting in transient [Ca2+]cyt elevations in the matrix of individual mitochondria (marks). This points to a high degree of coordination of cytoplasmic and mitochondrial Ca2+ signaling, and highly localized regulation of mitochondrial function that is coordinated with localized signaling activity of the cytosol. However, there is evidence that mitochondria play an important role in regulating + the extent of Ca2 cyt signal propagation by highly localized buffering. For example, Malli et al. (89) have shown, using DsRed and yellow chameleon Ca2+ indicators targeted to the mitochondria and ER, that localized [Ca2+]cyt elevations in response to histamine were only evident in mitochondrial-free zones and did not occur close to mitochondria. This heterogeneity of Ca2+ elevation could allow simultaneous activation of plasma membrane Ca2+-activated and Ca2+-inhibited channels in the same cell. Thus, by rapidly removing Ca2+ from the cytosol, mitochondria can
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play an important role in the spatiotemporal patterning of Ca2+ signals. Recently it was shown in HeLa cells that mitochondrial uptake required sustained release of Ca2+ from nearby locations on the ER membrane and balanced uptake of Ca2+ into the ER via the activity of plasma membrane Ca2+ channels (123). Although we can only speculate that similar mechanisms exist in plants (Figure 2), the data of Logan & Knight (84) are broadly consistent with results from mammalian cells. It will be interesting to determine whether the mean increases in [Ca2+]mit measured + with aequorin reect a heterogeneity of [Ca2 mit ] between individual mitochondria in plant cells. Polarized growing plant cells offer an obvious choice for investigating the role of mitochondria in [Ca2+]cyt spatial regulation. Regulating mitochondrial movements in growing pollen tubes and root hairs may be especially signicant. The apical [Ca2+]cyt gradient in growing lily pollen tubes is associated with large measured uxes of Ca2+ into the tube apex (59). These Ca2+ uxes show oscillations with the same frequency as oscillations in the apical [Ca2+]cyt gradient and growth. However, although it is clear that the [Ca2+]cyt gradient at the pollen tube apex is required for growth (59), the peak Ca2+ elevation at the growing tube apex lags behind the growth peak by approximately 4 s, and the maximum Ca2+ inux at the tube tip lags behind growth by approximately 15 s. Studies of mitochondria distribution in pollen tubes show that they are excluded from the extreme apex (28) but they also move with cytoplasmic streaming in a reverse fountain pattern (39) that continually delivers and removes mitochondria to the subapical region. Whether these mitochondria play a role in the periodic removal or buffering of [Ca2+]cyt in the pollen tube apex remains to be determined.
PROPAGATION OF PLANT Ca2+ SIGNALS

Experimental demonstration of the components required for propagated intracellular release provides compelling evidence that plant cells, like animal cells, can generate Ca2+ signals that propagate through the cytosol via intracellular Ca2+ release. Most recorded Ca2+ waves in animal cells fall into the fast Ca2+ wave category that propagate within a narrow range of velocities that may reect the highly conserved nature of the intracellular propagation mechanisms (64). However, slow Ca2+ waves also occur in animal cells, particularly those that propagate between cells and in developing systems (134), and it has been proposed that both fast and slow waves propagate via reaction-diffusion mechanisms (64, 134). In plants, very few intracellular propagating waves have been visualized directly, although Campbell et al. (15) showed the existence of intercellular waves that propagate slowly through tissue in whole Arabidopsis seedlings. Although [Ca2+]cyt elevations that appear to propagate as waves have been observed in guard cells, their velocity is approximately ten times slower than typical fast Ca2+ waves (49). So far the only recorded fast Ca2+ wave described in detail in a plant or algal cell is that of the osmotically induced wave in the Fucus rhizoid (48). Although this will likely be triggered by activating the mechanosensitive plasma membrane
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channels (127), it propagated by elemental sparks through ER-rich regions (48). Fast [Ca2+]cyt responses of higher plants, such as those in response to osmotic stimuli, that are likely to involve intracellular Ca2+ release (125) are potentially good candidates for studying plant Ca2+ waves. The question remains of how we explain the preponderance of relatively slow increases in cytosolic Ca2+ in terms of known mechanisms for Ca2+ elevation in plants. The rate of rise of [Ca2+]cyt and velocity of propagation in any cell depends on several factors, including the kinetics of receptor activation, rate of production and diffusion of second messengers such as InsP3, the rate of permeation of Ca2+ through channels (which also depends on their selectivity), the length of time the channels stay open (presumably a sustained elevation of Ca2+ requires a sustained increased channel-open probability), and the regulation of efux systems. The highly convoluted nature of the ER endomembrane system may allow Ca2+ release sites to be positioned in close proximity to one another, whereas the reaction-diffusion kinetics along the more linear vacuolar membrane may be signicantly different. Examples of signicant deviation from the typical animal reaction-diffusion model for Ca2+ waves do exist in plants. For example, a slowly propagating Ca2+ signal occurs in the maize egg during in vitro fertilization (8). In this system, elevation of Ca2+ is brought about by a slow wave of Ca2+ inux across the plasma membrane, which propagates around the egg. Another example is provided by cytoplasmic droplets from the giant alga Nitella that display Ca2+ spikes in response to hypo-osmotic treatment. The most likely explanation for this phenomenon is the activation of mechanosensitive Ca2+-permeable endomembrane channels (69).
[CA2+]cyt OSCILLATIONS AND TRANSIENTS

In animal cells, stimulus-induced [Ca2+]cyt oscillations are well documented and their mechanisms of generation and the potential for the information to be encoded in the frequency of [Ca2+]cyt oscillation was recently reviewed (10). [Ca2+]cyt oscillations and transients also occur in plant cells with, to the best of our knowledge, the rst report being IAA-stimulated [Ca2+]cyt oscillations in maize coleoptiles (37). The frequency, period, and amplitude of [Ca2+]cyt oscillations vary among different cell types and in response to different stimuli (36), and the possibility that [Ca2+]cyt oscillations might encode information has been discussed (36, 93, 114, 116, 117), as have mechanisms for generating [Ca2+]cyt oscillations (9, 55). To discuss how [Ca2+]cyt oscillations and transients are generated, and their possible roles in plant cell signaling, we focus on NF signaling in legume root hair cells and stimulus-response coupling in guard cells.
Calcium Oscillations in Legume Root Hair NF Signaling

NFs are bacterial lipochitooligosaccharide signals that play an important role in the early stages of nodule development. They stimulate root hair cells of susceptible plants to undergo morphological changes that lead to the entrapment and invasion
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of the bacteria. The role of NFs in this process has been comprehensively reviewed (16, 46, 81). Among the NFs induced changes in susceptible root hair are increases in [Ca2+]cyt, which include oscillations. There are two phases to NF-induced root hair [Ca2+]cyt elevations. The rst phase is rapid. In Medicago truncatula it consists of a rapid spike followed by a sustained [Ca2+]cyt increase or plateau that lasts for 34 min. Approximately 10 mins after NF application, the second phase occurs, which consists of calcium spiking in the nuclear region (7, 119). A typical spike in M. sativa or M. truncatula consists of a rapid increase of [Ca2+]cyt (approximately 500 nM) followed by a more gradual return to resting levels. This produces an asymmetric peak with a sharp rising phase and a slower recovery. The duration of the whole spike from onset to return to resting [Ca2+]cyt is between 30120 s, and the frequency of occurrence is typically approximately 1 min (7, 33). Because Ca2+ spiking is an example of an oscillatory phenomenon we refer to it as [Ca2+]cyt oscillation.
Generation of NF-Induced [Ca2+]cyt Oscillations in Root Hair Cells

In M. truncatula, (34) NF-induced [Ca2+]cyt oscillations were inhibited separately by 50 M 2-APB and 50 mM caffeine, which are putative antagonists of intracellular store-mediated Ca2+ release. 5 M cyclopiazonic acid (CPA), an inhibitor of type IIA calcium ATPases in plants, 10 M 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ), an inhibitor of mammalian sarco(endo)plasmic reticulum calcium ATPases (SERCA), and the phosphatidyl inositol specic phospholipase C (PI-PLC) inhibitor U73122 (20 M) also inhibited NF-induced root hair [Ca2+]cyt oscillations. Because both U73122 (121) and CPA (83) inhibit plant enzymes the data obtained by Engstrom and colleagues support a role for PI-PLC and Type IIA Calcium ATPases in NF generation of [Ca2+]cyt oscillations. No effects on [Ca2+]cyt oscillations were observed when the root hairs were treated with the putative plasma membrane calcium channel blockers verapamil (100 M) and LaCl3 (1 mM) (34). However, it might be unwise to rule out the involvement of plasmamembrane Ca2+ channels because it is possible that plants possess Ca2+ channels in the plasma membrane that are La3+- and verapamil-insensitive. Genetic approaches have also been used to identify components involved in the NF signaling pathway. Walker and colleagues (132) investigated the occurrence of NF-induced [Ca2+]cyt oscillations in the pea nodulation defective mutants sym8, sym10, and sym19. In all three of these mutants NF-induced [Ca2+]cyt oscillations were blocked, suggesting that the products of all three genes are upstream of and required for the generation of NF-induced [Ca2+]cyt oscillations. Long and colleagues (131) reached similar results and conclusions using the dmi1 and dmi2 nodulation mutants of M. truncatula. Shaw & Long (119) recently investigated whether the rst rapid NF-induced [Ca2+]cyt increase is required for generating NF-induced [Ca2+]cyt oscillations. When they challenged M. truncatula with 10 nM NF they observed the familiar
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two-phase Ca2+ response. However, when they applied 1 nM NF they did not observe any rapid NF-induced [Ca2+]cyt increase, although they saw the NF-induced [Ca2+]cyt oscillations. These results suggest that generating [Ca2+]cyt oscillations does not require the rst NF-induced [Ca2+]cyt elevation.
Stimulus-Induced Oscillations and Transients of Guard Cell [Ca2+]cyt

Stimulus-induced increases in guard cell [Ca2+]cyt are part of signal transduction network(s) responsible for coupling ABA and other external signals to alterations in stomatal aperture (11, 12, 57, 93, 117). Stimulus-induced oscillations in guard cell [Ca2+]cyt were observed when Commelina communis, Fura-2-loaded guard cells were bathed in 0.1 mM [Ca2+]ext (symmetrical oscillations with an average amplitude of approximately 430 nM and a mean period of 8.3 min) or 1.0 mM [Ca2+]ext (asymmetrical oscillations with an amplitude of 625850 nM and a mean period of 13.6 min) (94). Oscillations in guard cell [Ca2+]cyt were more extensively investigated in Arabidopsis using cameleon [Ca2+]cyt technology (5). Using this approach (3), 1 mM [Ca2+]ext induced oscillations with an average amplitude of approximately 160 nM and an average period of 2.6 min, whereas 10 mM [Ca2+]ext induced oscillations with an average amplitude of 1020 nM and a period of 6.6 min. In a separate study, 10 mM [Ca2+]ext induced [Ca2+]cyt oscillations with a period of approximately 8.2 min (2). The periods of the [Ca2+]ext-induced oscillations reported using the cameleon technique correlate well with data obtained in the same species using the uorescent ratiometric [Ca2+]cyt indicator Fura-2 (4). ABA can also induce [Ca2+]cyt oscillations in C. communis (121) and Arabidopsis. ABA-induced [Ca2+]cyt oscillations have been reported in different Arabidopsis ecotypes (Col, Ler, WS), and when they occur they are either in the form of regular oscillations with a magnitude of about 500 nM and a period of approximately 7.8 min or have no obvious periodicity. Schroeder and colleagues refer to these irregular increases as transients and we adopt this terminology here (2, 6, 61, 66, 72, 75, 76). Similar to earlier work (47, 90, 91), not all guard cells responded to ABA with detectable increases in [Ca2+]cyt. Transients were also observed when guard cells were challenged with a yeast elicitor (72), 100 M H2O2, and cold (3).
Generating Stimulus-Induced Oscillations and Transients in Guard Cell [Ca2+]cyt

Ca2+ inux across the plasma membrane and release from internal stores both contribute to the generation of [Ca2+]cyt oscillations. In C. communis the results of manganese quench experiments and application of verapamil suggests that plasma membrane calcium-permeable channels participate in the generation of [Ca2+]extinduced [Ca2+]cyt oscillations (94). A similar conclusion was reached from experiments in which Arabidopsis guard cells bathed in Ca2+-free media failed to exhibit [Ca2+]cyt transients when stimulated by a yeast elicitor or ABA (72).
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Although it is likely that more than one guard cell plasma membrane calcium channel is involved in the generation of [Ca2+]cyt oscillations (53, 72), most attention has focused on an HACC in Vicia faba (53) and Arabidopsis (where it is known as ICa) (108). In Vicia the open probability of the HACC is increased by ABA, the concentration of extracellular divalent cations (52, 53), and inhibitors of Type 1 and 2A phosphoprotein phosphatases (73). This latter result, coupled with the observation that ATP hydrolysis was required for channel activity, led Kohler & Blatt (73) to propose that phosphorylation of sites on or close to the channel facilitates opening. Increasing [Ca2+]cyt from 200 nM to 2 M reduces the open probability of the channel and provides a mechanism for feedback inhibition of calcium inux (53). In Arabidopsis the HACCs are permeable to several cations, including Mg2+, require cytosolic NAD(P)H, and are involved in the generation of [Ca2+]cyt increases by H2O2, ABA (108), and pathogenic elicitors (72). Schroeder and colleagues proposed a model for ABA action on Arabidopsis guard cells that involves ABA stimulating H2O2 production and the resulting H2O2 increasing the open probability of HACC (108). The suggestion that ABA action involves H2O2 is corroborated by investigations carried out in Vicia guard cells (144), and Schroeder and colleagues further suggest that ABA and a fungal elicitor signaling pathways converge on HACC (72). They also place the Type 2C phosphoprotein phosphatases encoded by the ABI1-1 and ABI2-1 genes upstream of ICa in ABA signaling (4, 99). The most recent work shows that although ABA-induced [Ca2+]cyt transients were not abolished, they were signicantly reduced in Arabidopsis NADPH oxidase catalytic subunit mutants (atrbohD/F double mutant), supporting a role for HACCs and ROS in Arabidopsis guard cell ABA signaling (76). However, the Blatt laboratorys (74) recent work in Vicia questions whether H2O2 really is a critical second messenger in guard cell ABA signaling. Kohler and colleagues (74) found that in Vicia although H2O2 regulated two key cellular components involved in guard cell turgor control (HACC and inward rectifying K+ channels) in a manner reminiscent of ABA, and H2O2 stimulates [Ca2+]cyt increases in C. communis (92) and Arabidopsis (108) it also depresses guard cell outwardrectifying K+ channels (74). This latter action is the direct opposite of what happens in guard cell ABA signaling. Ignoring possible species-specic differences as an explanation for these apparently conicting sets of data, it seems likely that, as guard cell ABA signaling is partially disrupted in the Arabidopsis AtrbohD/F double mutant (76), H2O2 and possibly other ROS are involved in some but not all aspects of guard cell ABA signaling. Finally, there is evidence that Type 2A phosphoprotein phosphatase (PP2A) activity is involved in the regulation of ABAinduced [Ca2+]cyt transients. In the loss of function rcn1 mutant, which encodes the regulatory subunit of a guard cellexpressed PP2A, stomatal responsiveness to ABA was greatly reduced, as was the probability of ABA-induced increases in [Ca2+]cyt (75). There are also data indicating that release of Ca2+ from internal stores contributes to the generation of stimulus induced [Ca2+]cyt transients and oscillations. There is evidence pointing to a role for the PI-PLCInsP3 system in generating
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[Ca2+]cyt oscillations and transients in guard cell ABA signaling. In both C. communis and Arabidopsis guard cells the pharmacological PI-PLC inhibitor U73122 interferes with ABAinduced alterations in stomatal aperture and ABA-induced [Ca2+]cyt oscillations (121) and transients (72). U73122 also interferes with ABAinduced K+ efux from guard cells (88). Is it possible to place PI-PLC in the sequence of events leading to the generation of ABA-induced [Ca2+]cyt oscillations and transients? Hunt and colleagues (62) present a model in which a primary ABA-generated increase in [Ca2+]cyt activates guard cell PI-PLC, which then generates InsP3, leading to Ca2+ release from internal stores. In this model PI-PLCInsP3 amplies the primary ABA-induced increase in [Ca2+]cyt and contributes to the rising phase of the oscillation. For this system to generate a series of oscillations, a separate mechanism for repriming [Ca2+]cyt to a level whereby it could activate PI-PLC would be required after the completion of each oscillation and the return of [Ca2+]cyt to resting levels. ABAinduced [Ca2+]cyt inux through the plasma membrane HACC (discussed above) might be a prime candidate to fulll such a role. Although PI-PLC plays a role in the generation of guard cell ABA-induced [Ca2+]cyt oscillations it is not apparently involved in [Ca2+]ext-induced [Ca2+]cyt oscillations in C. communis (121). Just as MacRobbie (88) showed that the mechanism used to generate ABAinduced increases in [Ca2+]cyt depends on the concentration of applied ABA, it is possible that other Ca2+-mobilizing messengers might be involved in ABA signaling. In guard (78) and other plant cells (1) cADPR induces Ca2+ release from vacuoles. There is evidence that cADPR might be involved in the generation of ABA-induced [Ca2+]cyt oscillations and transients. Microinjecting guard cells with cADPR induces increases in guard cell [Ca2+]cyt that include oscillations (78) and nicotinamide, which blocks cADPR synthesis and interferes with ABA-induced [Ca2+]cyt calcium transients (72). However, in contrast to the situation with the PI-PLCInsP3 system, it is unlikely that cADPR is involved in Ca2+ signal amplication or the phenomenon of CICR because cADPR-gated currents downregulate over the range of guard cell [Ca2+]cyt elevated by cADPR microinjection (78). This latter nding suggests that cADPR might be involved in the initial priming phase required in the generation of [Ca2+]cyt oscillations and transients. Understanding the contribution of cADPR to stimulus-induced [Ca2+]cyt oscillations and transients will be greatly facilitated with the identication of the enzyme systems responsible for its production and its intracellular receptors. Another molecule capable of generating increases in [Ca2+]cyt in animal and plant cells is sphingosine-1-phosphate (S1P) (138). When applied to C. communis guard cells 6 M S1P induced asymmetrical [Ca2+]cyt oscillations with a period of 3.8 min and peaks of up to 50 nM above resting levels of [Ca2+]cyt. 50 nM SIP induced symmetrical [Ca2+]cyt oscillations with a shorter period (2.8 min) but greater peak height (100 nM above resting levels of [Ca2+]cyt) (103). Recent work from the Assmann lab (23) resulted in the construction of a model for the involvement of S1P in guard cell ABA signaling, in which ABA activates sphingosine kinase in Arabidopsis guard cells resulting in the production of elevated
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SIP. The SIP then interacts with an unidentied receptor followed by interactions with the product of the GPA1 gene and generation of [Ca2+]cyt increases (23). Currently, we do not know whether other Ca2+-mobilizing messengers such as inositol hexakisphosphate (InsP6) (79) and NAADP (101) are involved in generating [Ca2+]cyt oscillations or transients. As with NF signaling in root hair cells, genetic approaches have also resulted in signicant insights into the generation of [Ca2+]cyt transients and oscillations. Schroeder and coworkers investigated whether the products of genes involved in ABA signaling are involved in the generation of [Ca2+]cyt oscillations and transients by examining ABA-induced [Ca2+]cyt elevations in the corresponding Arabidopsis mutants. Ten M ABA induced [Ca2+]cyt transients in 86% of wild-type guard cells but failed to induce [Ca2+]cyt increases in any abi2-1 guard cells (6). The result suggests that the product of the ABI2-1 gene (a type 2C phosphoprotein phosphatases) is required for the generating ABA-induced [Ca2+]cyt transients. Stomata of the Arabidopsis rcn1 mutant are less sensitive to ABA. 14% of rcn1 guard cells showed two or more [Ca2+]cyt transients when treated with 5 uM ABA, in contrast to the 44% of wild-type stomata that exhibited two or more [Ca2+]cyt transients. The conclusion from this work is that the type 2A phosphoprotein phosphatase encoded by the RCN1 gene is also involved in the generation of ABAinduced [Ca2+]cyt transients (75). Similar experimental approaches have shown that the ERA1 gene product (encoding a farnesyltransferase) (6), the catalytic subunits of NADPH oxidase encoded by AtRBOHD and AtRBOHF genes (76), the product of the GCA2 gene (2), and the mRNA cap binding protein encoded by ABH1 (61) can inuence ABA-induced [Ca2+]cyt transients.
Signicance of [Ca2+]cyt Oscillations and Transients in Plant Cell Signaling

In animal cells experimental evidence supports the hypothesis that information is encoded in [Ca2+]cyt oscillations (10). In plant cells it has been suggested that [Ca2+]cyt oscillations and transients might encode information that helps to dictate the nature of the downstream response (36, 55, 93, 114, 116, 117). We focus on three experimental systems, pollen tube growth, NF signaling in root hair cells, and stimulus-response coupling in guard cells, to assess the physiological signicance of [Ca2+]cyt oscillations and transients in plant cell signaling. The role of [Ca2+]cyt in the control of pollen tube growth was the subject of a recent comprehensive review (59). However, in brief, the growing pollen tube is an endogenous oscillator and accompanying growth oscillations are oscillations in [Ca2+]cyt, pHcyt, membrane potential, exo and endocytosis, cell wall thickness, and the uxes of extracellular Ca2+, H+, K+, and Cl. Because it has long been recognized that Ca2+ is essential for pollen tube growth, an attractive proposition was that [Ca2+]cyt oscillations might be the coordinator or pacemaker controlling growth. Current evidence suggests that this is unlikely for two reasons. First, [Ca2+]cyt oscillations follow rather than anticipate changes in pollen tube growth rate (95), and second, pollen tube [Ca2+]cyt oscillations have been observed in
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the absence of growth (96). Accordingly, the current situation in this system is that there are well-characterized [Ca2+]cyt oscillations but their physiological role remains to be established. Currently, a similar situation holds for NF-induced [Ca2+]cyt oscillations in legume root hairs, although this may change in the near future as a result of work by Shaw & Long (119). They showed that by judicial choice of NF concentration it is possible to induce [Ca2+]cyt oscillations in the absence of the rst NF-induced [Ca2+]cyt elevation. This seems to provide the ideal experimental system to address the question of the physiological role of [Ca2+]cyt oscillations. Given the perinuclear location of the [Ca2+]cyt oscillations, an obvious experiment to conduct would be a comparative analysis of 1 nM NF- (induces only [Ca2+]cyt oscillations) and 10 nM NF- (induces rst-phase [Ca2+]cyt elevations and second-phase oscillations) induced gene expression. Currently, the most compelling evidence that [Ca2+]cyt oscillations and transients might be responsible for encoding specic signaling information comes from guard cells. McAinsh and coworkers showed that that it is possible to correlate the pattern of [Ca2+]cyt oscillation with both the strength of the external stimulus and the extent of the nal response (stomatal closure) in [Ca2+]ext signaling (94). However, these data revealed a correlation rather than provided evidence of a causal relationship between [Ca2+]cyt oscillations and the control of stomatal aperture. Allen and colleagues (3) established a much stronger link between [Ca2+]cyt oscillations and the control of stomatal aperture using the Arabidopsis det3 mutant that exhibits a 60% reduction in expression of the C-subunit of the V-type H+-ATPase. The authors hypothesized that a reduction in V-type H+-ATPase activity might result in reduced Ca2+ sequestration into internal stores, and this in turn might affect stimulus-induced [Ca2+]cyt oscillations. Whatever the precise effects of the reduction in expression of the DET3 gene the authors observed, in contrast to wild-type guard cells, that det3 guard cells failed to exhibit [Ca2+]cyt oscillations and reductions in guard cell turgor in response to 1 and 10 mM [Ca2+]ext. When total guard cell [Ca2+]ext-stimulated [Ca2+]cyt was integrated over a 30-minute period it was signicantly higher in the det3 mutant than in wild type. This result clearly shows that the reason the det3 mutant failed to close its stomata in response to [Ca2+]ext was not due to an inability to increase [Ca2+]cyt, but rather due to the failure to generate [Ca2+]cyt oscillations that seemed to underlie the phenotype. Allen and colleagues also showed that experimentally induced [Ca2+]cyt oscillations brought about (rescued) closure in the det3 mutant. These data provide strong evidence that [Ca2+]cyt oscillations are required in the signaling pathway leading to stomatal closure. With evidence for the importance of [Ca2+]cyt oscillations in guard cell signaling the next question to address is what their specic role is in the control of stomatal aperture. One possibility is that [Ca2+]cyt oscillations are responsible for holding the guard cell in a steady low-turgor state while protecting it from the presumed deleterious effects of long-term exposure to elevated [Ca2+]cyt (94). However, although this might be benecial to the guard cell it is not a requirement in guard cell signaling because there are examples of closure-inducing,
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[Ca2+]cyt-elevating stimuli that do not bring about [Ca2+]cyt oscillations such as ABA (47, 91, 99), CO2 (133), and H2O2 (92). Instead, Schroeder and colleagues propose that [Ca2+]cyt controls stomatal aperture by two mechanisms, based on experiments carried out on the Arabidopsis gca2 mutant. They distinguish between short-term stomatal closure that occurs in response to elevated [Ca2+]cyt and long-term (steady-state) closure, the degree of which is programmed or controlled by [Ca2+]cyt oscillations within a dened range of frequency, transient number, duration, and amplitude (2). However, some additional factors need to be considered before any generalizations can be made about the role of [Ca2+]cyt oscillations in guard cells. Perhaps the most obvious issue to consider is that ABA-, CO2-, and H2O2-induced stomatal closure can occur in the absence of [Ca2+]cyt oscillations (47, 9092, 133). It is possible that the experiments in these studies were not long enough for the stomata to reach steady state and thus failed to exhibit [Ca2+]cyt oscillations. A further complication concerns the occurrence of spontaneous [Ca2+]cyt transients in guard cells (72). The lack of closure in the stomata exhibiting spontaneous [Ca2+]cyt transients is presumably due to the fact that the period of the spontaneous [Ca2+]cyt transients was shorter than ABA-induced [Ca2+]cyt oscillations and transients associated with closure. However, there are some difculties with this explanation because 10 mM [Ca2+]ext induces [Ca2+]cyt oscillations with a similar period to the spontaneous [Ca2+]cyt oscillations and 10 mM [Ca2+]ext closes stomata (3, 4). In summary, [Ca2+]cyt oscillations and transients are found in different plant cells, and their mechanism of generation and possible physiological signicance have been the subjects of considerable scrutiny in three well-studied plant signaling systems. Although we are learning much about the origin of [Ca2+]cyt oscillations and transients, pinning down their physiological role has proved more difcult. To date, the clearest evidence that stimulus-induced [Ca2+]cyt oscillations (as opposed to stimulus-induced nonoscillatory [Ca2+]cyt elevations) are required during signaling comes from the work performed on the det3 mutant (3). However, with the identication of new signaling mutants and ways of manipulating Ca2+ homeostasis it is likely that we are poised to learn much more about the possible functions of [Ca2+]cyt oscillations and transients in plant cell signaling.
CONCLUSIONS AND FUTURE PERSPECTIVES

It was recently proposed, on the basis of a well-argued case, that many (but not all) Ca2+ signals act as simple on-off binary switches and thus the Ca2+ signal is an unlikely mechanism for ensuring delity in plant signaling systems (118). However, as Scrase-Field & Knight (118) acknowledge and we discuss here, the guard cell provides examples of where Ca2+ signals do appear to encode the information for dening the outcome of responses (2, 3). The question that we now need to address is whether the guard cell is unique in using the Ca2+ signal
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to help provide delity in stimulus-response coupling, or whether it exists in other cells but we have failed to detect it so far. Unless the symplastic isolation of mature guard cells is a factor, there is no obvious reason why stomata should have evolved a unique method of signaling. Guard cells have provided important insights into signaling because it is possible to investigate the effects of different stimuli on Ca2+ dynamics at the single-cell level while monitoring a robust readout (changes in stomatal aperture). When the Schroeder lab combined the experimental advantages of the guard cell with the use of mutants the denitive evidence that Ca2+ signals can encode information was obtained. Before we conclude that information-encoding Ca2+ signals are unique to guard cells we need to thoroughly investigate whether they occur in other cell types. In this respect, NF signaling holds great promise, especially if a robust readout, such as an alteration in gene expression, can be identied similar to the way Shaw & Long (119) were able to experimentally isolate NF-induced [Ca2+]cyt oscillations from other NF-induced [Ca2+]cyt elevations. The technology for addressing whether the binary switch or the Ca2+ signal model predominates in plant cell signaling is advancing on several fronts. In this review we identify a range of targets for potential disruption and modication. The availability of mutants displaying lesions in these targets is increasing and should enable investigation of the effect of manipulating Ca2+ signals on stimulus-induced responses. Signicant advances are also being made in the use of FRET-based Ca2+ indicators such as cameleons and pericams (100, 142) that can be expressed in a targeted manner. These advances in reporter technology are being matched by fast improving camera and scanning-based imaging technology, and several options are now available for monitoring Ca2+ from total internal reection uorescence (TIRF) for near-membrane fast events in wall-free cells to multiphoton microscopy for deeper penetration and improved signal-to-noise ratio (39) in whole tissues and plants. Combining these techniques with robust cellular or tissue responses will allow the global signicance of Ca2+ signals to be assessed. Overall, the eld is poised for exciting developments and insights over the next few years. ACKNOWLEDGMENTS The authors acknowledge the support of the BBSRC and NERC for funding signaling research in their laboratories. NOTE ADDED IN PROOF Recently, a specic receptor for apoplastic Ca2+ that is expressed in guard cells and is required for oscillatory elevations in guard cell Ca2+ and stomatal closure + in response to elevated [Ca2 ext ] was cloned from Arabidopsis (53a). This suggests + a specic external messenger role for [Ca2 ext ]. It will be interesting to determine the downstream components of this signal transduction pathway.
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The Annual Review of Plant Biology is online at http://plant.annualreviews.org
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CALCIUM SIGNALING
C-1
Figure 1 Schematic representation of complex interactions between channels, pumps, transporters, and Ca2+-binding proteins in bringing about [Ca2+]cyt homeostasis and stimulus-specific elevations (as represented by the solid black trace) or oscillations of [Ca2+]cyt. Light arrows indicate hypothetical actions in raising or lowering [Ca2+]cyt at particular points in a Ca2+ transient. The assignment of Ca2+ pumps and transporters to different membranes is indicative rather than exhaustive. The combined activity of channels and pumps contributes to resting [Ca2+]cyt. NSCCs and HACCs may be particularly involved in maintaining steady Ca2+ influx. The chloroplast and nucleus are not shown. ACA,ECA, Ca2+-ATPAses; CAX, H+/Ca2+ exchangers; NSCC, nonselective cation channels; DACC, depolarization-activated Ca2+ channels; HACC, hyperpolarization-activated Ca2+ channels; GLR, glutamate receptor channels; CNGC, cyclic nucleotide gated channels; PM, plasma membrane; mit, mitochondria; ER, endoplasmic reticulum; CRT, calreticulin; VCaB, vacuolar Ca2+-binding protein.
C-2
HETHERINGTON
BROWNLEE
Figure 2 Hypothetical scheme for Ca2+ cycling between the cytosol, the ER, and mitochondria. [Ca2+]cyt elevations arising via influx through plasma membrane (PM) or endoplasmic reticulum (ER) channels (e.g., in response to touch, osmotic, or cold) are offset by mitochondrial and ER uptake or reuptake. Ca2+ efflux from mitochondria is removed from the cytosol by adjacent ER. [Ca2+]mit elevations stimulate ATP production, which may be used to fuel Ca2+ efflux via Ca2+- ATPase activity. Green arrows represent the proposed Ca2+ cycling pathway.
Annual Review of Plant Biology Volume 55, 2004
CONTENTS
AN UNFORESEEN VOYAGE TO THE WORLD OF PHYTOCHROMES,
Masaki Furuya
ALTERNATIVE NAD(P)H DEHYDROGENASES OF PLANT MITOCHONDRIA, Allan G. Rasmusson, Kathleen L. Soole,

and Thomas E. Elthon 23 41
DNA METHYLATION AND EPIGENETICS, Judith Bender PHOSPHOENOLPYRUVATE CARBOXYLASE: A NEW ERA OF STRUCTURAL BIOLOGY, Katsura Izui, Hiroyoshi Matsumura,
Tsuyoshi Furumoto, and Yasushi Kai
69 85
METABOLIC CHANNELING IN PLANTS, Brenda S.J. Winkel RHAMNOGALACTURONAN II: STRUCTURE AND FUNCTION OF A BORATE CROSS-LINKED CELL WALL PECTIC POLYSACCHARIDE,
Malcolm A. ONeill, Tadashi Ishii, Peter Albersheim, and Alan G. Darvill
109
NATURALLY OCCURRING GENETIC VARIATION IN ARABIDOPSIS THALIANA, Maarten Koornneef, Carlos Alonso-Blanco, and
Dick Vreugdenhil 141
SINGLE-CELL C4 PHOTOSYNTHESIS VERSUS THE DUAL-CELL (KRANZ) PARADIGM, Gerald E. Edwards, Vincent R. Franceschi,
and Elena V. Voznesenskaya 173 197 225 263 289 315
MOLECULAR MECHANISM OF GIBBERELLIN SIGNALING IN PLANTS,

Tai-ping Sun and Frank Gubler
PHYTOESTROGENS, Richard A. Dixon DECODING Ca2+ SIGNALS THROUGH PLANT PROTEIN KINASES,
Jeffrey F. Harper, Ghislain Breton, and Alice Harmon
PLASTID TRANSFORMATION IN HIGHER PLANTS, Pal Maliga SYMBIOSES OF GRASSES WITH SEEDBORNE FUNGAL ENDOPHYTES,
Christopher L. Schardl, Adrian Leuchtmann, Martin J. Spiering
TRANSPORT MECHANISMS FOR ORGANIC FORMS OF CARBON AND NITROGEN BETWEEN SOURCE AND SINK, Sylvie Lalonde,
Daniel Wipf, and Wolf B. Frommer 341
vii
viii
CONTENTS
REACTIVE OXYGEN SPECIES: METABOLISM, OXIDATIVE STRESS, AND SIGNAL TRANSDUCTION, Klaus Apel and Heribert Hirt THE GENERATION OF Ca2+ SIGNALS IN PLANTS,
Alistair M. Hetherington and Colin Brownlee
373 401 429
BIOSYNTHESIS AND ACCUMULATION OF STEROLS, Pierre Benveniste HOW DO CROP PLANTS TOLERATE ACID SOILS? MECHANISMS OF ALUMINUM TOLERANCE AND PHOSPHOROUS EFFICIENCY,
eros Leon V. Kochian, Owen A. Hoekenga, and Miguel A. Pin
459 495 521 537 555
VIGS VECTORS FOR GENE SLIENCING: MANY TARGETS, MANY TOOLS, Dominique Robertson GENETIC REGULATION OF TIME TO FLOWER IN ARABIDOPSIS THALIANA,
Yoshibumi Komeda
VISUALIZING CHROMOSOME STRUCTURE/ORGANIZATION,

Eric Lam, Naohiro Kato, and Koichi Watanabe
THE UBIQUITIN 26S PROTEASOME PROTEOLYTIC PATHWAY,

Jan Smalle and Richard D. Vierstra
RISING ATMOSPHERIC CARBON DIOXIDE: PLANTS FACE THE FUTURE,

Stephen P. Long, Elizabeth A. Ainsworth, Alistair Rogers, and Donald R. Ort 591
INDEXES
Subject Index Cumulative Index of Contributing Authors, Volumes 4555 Cumulative Index of Chapter Titles, Volumes 4555 629 661 666
ERRATA
An online log of corrections to Annual Review of Plant Biology chapters may be found at http://plant.annualreviews.org/

T G Ca S P: HE Eneration of 2+ Ignals in Lants

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Annu. Rev. Plant Biol. 2004. 55:40127 doi: 10.1146/annurev.arplant.55.031903.141624 Copyright c 2004 by Annual Reviews.

THE GENERATION OF Ca2+ SIGNALS IN PLANTS

BROWNLEE 412 413 413 416 418

DISSECTING THE COMPONENTS OF Ca2+ SIGNALS Entry Points: Ca2+-Permeable Channels

Plasma Membrane Depolarization-Activated Ca2+ Channels (DACCs)

Plasma Membrane Hyperpolarization-Activated Ca2+ Channels (HACCs)

Plasma Membrane NSCCs Indicate Diverse Functions Including Ca2+ Signaling

Endomembrane Ca2+ Channels: Unidentied Players with Important Roles

Shaping the Ca2+ Signal: Pumping and Binding!

Functional Roles: Evidence from Disruption Studies

ER Ca2+ Binding and Homeostasis

Mitochondria: Energetic Players in a Dynamic Ca2+ Network

Mitochondria as Ca2+ Buffers: Marks and Sparks

PROPAGATION OF PLANT Ca2+ SIGNALS

[CA2+]cyt OSCILLATIONS AND TRANSIENTS

Calcium Oscillations in Legume Root Hair NF Signaling

Generation of NF-Induced [Ca2+]cyt Oscillations in Root Hair Cells

Stimulus-Induced Oscillations and Transients of Guard Cell [Ca2+]cyt

Generating Stimulus-Induced Oscillations and Transients in Guard Cell [Ca2+]cyt

Signicance of [Ca2+]cyt Oscillations and Transients in Plant Cell Signaling

CONCLUSIONS AND FUTURE PERSPECTIVES

The Annual Review of Plant Biology is online at http://plant.annualreviews.org

Annual Review of Plant Biology Volume 55, 2004

ALTERNATIVE NAD(P)H DEHYDROGENASES OF PLANT MITOCHONDRIA, Allan G. Rasmusson, Kathleen L. Soole,

MOLECULAR MECHANISM OF GIBBERELLIN SIGNALING IN PLANTS,

373 401 429

459 495 521 537 555

VISUALIZING CHROMOSOME STRUCTURE/ORGANIZATION,

THE UBIQUITIN 26S PROTEASOME PROTEOLYTIC PATHWAY,

RISING ATMOSPHERIC CARBON DIOXIDE: PLANTS FACE THE FUTURE,

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