《细胞生物学》课程教学资源（文献资料）Calcium at the Crossroads of Signaling

ThePlantCell,S401-S417,Supplement2002,www.plantcell.org2002AmericanSocietyofPlantBiologistsCalcium attheCrossroads ofSignalingDale Sanders,a,1 Jerome Pelloux,a Colin Brownlee,b and Jeffrey F.Harperc Biology Department, University of York, York YO10 5YW, United Kingdomb Marine Biological Association, The Laboratory, Citadel Hill, Plymouth PL12PB, United KingdomcScrippsResearchlnstitute,10550NorthTorreyPinesRoad,LaJolla,California92037INTRODUCTIONTheSpecificityQuestionAn all-pervading question during the last decade of calciumCalcium Signals:ACentral Paradigm insignalingresearchhasrevolvedaroundtheissueof specificityStimulus-Response Coupling(McAinsh and Hetherington, 1998). How can a simple non-protein messenger be involved in so many signal transduc-Cells must respond to an array of environmental and devel-tionpathwaysandyetstillconveystimulusspecificitywithinaopmental cues.The signaling networks that have evolved tovarietyofpathways?Ostensiblythereareanumberreason-generate appropriate cellular responses are varied and areable nonexclusive answers to this question. First, the Ca2+normalycomposedofelementsthatincludeasequenceofsignal itself might be a necessary but insufficient trigger forreceptors, nonprotein messengers, enzymes and transcrip-the response, with effective signal transduction occurringtion factors. Receptors are normally highly specific for theonly should another signal change in parallel. Second, speci-physiological stimulus, and therefore are disparate in theirficitymightbeencodedbythespatialpropertiesoftheCa2+identities.Likewise enzymes and transcriptionfactors tendsignal,eitherbecausethesignaliscompartmentallylocalizedtowardspecificity,andthisfactisreflectedinabundanceat(for example, to the nucleus, rather than the cytosol) or be-the genome level. The Arabidopsis genome, for example,cause the source of the Ca2+ signal (from outside the cell orpotentially encodes in the region of 1000 protein kinases,from intracellular stores) can selectively trigger response ele-300proteinphosphatases,and1500transcriptionfactorsments. Third, the dynamic properties of the Ca2+ signal mightBy contrast, nonprotein messengers are relatively few. Theydetermine the efficacy with which the response is elicited.include cyclic nucleotides (Newton et al., 1999), hydrogenFourth, of course, the appropriate response elements mustions (Guern et al.,1991), active oxygen species (Vanbe present in the particular cell type in which the Ca2+ signalBreusegemetal.,2001),lipids(NgandHetherington,2001;arises. Since Ca2+ signaling was last reviewed in this journalNurnbergerand Scheel,2001;Munne-BoschandAlegre,(Sanders et al.,1999),remarkable advances have been made2002), and, above all, calcium.in addressing this central problem of specificity, in manyChanges in cytosolic free calcium ([Ca2+J.) are apparentcases thanks to the insights provided bygenetic approaches.duringthetransductionofaverywidevarietyofabioticandThus, while alluding briefly to the earlier literature, the presentbiotic signals. The spectrum of stimuli that evokes rapidreview will focus on developments in our understanding thatchanges in [Ca2+].has been cataloged in a number of re-haveoccurredoverthepastfouryears.cent reviews (Sanders et al., 1999; Knight, 2000; Anil andRao, 2001; Knight and Knight, 2001; Rudd and Franklin-Tong, 2001). Abiotic stimuli include light-with red, blue,ELEMENTSENCODINGCALCIUMSIGNALSand UV/B irradiation each acting via different receptors andleading to distinct developmental responses (Shacklock etal., 1992; Baum et al., 1999; Frohnmeyer et al., 1999), lowCalcium signals are generated through the opening of ionand high temperature, touch, hyperosmotic stress, and oxi-channels that allow the downhill flow of Ca2+ from a com-dativestress.Biotic stimuliincludethehormonesabscissicpartment in which the ion is present at relatively high elec-acid (ABA) and gibberellin, fungal elicitors, and nodulationtrochemical potential (either outside the cell,or from an(Nod) factors.intracellular store)toone in which Ca2+isat lower potential.There has, in the past, been a tendency to refer to suchchannels as “Ca2+ channels," although we prefer the term"Ca2+-permeable channels"because this reflects the likely1Towhomcorrespondenceshouldbeaddressed.E-mailds10importance of nonselective cation channels in generatingyork.ac.uk; fax 44-1904-434317.plant Ca2+ signals.Maintenanceof low Ca2+electrochemicalArticle, publication date, and citation information can be found atwww.plantcell.org/cgi/doi/10.1105/tpc.002899.activity in the Ca2+-responsive compartment is achieved by

The Plant Cell, S401–S417, Supplement 2002, www.plantcell.org © 2002 American Society of Plant Biologists Calcium at the Crossroads of Signaling Dale Sanders,a,1 Jérôme Pelloux,a Colin Brownlee,b and Jeffrey F. Harperc a Biology Department, University of York, York YO10 5YW, United Kingdom b Marine Biological Association, The Laboratory, Citadel Hill, Plymouth PL1 2PB, United Kingdom c Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037 INTRODUCTION Calcium Signals: A Central Paradigm in Stimulus–Response Coupling Cells must respond to an array of environmental and devel￾opmental cues. The signaling networks that have evolved to generate appropriate cellular responses are varied and are normally composed of elements that include a sequence of receptors, nonprotein messengers, enzymes and transcrip￾tion factors. Receptors are normally highly specific for the physiological stimulus, and therefore are disparate in their identities. Likewise enzymes and transcription factors tend toward specificity, and this fact is reflected in abundance at the genome level. The Arabidopsis genome, for example, potentially encodes in the region of 1000 protein kinases, 300 protein phosphatases, and 1500 transcription factors. By contrast, nonprotein messengers are relatively few. They include cyclic nucleotides (Newton et al., 1999), hydrogen ions (Guern et al., 1991), active oxygen species (Van Breusegem et al., 2001), lipids (Ng and Hetherington, 2001; Nurnberger and Scheel, 2001; Munne-Bosch and Alegre, 2002), and, above all, calcium. Changes in cytosolic free calcium ([Ca2]c) are apparent during the transduction of a very wide variety of abiotic and biotic signals. The spectrum of stimuli that evokes rapid changes in [Ca2]c has been cataloged in a number of re￾cent reviews (Sanders et al., 1999; Knight, 2000; Anil and Rao, 2001; Knight and Knight, 2001; Rudd and Franklin￾Tong, 2001). Abiotic stimuli include light—with red, blue, and UV/B irradiation each acting via different receptors and leading to distinct developmental responses (Shacklock et al., 1992; Baum et al., 1999; Frohnmeyer et al., 1999), low and high temperature, touch, hyperosmotic stress, and oxi￾dative stress. Biotic stimuli include the hormones abscissic acid (ABA) and gibberellin, fungal elicitors, and nodulation (Nod) factors. The Specificity Question An all-pervading question during the last decade of calcium signaling research has revolved around the issue of specificity (McAinsh and Hetherington, 1998). How can a simple non￾protein messenger be involved in so many signal transduc￾tion pathways and yet still convey stimulus specificity within a variety of pathways? Ostensibly there are a number reason￾able nonexclusive answers to this question. First, the Ca2 signal itself might be a necessary but insufficient trigger for the response, with effective signal transduction occurring only should another signal change in parallel. Second, speci￾ficity might be encoded by the spatial properties of the Ca2 signal, either because the signal is compartmentally localized (for example, to the nucleus, rather than the cytosol) or be￾cause the source of the Ca2 signal (from outside the cell or from intracellular stores) can selectively trigger response ele￾ments. Third, the dynamic properties of the Ca2 signal might determine the efficacy with which the response is elicited. Fourth, of course, the appropriate response elements must be present in the particular cell type in which the Ca2 signal arises. Since Ca2 signaling was last reviewed in this journal (Sanders et al., 1999), remarkable advances have been made in addressing this central problem of specificity, in many cases thanks to the insights provided by genetic approaches. Thus, while alluding briefly to the earlier literature, the present review will focus on developments in our understanding that have occurred over the past four years. ELEMENTS ENCODING CALCIUM SIGNALS Calcium signals are generated through the opening of ion channels that allow the downhill flow of Ca2 from a com￾partment in which the ion is present at relatively high elec￾trochemical potential (either outside the cell, or from an intracellular store) to one in which Ca2 is at lower potential. There has, in the past, been a tendency to refer to such channels as “Ca2 channels,” although we prefer the term “Ca2-permeable channels” because this reflects the likely importance of nonselective cation channels in generating plant Ca2 signals. Maintenance of low Ca2 electrochemical activity in the Ca2-responsive compartment is achieved by 1 To whom correspondence should be addressed. E-mail ds10@ york.ac.uk; fax 44-1904-434317. Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.002899.

S402ThePlant Cellthe ATP-or proton motive force-driven removal of Ca2+ oncium-permeable channels that are activated by membranepumps or carriers (transporters), respectively. As shown indepolarization (reviewed by White, 2000). It has been specu-Figure 1, the interplay between influx through channels andlated that this form of voltage gating might endow sucheffluxfrompumpsandcarriers willdeterminetheformofachannels with a pivotal role at an early stage in signal trans-Ca2+ spike that is potentially specific to relevant decoders.duction (Ward et al., 1995). Thus, perception of a range ofFigure 2 shows the location of channels, pumps, and carri-stimuli results in membranedepolarization,possiblyas a re-ers involved in Ca2+ transport for a generalized Arabidopsissult of the activation of anion channels,and the resultantopening of depolarization-activated Ca2+-permeable chan-cell, as the basis for the discussion below.nelscouldleadtoelevationof[Ca2+lcWhile depolarization-activation of Ca2+-permeable chan-Calcium-PermeablelonChannelsnels is a recurring theme in a number of biological systems,recent simultaneousand independent studies havefollowedThe importance of the cellular location of ion channels in de-pioneering work by Gelli et al. (1997) and Gelli and Blumwaldtermining stimulus specificity is emphasized by a study of(1997)on tomatocell suspensions,reportingthepresenceCa2+-mediated stomatal closure in tobacco (Wood et al.,inplantplasma membranes of Ca2+-permeablechannels2000).Removalof extracellularCa2+withthechelatorEGTAthat are activated by membrane hyperpolarization. Suchorblockageofentrywithanumberofionchannelblockerschannels have a high selectivity for Ca2+ over K+ and Cl-suggested that low temperature-induced closure involves(Gelli and Blumwald, 1997; Hamilton et al., 2000; Very andprimarily entry of Ca2+ across the plasma membrane, whileDavies, 2000). It has been known for some time that inintracellular mobilization appears to dominate if stomatalguardcells,membranehyperpolarizationisdirectlyasso-closure is initiated with ABA or mechanical stimulation.ciated with the elevation of [Ca2+]。that follows ABA appli-Calcium-permeable channels have been investigatedcation (Grabov and Blatt, 1998). The observation thatwithelectrophysiological,biochemical,and molecularap-hyperpolarization-activated Ca2+-permeable channels in theproaches, and these are now beginning to yield comple-plasmamembrane of guard cellsare opened by ABA evenmentary insights into the nature and control of channels thatinexcisedmembranepatches impliesa very closephysicalunderliethegenerationofCa2+signals.coupling between the channels and the sites of ABA per-ception (Hamilton et al., 2000). Channel opening might alsobesubjecttonegativefeedbackcontroltopreventexces-PlasmaMembranesive Ca2+ entry, since activity decreases around ten-foldover the range of [Ca2+]。from 0.2 to 2 μM. In root hairs,Electrophysiological studies during the past decade havechannel activity is present at the tips of growing cells, butrevealed thepresence in plant plasma membranes of cal-not detectable in subapical regions or at the tips of maturecells (Very and Davies, 2000),an observation consistentwith the notion that these channels play a pivotal role in theStimulusgeneration of the tip-to-base Ca2+ gradient that is essential+for maintainingpolarization intip-growing systems (includ-ing pollen tubes and rhizoid cells). Intriguingly, the root hairchannels are,in contrast to their counterparts in guard cells,Influxactivated by elevation of [Ca2+J, suggesting that they mightCa-Spikeplay a self-sustaining role in maintaining the tip-to-baseEffluxCa2+ gradient. Hyperpolarization-activated Ca2+-permeable★channelshavealsobeenreported inthegrowingrootapexDecoders25,000genesof Arabidopsis roots, but not in other more mature cells+(Kiegle et al., 2000a), possibly suggesting a role for these★channels in cell division and elongation.ABA-inducedstomatalclosure involvestheproductionofResponsereactive oxygen species, notably hydrogen peroxide (Pei etThrough Regulational., 2000; Zhang et al., 2001), and hyperpolarization-acti-ofEnzymesandStructuresvated Ca2+-permeable channels play a critical role in thisreponse.In Arabidopsis guard cells, hydrogen peroxideFigure 1. Decoding Calcium Signals Leads to a Specific Responseat the Cellular Level.stimulates hyperpolarization-activated Ca2+-permeable chan-nels, and thereby an increase in [Ca2+l。 (Pei et al., 2000)Various feedback mechanisms from the calcium sensor (or"de-This process requires cytosolic NAD(P)H, suggesting thatcoder') are possible. These could include the regulation of calciumNAD(P)H oxidases could be part of the ABA signaling cas-spikes via the control of calcium permeable channel gating (e.g-cade (Murata et al., 2001).through EF binding hands, or via Ca2+/CaM binding) or via control ofIt is unlikely that voltage-gated pathways comprise thepump activity

S402 The Plant Cell the ATP- or proton motive force–driven removal of Ca2 on pumps or carriers (transporters), respectively. As shown in Figure 1, the interplay between influx through channels and efflux from pumps and carriers will determine the form of a Ca2 spike that is potentially specific to relevant decoders. Figure 2 shows the location of channels, pumps, and carri￾ers involved in Ca2 transport for a generalized Arabidopsis cell, as the basis for the discussion below. Calcium-Permeable Ion Channels The importance of the cellular location of ion channels in de￾termining stimulus specificity is emphasized by a study of Ca2-mediated stomatal closure in tobacco (Wood et al., 2000). Removal of extracellular Ca2 with the chelator EGTA or blockage of entry with a number of ion channel blockers suggested that low temperature–induced closure involves primarily entry of Ca2 across the plasma membrane, while intracellular mobilization appears to dominate if stomatal closure is initiated with ABA or mechanical stimulation. Calcium-permeable channels have been investigated with electrophysiological, biochemical, and molecular ap￾proaches, and these are now beginning to yield comple￾mentary insights into the nature and control of channels that underlie the generation of Ca2 signals. Plasma Membrane Electrophysiological studies during the past decade have revealed the presence in plant plasma membranes of cal￾cium-permeable channels that are activated by membrane depolarization (reviewed by White, 2000). It has been specu￾lated that this form of voltage gating might endow such channels with a pivotal role at an early stage in signal trans￾duction (Ward et al., 1995). Thus, perception of a range of stimuli results in membrane depolarization, possibly as a re￾sult of the activation of anion channels, and the resultant opening of depolarization-activated Ca2-permeable chan￾nels could lead to elevation of [Ca2]c. While depolarization-activation of Ca2-permeable chan￾nels is a recurring theme in a number of biological systems, recent simultaneous and independent studies have followed pioneering work by Gelli et al. (1997) and Gelli and Blumwald (1997) on tomato cell suspensions, reporting the presence in plant plasma membranes of Ca2-permeable channels that are activated by membrane hyperpolarization. Such channels have a high selectivity for Ca2 over K and Cl (Gelli and Blumwald, 1997; Hamilton et al., 2000; Véry and Davies, 2000). It has been known for some time that in guard cells, membrane hyperpolarization is directly asso￾ciated with the elevation of [Ca2]c that follows ABA appli￾cation (Grabov and Blatt, 1998). The observation that hyperpolarization-activated Ca2-permeable channels in the plasma membrane of guard cells are opened by ABA even in excised membrane patches implies a very close physical coupling between the channels and the sites of ABA per￾ception (Hamilton et al., 2000). Channel opening might also be subject to negative feedback control to prevent exces￾sive Ca2 entry, since activity decreases around ten-fold over the range of [Ca2]c from 0.2 to 2 M. In root hairs, channel activity is present at the tips of growing cells, but not detectable in subapical regions or at the tips of mature cells (Véry and Davies, 2000), an observation consistent with the notion that these channels play a pivotal role in the generation of the tip-to-base Ca2 gradient that is essential for maintaining polarization in tip-growing systems (includ￾ing pollen tubes and rhizoid cells). Intriguingly, the root hair channels are, in contrast to their counterparts in guard cells, activated by elevation of [Ca2]c, suggesting that they might play a self-sustaining role in maintaining the tip-to-base Ca2 gradient. Hyperpolarization-activated Ca2-permeable channels have also been reported in the growing root apex of Arabidopsis roots, but not in other more mature cells (Kiegle et al., 2000a), possibly suggesting a role for these channels in cell division and elongation. ABA-induced stomatal closure involves the production of reactive oxygen species, notably hydrogen peroxide (Pei et al., 2000; Zhang et al., 2001), and hyperpolarization-acti￾vated Ca2-permeable channels play a critical role in this reponse. In Arabidopsis guard cells, hydrogen peroxide stimulates hyperpolarization-activated Ca2-permeable chan￾nels, and thereby an increase in [Ca2]c (Pei et al., 2000). This process requires cytosolic NAD(P)H, suggesting that NAD(P)H oxidases could be part of the ABA signaling cas￾cade (Murata et al., 2001). It is unlikely that voltage-gated pathways comprise the Figure 1. Decoding Calcium Signals Leads to a Specific Response at the Cellular Level. Various feedback mechanisms from the calcium sensor (or “de￾coder”) are possible. These could include the regulation of calcium spikes via the control of calcium permeable channel gating (e.g., through EF binding hands, or via Ca2/CaM binding) or via control of pump activity.

S403CalciumattheCrossroadsofSignalingNSCCytosolAD SmallvacuoleCa"*-Ca2ACA4ATPCentralvacuoleADPADPCa2+A?ACA8CaCa2tA2+CaACAXATPATPADRDACATPH?HACCAX1Ca3CaCNGCXInsp...InsP.RCADPR-?TPC1RyRGolgiCa....42GLRXADPSV ChannelCa*4TACAXATPVVCaChannelCaatACA1ERInsR.@ADPATE一InsP.RATPCa2ACA2CADPRRYRADPCaeaCatECA14NAADP-ATPFigure 2.Schematic Representation of Major Identified Ca2+Transport Pathways in Arabidopsis Cell MembranesBlue circles represent energized transport systems. ACA1, ACA4, ACA8 are autoinhibited calcium ATPases identified at a molecular level. Thedirection of Ca2+ pumping for ACA1 is hypothetical. ECA is an ER-type calcium ATPase. ACAx in the central vacuole and in the Golgi has notbeen identified at a molecular level. CAX1 is a Ca?+/H+ antiporter expected to be localized at the vacuolar membrane. Red squares representCa2+-permeable channels. At the plasma membrane, nonselective cation (NSC) channels, depolarization activated channels (DACs) and hyper-polarization activated channels (HACs) have been characterized at an electrophysiological but not at a molecular level. A two-pore channel(TPC1) has been shown to complement a yeast mutant deficient in Ca2+ uptake, but channel location is hypothetical. Using electrophysiolocaltechniques, cyclic nucleotide gated channels (CNGC1 and CNGC2) were shown to be permeable to calcium. Plasma membrane location isagain hypothetical. Glutamate receptors (GLRs) might be involved in the increase of cytosolic calcium concentrations and have been identifiedat a molecular level. The channels identified at endomembranes have been characterized at electrophysiological and biochemical but not molec-ular levels. InsPgR, putative InsgP receptor; RyR, putative ryanodine receptor activated by cADPR; NAADP-activated channels also reside in theER as shown. SV channel, slowty activating vacuolar channel; WVCa channel, vacuolar voltage-gated Ca2+ channel.sole route for Ca2+ entry across the plasma membrane.variety of functions in addition to that of Ca2+ uptake, in-Channels that discriminate poorly between mono-and diva-cluding the uptake of cations for general nutritional purposes,lent cations and that exhibit at best only very weak voltage-and thepresence of various classes of channel is suggesteddependence are ubiquitous in plant cells (Demidchik et al.,by reports of diverse modes of regulation, including cyclic2002). These nonselective cation channels probably fulfll anucleotides (MaathuisandSanders,2001)andextracellular

Calcium at the Crossroads of Signaling S403 sole route for Ca2 entry across the plasma membrane. Channels that discriminate poorly between mono- and diva￾lent cations and that exhibit at best only very weak voltage￾dependence are ubiquitous in plant cells (Demidchik et al., 2002). These nonselective cation channels probably fulfill a variety of functions in addition to that of Ca2 uptake, in￾cluding the uptake of cations for general nutritional purposes, and the presence of various classes of channel is suggested by reports of diverse modes of regulation, including cyclic nucleotides (Maathuis and Sanders, 2001) and extracellular Figure 2. Schematic Representation of Major Identified Ca2 Transport Pathways in Arabidopsis Cell Membranes. Blue circles represent energized transport systems. ACA1, ACA4, ACA8 are autoinhibited calcium ATPases identified at a molecular level. The direction of Ca2 pumping for ACA1 is hypothetical. ECA is an ER-type calcium ATPase. ACAx in the central vacuole and in the Golgi has not been identified at a molecular level. CAX1 is a Ca2/H antiporter expected to be localized at the vacuolar membrane. Red squares represent Ca2-permeable channels. At the plasma membrane, nonselective cation (NSC) channels, depolarization activated channels (DACs) and hyper￾polarization activated channels (HACs) have been characterized at an electrophysiological but not at a molecular level. A two-pore channel (TPC1) has been shown to complement a yeast mutant deficient in Ca2 uptake, but channel location is hypothetical. Using electrophysiolocal techniques, cyclic nucleotide gated channels (CNGC1 and CNGC2) were shown to be permeable to calcium. Plasma membrane location is again hypothetical. Glutamate receptors (GLRs) might be involved in the increase of cytosolic calcium concentrations and have been identified at a molecular level. The channels identified at endomembranes have been characterized at electrophysiological and biochemical but not molec￾ular levels. InsP3R, putative Ins3P receptor; RyR, putative ryanodine receptor activated by cADPR; NAADP-activated channels also reside in the ER as shown. SV channel, slowly activating vacuolar channel; VVCa channel, vacuolar voltage-gated Ca2 channel.

S404ThePlant CellpH (Demidchik and Tester, 2002). It will therefore be impor-ion channels that are inhibited by cyclic nucleotides (cAMPtant to identify these channels at a molecular level beforeand cGMP) has been recorded in Arabidopsis root cellsdrawing conclusions concerning their specific roles in Ca2+.(Maathuis and Sanders, 2001). It is possible that this activitybased signal transduction.relatestoCNGC isoformsotherthanthosethathavebeenThemolecularbasisofplasmamembraneCa2+-perme-analyzed in heterologous systems. A number of reportsable channel activity is only just becomingapparent, andthere a number of intriguing candidate genes. A uniquegeneinArabidopsis,TPC1(At4g03560),encodesachan-Anel with two Shaker-like domains (i.e., 2 × 6 transmem-AArabidopsis Two-Pore Calcium Channcl (TPCI)brane spans,each of which contains a putative“pore"region)connected by a hydrophilic domainthat includestwo EF hands (Figure 3A). The general structure resem-bles that of the pore-forming subunits of mammalian andyeast Ca2+ channels that contain four Shaker-like do-1IICytosolmains, and there is some sequence similarity.TPC1 ex-?COOH Ca?pression enhances Ca2+ uptake in a yeast Ca2+-channelNIL,EF-binding handsmutant (Furuichi et al., 2001). There are indications thatTPC1, which is expressed ubiquitously,forms a depolar-ization-activated channel because overexpression and an-BArabidopsis Cyelie Nucleotide Gated Channel (CNGC)tisense expression appear,respectively,toenhanceandsuppress an increase in [Ca2+l。that occurs as a result ofsugar-induced membrane depolarization.However,firmconclusions regarding voltage gating await electrophysio-logical characterization,and there are as yet no indicationsas to the physiological role(s) of TPC1.CytosCaM.Ca?The Arabidopsis genome also appears to encode noelic Nucleotidefewerthan20membersofacyclicnucleotide-gatedchan-lindingBindingNH,nel (CNGC) family (Maser et al., 2001; http://plantst.sdsc.COOHedu/plantst/htm/1.A.1.shtml).The general structure is showninFigure3B.AShaker-likedomainissupplementedattheCCCArabidopsis Glutamate receptor protein (AtGLR)terminus with overlapping calmodulin and cyclic nucleotidebinding domains (Arazi et al.,2000; Kohler and Neuhaus,2000).MammalianorthologsformtetramericchannelsthatPlasma membrane signalNH,are weakly selective among Group I and Group Il cations.sequenceS1S2 (Gln2)AtCNGC2 has been analyzed electrophysiologically and(GHI)shown to conduct a number of cations, including Ca2+, inresponse to cAMP (Leng et al., 1999, 2002). A tobaccoM2CNGC (NtCBP4) is localized at theplasma membrane andM4when overexpressed confers Pb2+hypersensitivity (Arazi etCylosolal.,1999).Conversely,expressionofaC-terminal truncatedCOOHNtCBP4 confers Pb2+ tolerance, as does disruption of theAtCNGC1 gene (Sunkar et al., 2000). Since Pb2+ has noFigure 3.Topology Models of Putative Plasma Membrane ProteinsInvolved in Calcium Influx in the Cytosol in Arabidopsis.knownphysiological role in plantsbut isknowntopermeatesome Ca2+-permeable channels, these observations are(A) The two-pore channel (TPC1) is composed of two EF calciumconsistent withNtCBP4andAtCNGC1providingarouteforbinding hands, which could be involved in the feedback control ofCa2+ entry across the plasma membrane in planta. Intrigu-the channel activity via cytosolic calcium concentration. The poreloop (P) is localized between the 5th and 6th transmembrane do-ingly, cngc2 mutants of Arabidopsis are defective in themains of each repeat. The 4th transmembrane domain in each re-hypersensitive response that follows pathogen infection, al-peat is enriched in basic residues, which might suggest that thethough these plants nevertheless exhibit gene-for-genechannel is voltage gated.resistance(Cloughetal.,2000),implyingthatAtCNGC2 is(B) CNGC structure also contains a P loop and, unlike counterpartsnot an essential component in defense responses. Rather,in animals, overlapping of the calmodulin and cyclic nucleotide bind-expression analyses of AtCNGC2 support the notion thating domains at the C terminus of the protein.this channel might play a role in senescence or develop-(C) GLR structure is similar to that of animal ionotropic glutamate re-mentally regulated cell death (Kohler et al., 2001). To date,ceptors and is composed of four membrane-localized domains amongthereareno reportsofcyclicnucleotide-activatedchannelwhich M2 is predicted not to span the membrane. Two glutamateactivityinplanta,althoughthepresenceofnonselectivecat-binding domains (GlnH) are localized on the outside of the membrane

S404 The Plant Cell pH (Demidchik and Tester, 2002). It will therefore be impor￾tant to identify these channels at a molecular level before drawing conclusions concerning their specific roles in Ca2- based signal transduction. The molecular basis of plasma membrane Ca2-perme￾able channel activity is only just becoming apparent, and there a number of intriguing candidate genes. A unique gene in Arabidopsis, TPC1 (At4 g03560), encodes a chan￾nel with two Shaker-like domains (i.e., 2  6 transmem￾brane spans, each of which contains a putative “pore” region) connected by a hydrophilic domain that includes two EF hands (Figure 3A). The general structure resem￾bles that of the pore-forming subunits of mammalian and yeast Ca2 channels that contain four Shaker-like do￾mains, and there is some sequence similarity. TPC1 ex￾pression enhances Ca2 uptake in a yeast Ca2-channel mutant (Furuichi et al., 2001). There are indications that TPC1, which is expressed ubiquitously, forms a depolar￾ization-activated channel because overexpression and an￾tisense expression appear, respectively, to enhance and suppress an increase in [Ca2]c that occurs as a result of sugar-induced membrane depolarization. However, firm conclusions regarding voltage gating await electrophysio￾logical characterization, and there are as yet no indications as to the physiological role(s) of TPC1. The Arabidopsis genome also appears to encode no fewer than 20 members of a cyclic nucleotide-gated chan￾nel (CNGC) family (Maser et al., 2001; http://plantst.sdsc. edu/plantst/html/1.A.1.shtml). The general structure is shown in Figure 3B. A Shaker-like domain is supplemented at the C terminus with overlapping calmodulin and cyclic nucleotide binding domains (Arazi et al., 2000; Kohler and Neuhaus, 2000). Mammalian orthologs form tetrameric channels that are weakly selective among Group I and Group II cations. AtCNGC2 has been analyzed electrophysiologically and shown to conduct a number of cations, including Ca2, in response to cAMP (Leng et al., 1999, 2002). A tobacco CNGC (NtCBP4) is localized at the plasma membrane and when overexpressed confers Pb2 hypersensitivity (Arazi et al., 1999). Conversely, expression of a C-terminal truncated NtCBP4 confers Pb2 tolerance, as does disruption of the AtCNGC1 gene (Sunkar et al., 2000). Since Pb2 has no known physiological role in plants but is known to permeate some Ca2-permeable channels, these observations are consistent with NtCBP4 and AtCNGC1 providing a route for Ca2 entry across the plasma membrane in planta. Intrigu￾ingly, cngc2 mutants of Arabidopsis are defective in the hypersensitive response that follows pathogen infection, al￾though these plants nevertheless exhibit gene-for-gene resistance (Clough et al., 2000), implying that AtCNGC2 is not an essential component in defense responses. Rather, expression analyses of AtCNGC2 support the notion that this channel might play a role in senescence or develop￾mentally regulated cell death (Kohler et al., 2001). To date, there are no reports of cyclic nucleotide–activated channel activity in planta, although the presence of nonselective cat￾ion channels that are inhibited by cyclic nucleotides (cAMP and cGMP) has been recorded in Arabidopsis root cells (Maathuis and Sanders, 2001). It is possible that this activity relates to CNGC isoforms other than those that have been analyzed in heterologous systems. A number of reports Figure 3. Topology Models of Putative Plasma Membrane Proteins Involved in Calcium Influx in the Cytosol in Arabidopsis. (A) The two-pore channel (TPC1) is composed of two EF calcium binding hands, which could be involved in the feedback control of the channel activity via cytosolic calcium concentration. The pore loop (P) is localized between the 5th and 6th transmembrane do￾mains of each repeat. The 4th transmembrane domain in each re￾peat is enriched in basic residues, which might suggest that the channel is voltage gated. (B) CNGC structure also contains a P loop and, unlike counterparts in animals, overlapping of the calmodulin and cyclic nucleotide bind￾ing domains at the C terminus of the protein. (C) GLR structure is similar to that of animal ionotropic glutamate re￾ceptors and is composed of four membrane-localized domains among which M2 is predicted not to span the membrane. Two glutamate binding domains (GlnH) are localized on the outside of the membrane.

S405CalciumattheCrossroadsofSignalingofother intracellular Ca2+ stores,notablytheendoplasmichavelinkedcyclicnucleotideswithCa2+signaling(Bowlerreticulum (ER). Voltage-dependent Ca2+-selective channelset al.,1994;Volotovskietal.,1998;Jin andWu,1999;Moutinho et al., 2001), and it is possible that CNGCs pro-have been identified in the ER (Klusener et al., 1995;videanessential linkbetweenthetwomessengersKlusenerandWeiler,1999),andthedemonstrationofhigh-Glutamatereceptors (GLRs)comprisea furtherclass ofaffinity InsPs binding sites on the ER is also suggestive of thepresence of InsP3-gated Ca2+ release channels (Martinec etion channel that might provide acalcium-permeable path-al.,2000).Calcium release assayshave also revealedthewayacrosstheplasmamembrane.Ageneralizedstructureis shown in Figure 3C. In animals, GLRs are neurotransmit-presence of cADPR-mobilizable Ca2+ in ER vesicles (Navazioter gated and form nonselective cation channels in theet al.,2001),and have identified as well a novel and discretepostsynaptic membrane.In Arabidopsis,the GLR familyCa2+releasepathwayactivatedbytheNADP metabolitenico-comprises 30 genes (Lacombe et al., 2001; http://plantst.tinic acid adenine dinucleotide phosphate (NAADP; Navaziosdsc.edu/plantst/htm/1.A.10.shtml). Glutamatetriggers inet al.,2000). Interestingly,NAADP does not effect Ca2+ mo-Arabidopsis roots a large transient elevation in [Ca2+].and abilizationfromvacuolarmembranevesicles.membrane depolarization, both of which are sensitive to theDistinct roles for some of these ligands are emerging.ForInsPg,these roles includetransduction of salt andhyperos-Ca2+antagonistLa3+(DennisonandSpalding,2000).There-sponse is relatively specifictoglutamate.Overexpression ofmotic stress signals (Drobak and Watkins, 2000; DeWald etthe AtGLR2 gene leads to Ca2+ deficiency symptoms andal., 2001) as well as involvement in gravitropism (Perera etotherionicdefectsthatcanbealleviatedbyincreasingex-al.,1999),whileforcADPR,mediationinactivationofplantternal Ca2+ concentration (Kim et al., 2001).Expressiondefense genes seems likely (Durner et al., 1998), as well asanalysis suggests that AtGLR2 might be involved in unload-in ABA signal transduction (Wu et al., 1997; McAinsh anding calcium from the xylem vessels (Kim et al., 2001). It isHetherington, 1998)possible that physiological activation of GLRs involvesThe full significance of this plethora of endomembraneCa2+ releasepathways has yet to be assessed rigorously.openingofnonselectiveanionchannelsattheplasmamem-brane,althoughaplasma membranelocationforplantGLRsHowever,todate,nogenes encodingendomembraneCa2hasyettobedemonstratedreleasechannelshavebeenidentified,anduntiltheencodedchannels are characterized and localized, it is difficult tospeculateonthesignificanceoftheintracellulardistribution.EndomembranesThe large lytic vacuole of mature plant cells is unquestion-CalciumEfflux through H+/Ca2+Antiporters andably the principal intracellular Ca2+ store,and accordingly,aCalciumATPasesnumber of Ca2+ release channels havebeen reported to re-The transport systems that energize efflux from the cytosolsideinthevacuolarmembrane(Sandersetal.,1999).Twoofthesechannelsareligandgatedrespectivelybyinositolprovidethreecriticalhousekeepingfunctions.First,follow-trisphosphate and by cyclic ADP-ribose. Two further chan-ing a calcium release, efflux systems restore [Ca2+]。to rest-ing levels, thereby terminating a Ca2+ signal. Second, theyneltypesaregatedbyvoltage,onebymembranehyperpo-larization and a second by membrane depolarization.Thisload Ca2+intocompartments suchas the ERandvacuoletosecond class of channel is known as the slowly activatingbe used as sources for a regulated Ca2+ release.Third, theyvacuolar(SV)channel inreferencetoitsvoltage-activationsupply Ca2+ to various organelles to support specific bio-chemical functions. For example, high levels of Ca2+ in thekinetics (Hedrich and Neher, 1987), and it is activated byER are required for proper protein processing through therises in [Ca2+Je over the physiological range. This responsepotentiallyendowsthechannelwiththecapacitytocatalyzesecretory pathway (e.g., Durr et al., 1998).Ca2+-induced Ca2+release (CICR)(Ward and Schroeder,A fundamental question is whether, in addition to their1994:Bewellet al.,1999).Although the response to Ca2+housekeepingfunctions,anyoftheseeffluxpathwayshelpalone would not permit CICR in vivo (Pottosin et al., 1997),shape thedynamic form of acalcium spike and therebyhelpthe presence of Mg2+ ions at physiological concentrationsdefine the information encoded in the signal. If efflux is sub-potentiates the Ca2+ response, thereby suggesting a bonaject to regulation, then elucidating the signals that controlfide role in CICR (Pei et al., 1999; Carpaneto et al., 2001). Inthese efflux systems will be equally important in identifyingaddition to regulation by[Ca2+]。and by phosphorylationthe signals that open various calcium channels. Pioneeringstate (Allen and Sanders, 1995), SV channels are also po-work in two nonplant systems (Xenopus oocytes and Dic-tently downregulated by 14-3-3 proteins (van den Wijngaardtyostelium)has demonstrated that increasing the abundanceet al., 2001), implying a central role for SV channels in coor-or activity ofa Ca2+pump can indeed alter signal transduc-dination of signaling events.tion (Camacho and Lechleiter, 1993; Lechleiter et al., 1998;Themerepresenceofa largeintracellularCa2+storedoesRoderick et al., 2000).Thus, the potential signaling impor-not, of course, guarantee that it is mobilized in signalingtance of efflux systems in plants must be seriously consid-events, and recent attention has highlighted the importanceered

Calcium at the Crossroads of Signaling S405 have linked cyclic nucleotides with Ca2 signaling (Bowler et al., 1994; Volotovski et al., 1998; Jin and Wu, 1999; Moutinho et al., 2001), and it is possible that CNGCs pro￾vide an essential link between the two messengers. Glutamate receptors (GLRs) comprise a further class of ion channel that might provide a calcium-permeable path￾way across the plasma membrane. A generalized structure is shown in Figure 3C. In animals, GLRs are neurotransmit￾ter gated and form nonselective cation channels in the postsynaptic membrane. In Arabidopsis, the GLR family comprises 30 genes (Lacombe et al., 2001; http://plantst. sdsc.edu/plantst/html/1.A.10.shtml). Glutamate triggers in Arabidopsis roots a large transient elevation in [Ca2]c and a membrane depolarization, both of which are sensitive to the Ca2 antagonist La3 (Dennison and Spalding, 2000). The re￾sponse is relatively specific to glutamate. Overexpression of the AtGLR2 gene leads to Ca2 deficiency symptoms and other ionic defects that can be alleviated by increasing ex￾ternal Ca2 concentration (Kim et al., 2001). Expression analysis suggests that AtGLR2 might be involved in unload￾ing calcium from the xylem vessels (Kim et al., 2001). It is possible that physiological activation of GLRs involves opening of nonselective anion channels at the plasma mem￾brane, although a plasma membrane location for plant GLRs has yet to be demonstrated. Endomembranes The large lytic vacuole of mature plant cells is unquestion￾ably the principal intracellular Ca2 store, and accordingly, a number of Ca2 release channels have been reported to re￾side in the vacuolar membrane (Sanders et al., 1999). Two of these channels are ligand gated respectively by inositol trisphosphate and by cyclic ADP-ribose. Two further chan￾nel types are gated by voltage, one by membrane hyperpo￾larization and a second by membrane depolarization. This second class of channel is known as the slowly activating vacuolar (SV) channel in reference to its voltage-activation kinetics (Hedrich and Neher, 1987), and it is activated by rises in [Ca2]c over the physiological range. This response potentially endows the channel with the capacity to catalyze Ca2-induced Ca2 release (CICR) (Ward and Schroeder, 1994; Bewell et al., 1999). Although the response to Ca2 alone would not permit CICR in vivo (Pottosin et al., 1997), the presence of Mg2 ions at physiological concentrations potentiates the Ca2 response, thereby suggesting a bona fide role in CICR (Pei et al., 1999; Carpaneto et al., 2001). In addition to regulation by [Ca2]c and by phosphorylation state (Allen and Sanders, 1995), SV channels are also po￾tently downregulated by 14-3-3 proteins (van den Wijngaard et al., 2001), implying a central role for SV channels in coor￾dination of signaling events. The mere presence of a large intracellular Ca2 store does not, of course, guarantee that it is mobilized in signaling events, and recent attention has highlighted the importance of other intracellular Ca2 stores, notably the endoplasmic reticulum (ER). Voltage-dependent Ca2-selective channels have been identified in the ER (Klusener et al., 1995; Klusener and Weiler, 1999), and the demonstration of high￾affinity InsP3 binding sites on the ER is also suggestive of the presence of InsP3-gated Ca2 release channels (Martinec et al., 2000). Calcium release assays have also revealed the presence of cADPR-mobilizable Ca2 in ER vesicles (Navazio et al., 2001), and have identified as well a novel and discrete Ca2 release pathway activated by the NADP metabolite nico￾tinic acid adenine dinucleotide phosphate (NAADP; Navazio et al., 2000). Interestingly, NAADP does not effect Ca2 mo￾bilization from vacuolar membrane vesicles. Distinct roles for some of these ligands are emerging. For InsP3, these roles include transduction of salt and hyperos￾motic stress signals (Drobak and Watkins, 2000; DeWald et al., 2001) as well as involvement in gravitropism (Perera et al., 1999), while for cADPR, mediation in activation of plant defense genes seems likely (Durner et al., 1998), as well as in ABA signal transduction (Wu et al., 1997; McAinsh and Hetherington, 1998). The full significance of this plethora of endomembrane Ca2 release pathways has yet to be assessed rigorously. However, to date, no genes encoding endomembrane Ca2 release channels have been identified, and until the encoded channels are characterized and localized, it is difficult to speculate on the significance of the intracellular distribution. Calcium Efflux through H/Ca2 Antiporters and Calcium ATPases The transport systems that energize efflux from the cytosol provide three critical housekeeping functions. First, follow￾ing a calcium release, efflux systems restore [Ca2]c to rest￾ing levels, thereby terminating a Ca2 signal. Second, they load Ca2 into compartments such as the ER and vacuole to be used as sources for a regulated Ca2 release. Third, they supply Ca2 to various organelles to support specific bio￾chemical functions. For example, high levels of Ca2 in the ER are required for proper protein processing through the secretory pathway (e.g., Durr et al., 1998). A fundamental question is whether, in addition to their housekeeping functions, any of these efflux pathways help shape the dynamic form of a calcium spike and thereby help define the information encoded in the signal. If efflux is sub￾ject to regulation, then elucidating the signals that control these efflux systems will be equally important in identifying the signals that open various calcium channels. Pioneering work in two nonplant systems (Xenopus oocytes and Dic￾tyostelium) has demonstrated that increasing the abundance or activity of a Ca2 pump can indeed alter signal transduc￾tion (Camacho and Lechleiter, 1993; Lechleiter et al., 1998; Roderick et al., 2000). Thus, the potential signaling impor￾tance of efflux systems in plants must be seriously consid￾ered.

S406ThePlant CellH+/Ca2+-antiport activity conducted with oat root vacuolesCalciumExchangers(Schumaker and Sze, 1986).The ionic specificity of CAX1 isH+/Ca2+ antiporters can in principle drive "uphill" transportpartially determined by a 9-amino-acid sequencefollowing theof Ca2+,in which a proton motive force is maintained, al-first predicted transmembrane domain (Shigaki et al., 2001).though in plants this usually requires an H+/Ca2+ stoichiom-In Arabidopsis, there are 12 genes predicted to encodeetry of at least three (Blackford et al., 1990). The first plantantiporters closely related to CAX1 (Maser et al., 2001; http://H+/Ca2+antiportertobeclonedandfunctionallyexpressedplantst.sdsc.edu/plantst/html/2.A.19.shtml).Whetherallofthese related antiporters can transportcalciumhas notbeenwasCAX1(calciumexchanger 1;Hirschietal.,1996;Hirschi,2001)and its projected membrane topology is shown in Fig-determined.CAX2appearstotransportMninadditiontoCaure 4A The gene was identified by its ability to restore(Hirschi, et al., 2000). Although CAX1 is expected to be lo-growth on high-Ca2+ media to a yeast mutant defective incalized to the plant vacuole, there is evidence for H+/Ca2+vacuolarCa2+transport.CAX1appearstotransportCa2+withantiporters in other locations, such as the plasma mem-brane (Kasai and Muto,1990).The subcellular locations ofa low affinity (Km is ~13 μM), consistent with kinetic studies onAArabidopsis Calcium-Proton Exchanger (CAX1)H+Ca2+Auto-COOHInhibitorCytosolNE,B Arabidopsis Autoinhibited Calcium ATPase (ACAtype ATPase)410COOHCa2tCytosolAuto-VInhibitorAsp-PATP-bindingCaM-晨BindingTNH,Figure 4. Topology Models of Systems Catalyzing Ca2+ Efflux from the Cytosol.(A) Ca2+/H+ antiporters. The topology of CAX1 is based on speculation from hydropathy analyses. The number of transmembrane domains pre-dicted varies from eight to eleven. CAX1 has recently been shown to have an N-terminal autoinhibitor. The blue highlight indicates the position ofa 9-amino-acid sequence implicated in providing transport specificity for cations.(B) Ca2+-ATPase (ACA type). The topology of calcium pumps is well-supported by homology modeling based on a crystal structure of a mam-malian sacro(endo)plasmic reticulum-type Ca2+ ATPase pump. ECA-type calcium pumps are predicted to have similar topologies, but lack thedistinguishing feature of an N-terminal auotoinhibitor and calmodulin binding site

S406 The Plant Cell Calcium Exchangers H/Ca2 antiporters can in principle drive “uphill” transport of Ca2, in which a proton motive force is maintained, al￾though in plants this usually requires an H/Ca2 stoichiom￾etry of at least three (Blackford et al., 1990). The first plant H/Ca2 antiporter to be cloned and functionally expressed was CAX1 (calcium exchanger 1; Hirschi et al., 1996; Hirschi, 2001) and its projected membrane topology is shown in Fig￾ure 4A. The gene was identified by its ability to restore growth on high-Ca2 media to a yeast mutant defective in vacuolar Ca2 transport. CAX1 appears to transport Ca2 with a low affinity (Km is 13 M), consistent with kinetic studies on H/Ca2-antiport activity conducted with oat root vacuoles (Schumaker and Sze, 1986). The ionic specificity of CAX1 is partially determined by a 9-amino-acid sequence following the first predicted transmembrane domain (Shigaki et al., 2001). In Arabidopsis, there are 12 genes predicted to encode antiporters closely related to CAX1 (Maser et al., 2001; http:// plantst.sdsc.edu/plantst/html/2.A.19.shtml). Whether all of these related antiporters can transport calcium has not been determined. CAX2 appears to transport Mn in addition to Ca (Hirschi, et al., 2000). Although CAX1 is expected to be lo￾calized to the plant vacuole, there is evidence for H/Ca2 antiporters in other locations, such as the plasma mem￾brane (Kasai and Muto, 1990). The subcellular locations of Figure 4. Topology Models of Systems Catalyzing Ca2 Efflux from the Cytosol. (A) Ca2/ H antiporters. The topology of CAX1 is based on speculation from hydropathy analyses. The number of transmembrane domains pre￾dicted varies from eight to eleven. CAX1 has recently been shown to have an N-terminal autoinhibitor. The blue highlight indicates the position of a 9-amino-acid sequence implicated in providing transport specificity for cations. (B) Ca2-ATPase (ACA type). The topology of calcium pumps is well-supported by homology modeling based on a crystal structure of a mam￾malian sacro(endo)plasmic reticulum-type Ca2 ATPase pump. ECA-type calcium pumps are predicted to have similar topologies, but lack the distinguishing feature of an N-terminal auotoinhibitor and calmodulin binding site.

CalciumattheCrossroadsofSignalingS407all twelve Arabidopsis CAX1-related isoforms still need toACA2 (ER; Liang et al., 1997; Hong, et al., 1999) and ACA4bedetermined(small vacuoles; Geisler et al., 2000b). In addition, evidenceRecently,the activityofCAX1wasshown tobe regulatedbysuggests that ACA1 is located in the plastid inner envelopeanN-terminalautoinhibitor(PittmanandHirschi,2001).Theex-membrane (Huang et al., 1993).Thesubcellularlocationspression of a "deregulated" CAX1 in tobacco resulted in plantshave not been determined for ACA7, 9, 10, 11, 12, and 13.with increased accumulation of Ca (Hirschi, 1999), consistentwiththeincreased activity ofanantiporterthatfunctionstosequester calcium into an endomembrane compartment. In-Functional Overlap?terestingly,the plants displayed growth phenotypes that mim-icked calcium deficiency symptoms. In addition, the plantsGiven the probability of at least 26 calcium pumps and anti-displayed hypersensitivity to K and Mgand increased sensitiv-porters in Arabidopsis, it is likely that multiple efflux systemsity to various stresses, including cold. Thus, aspects of plantlocated in the same membrane system will be found.For ex-development and stress tolerance are dependenton regulationample, in vacuoles, there is evidenceforboth an H+/Ca2+an-of CAX1 activity.An important challenge is to understand howtiporter (such as CAX1p) and an autoinhibited calcium pump,theactivityof CAX1and related antiporters is controlled.suchasACA4.IntheER,thereis evidencefortwo differenttypes of calcium pumps, ECA1andACA2.An importantchal-lenge is to delineate the specific and redundant functions forPumpseach of the different efflux pathways (Harper, 2001)CalciumpumpsbelongtothesuperfamilyofP-typeATPasesthat directly use ATP to drive ion translocation. Two distinctRegulationCa2+pumpfamilieshavebeenproposedonthebasisofpro-tein sequence identities (Geisler et al., 2000a; Axelsen andIt is now clear that transport activities are regulated forPalmgren,2001;http://biobase.dk/~axe/Patbase.html).Mem-CAX1 and most members of the ACA-type calcium pumps,bers of the type lIA and lB families, respectively, include theas indicated by experimental evidence and structural anal-ER-typecalciumATPases (ECAs)andtheautoinhibited cal-ogy.However, in plants, there is no experimental evidencecium ATPases (ACAs). The ACAs are distinguished fromfortheregulationofECAactivity.lntheory;theplantECAECAsbythreebiochemicalfeatures:1)Thepresenceofanpathwaymayprovideaconstitutive"house-keeping"activ-N-terminal autoinhibitor, 2) direct activation through bindingity that simply “cleans up" at a constant rate after any cal-Ca/calmodulin,and3)insensitivitytoinhibitionbycyclopiaz-cium release. Nevertheless, there are two rationales foronic acid and thapsigargin (Figure 4B; Sze et al., 2000). Inter-expecting some kind of regulatory control. First, the mostestingly,a pump showing mixed characteristics of both ECAclosely related ER-type calcium pumps in animal systemsand ACA pumps was identified in maize (Subbaiah and Sachs,are highly regulated. In animals, the activity of the sacro-2000).However,a corresponding"chimeric gene"has not(endo)plasmic reticulum-type Ca2+ ATPase pump is tightlybeen found in the Arabidopsis genome, suggesting that thisregulated by the phosphorylation status of an inhibitory sub-unusual pump is not common to all land plants.unit, phospholamban (East, 2000). In addition, there is evi-In Arabidopsis, there are four ECA- and ten ACA-type cal-dence thattheanimal ER-typepumps canbe regulated by aciumpumps(Axelsen andPalmgren,2001).IsoformECA1feedback system that maintains an appropriate Ca2+ loadappears to be located in the ER, as determined by mem-within the ER lumen (Bhogal and Colyer, 1998; Mogami et al.,brane fractionation and immunodetection (Liang etal.,1997;1998).Second, the observation in plants of theregulation ofHong et al., 1999). However, the potential for other isoformsbothCAX1and ACApathways supportsa speculation thattargetingto non-ER locations mustbe considered.In to-all major efflux pathways in plant cells are carefully regu-mato,there is evidence from membrane fractionation andlated. Assuming that ECAs are regulated, an important chal-immunodetection suggesting that related ER-type calciumlenge is to determine for each specific efflux pathway whetherpumps (LCA1-related) are present in the vacuolar andthe transporter's regulation is as a tuning mechanism toplasma membranes (Ferrol and Bennett, 1996)control the magnitude or duration of a calcium spike (i.e.,aTheACA subgroup ofplant Ca2+ATPases ismost closelysignalingfunction),orasfeedbackcontroltoadjustthedis-related to the plasma membrane-type pumps found in ani-tribution andlevels ofcalcium at thecell surfaceor indiffer-mals. However, they form a distinct subgroup distinguishedent endomembrane compartments (ie., a nutritional function).by two features: 1)a unique structural arrangement with theautoinhibitory domain at the N terminus instead of the C terminus, and 2) representatives that target to membranesDECODINGCALCIUMSIGNALSother than the plasma membrane (i.e., endomembranes).WhileACA8 has been found at the plasma membraneThe initial perception of a calcium signal occurs through the(Bonzaetal.,2000),asexpectedonthebasisoftheanimalbinding of calcium to many different calcium sensors. Sensorsparadigm,endomembrane locationshavebeenidentifiedfor

Calcium at the Crossroads of Signaling S407 all twelve Arabidopsis CAX1–related isoforms still need to be determined. Recently, the activity of CAX1 was shown to be regulated by an N-terminal autoinhibitor (Pittman and Hirschi, 2001). The ex￾pression of a “deregulated” CAX1 in tobacco resulted in plants with increased accumulation of Ca (Hirschi, 1999), consistent with the increased activity of an antiporter that functions to sequester calcium into an endomembrane compartment. In￾terestingly, the plants displayed growth phenotypes that mim￾icked calcium deficiency symptoms. In addition, the plants displayed hypersensitivity to K and Mg and increased sensitiv￾ity to various stresses, including cold. Thus, aspects of plant development and stress tolerance are dependent on regulation of CAX1 activity. An important challenge is to understand how the activity of CAX1 and related antiporters is controlled. Pumps Calcium pumps belong to the superfamily of P-type ATPases that directly use ATP to drive ion translocation. Two distinct Ca2 pump families have been proposed on the basis of pro￾tein sequence identities (Geisler et al., 2000a; Axelsen and Palmgren, 2001; http://biobase.dk/~axe/Patbase.html). Mem￾bers of the type IIA and IIB families, respectively, include the ER-type calcium ATPases (ECAs) and the autoinhibited cal￾cium ATPases (ACAs). The ACAs are distinguished from ECAs by three biochemical features: 1) The presence of an N-terminal autoinhibitor, 2) direct activation through binding Ca/calmodulin, and 3) insensitivity to inhibition by cyclopiaz￾onic acid and thapsigargin (Figure 4B; Sze et al., 2000). Inter￾estingly, a pump showing mixed characteristics of both ECA and ACA pumps was identified in maize (Subbaiah and Sachs, 2000). However, a corresponding “chimeric gene” has not been found in the Arabidopsis genome, suggesting that this unusual pump is not common to all land plants. In Arabidopsis, there are four ECA- and ten ACA-type cal￾cium pumps (Axelsen and Palmgren, 2001). Isoform ECA1 appears to be located in the ER, as determined by mem￾brane fractionation and immunodetection (Liang et al., 1997; Hong et al., 1999). However, the potential for other isoforms targeting to non-ER locations must be considered. In to￾mato, there is evidence from membrane fractionation and immunodetection suggesting that related ER-type calcium pumps (LCA1-related) are present in the vacuolar and plasma membranes (Ferrol and Bennett, 1996). The ACA subgroup of plant Ca2ATPases is most closely related to the plasma membrane–type pumps found in ani￾mals. However, they form a distinct subgroup distinguished by two features: 1) a unique structural arrangement with the autoinhibitory domain at the N terminus instead of the C ter￾minus, and 2) representatives that target to membranes other than the plasma membrane (i.e., endomembranes). While ACA8 has been found at the plasma membrane (Bonza et al., 2000), as expected on the basis of the animal paradigm, endomembrane locations have been identified for ACA2 (ER; Liang et al., 1997; Hong, et al., 1999) and ACA4 (small vacuoles; Geisler et al., 2000b). In addition, evidence suggests that ACA1 is located in the plastid inner envelope membrane (Huang et al., 1993). The subcellular locations have not been determined for ACA7, 9, 10, 11, 12, and 13. Functional Overlap? Given the probability of at least 26 calcium pumps and anti￾porters in Arabidopsis, it is likely that multiple efflux systems located in the same membrane system will be found. For ex￾ample, in vacuoles, there is evidence for both an H/Ca2 an￾tiporter (such as CAX1p) and an autoinhibited calcium pump, such as ACA4. In the ER, there is evidence for two different types of calcium pumps, ECA1 and ACA2. An important chal￾lenge is to delineate the specific and redundant functions for each of the different efflux pathways (Harper, 2001). Regulation It is now clear that transport activities are regulated for CAX1 and most members of the ACA-type calcium pumps, as indicated by experimental evidence and structural anal￾ogy. However, in plants, there is no experimental evidence for the regulation of ECA activity. In theory, the plant ECA pathway may provide a constitutive “house-keeping” activ￾ity that simply “cleans up” at a constant rate after any cal￾cium release. Nevertheless, there are two rationales for expecting some kind of regulatory control. First, the most closely related ER-type calcium pumps in animal systems are highly regulated. In animals, the activity of the sacro- (endo)plasmic reticulum-type Ca2 ATPase pump is tightly regulated by the phosphorylation status of an inhibitory sub￾unit, phospholamban (East, 2000). In addition, there is evi￾dence that the animal ER-type pumps can be regulated by a feedback system that maintains an appropriate Ca2 load within the ER lumen (Bhogal and Colyer, 1998; Mogami et al., 1998). Second, the observation in plants of the regulation of both CAX1 and ACA pathways supports a speculation that all major efflux pathways in plant cells are carefully regu￾lated. Assuming that ECAs are regulated, an important chal￾lenge is to determine for each specific efflux pathway whether the transporter’s regulation is as a tuning mechanism to control the magnitude or duration of a calcium spike (i.e., a signaling function), or as feedback control to adjust the dis￾tribution and levels of calcium at the cell surface or in differ￾ent endomembrane compartments (i.e., a nutritional function). DECODING CALCIUM SIGNALS The initial perception of a calcium signal occurs through the binding of calcium to many different calcium sensors. Sensors

S408ThePlant Celladdition, there are two closely related PPCKs (phospho-can be divided into two types, sensor relays and sensor re-enolpyruvate caboxylasekinases)andPPRKs (PPCK-relatedsponders. Sensor relays, such as calmodulin, undergo acalcium-induced conformational change (sensing)that is re-kinases) that have either no C-terminal domain or one withlayed to an interactingpartner.The interacting partner thenno significant similarityto EF hand-containing proteins.What appearsto bemissingfromArabidopsis arepoten-respondswithsomechangeinitsenzymeactivityorstruc-tial homologs of a CaMk (calmodulin-dependent protein ki-ture(forexample,calmodulinstimulationofanACAcalciumpump activity). This type of sensor includes calmodulin andnase)and proteinkinaseC,which representthe twomajorcalcineurin B-like proteins that are reviewed separately intypes of calcium-regulated kinases that function in animalthis issue by Luan et al. (2002).systems (Satterlee and Sussman, 1998).In addition, an Ara-In contrast,sensor responders undergo a calcium-inducedbidopsishomologhasnotbeenfoundfora calcium/cal-conformational change (sensing) that alters the protein'smodulin-dependent protein kinase, a kinase identified in lillyown activity or structure (e.g.,intramolecularactivation ofa(Patil et al., 1995) and tobacco (Liu et al., 1998) that can beCa2+-dependent protein kinase [CDPK]. By definition,regulated by both calmodulin (analogous to a CaMK)and asensor relays function through bimolecular interactions,visinin-like regulatory domain (analogous toa CDPK)whereassensorrespondersfunctionthroughintramolecularThere is evidence for cytosolic-,nuclear-,cytoskeletal-,interactions. These two different modes of decoding cal-and membrane-associated CDPKs (e.g.,Schaller et al.,1992; Satterlee and Sussman, 1998; Martin and Busconi,ciumsignalsareusedextensivelyinplantstoprovidemanypathways by which calcium can trigger a diverse number of2000;PatharkarandCushman,2000:Romeisetal..2000;Lu and Hrabak, 2002), raising the expectation that CDPKsresponses.couldparticipateinregulatingallaspectsofcellphysiologyWhilenoneof the CDPKs appear tobe integral membraneCalcium-RegulatedKinasesproteins, 24 of the 34 Arabidopsis CDPKs have potentialmyristoylation sites at the beginning of their highly variableKinases represent an important pathway by which calciumN-terminaldomains.Myristoylationandpalmitoylationatthesignals are decoded and propagated downstream intoNterminushavebeen implicated inCDPK-membraneasso-changes in structure and enzyme activity (Harmon et al.,ciations (Ellard-lvey et al., 1999; Martin and Busconi, 2000;2000).In Arabidopsis, the kinases shown to be involvedLuand Hrabak,2002)indecoding calciumsignals all belongto the CDPK/SNF1-ThepotentialfordifferentCDPKs to senseand respondtorelated kinase family (http://plantsP.sdsc.edu). Of the 84 mem-differentcalciumspikesisprovidedbythefunctionaldiver-bers ofthisfamily,59arepredicted toberegulated bycalciumgence observed in the EF handsof different calmodulin-likebased onstructural similarities tobiochemicallycharacter-regulatory domains. Evidence that such differences canizedmembers,andofthese34areCDPKsthatbindcalciuminfluence calcium activation thresholds was provided bydirectly through their calmodulin-like regulatory domainbiochemicalcharacterizationofthreedifferentsoybeaniso-(sensor-responders).The remaining 25 comprise the SNF1-forms (Lee et al.,1998).Each ofthe three isoforms displayedrelated kinase 3subgroup,membersofwhich appear to in-a different calcium thresholdfor half-maximal activation, withteract with a diverse collection of calcineurin B-like sensorthe greatest difference between two isoforms being morerelays and to play a role in a number of signaling processes,than ten-fold (using the same syntide-2 as protein sub-including those related to salt tolerance (Halfter et al.,2000;strate). Interestingly,the protein substrate itself (e.g, histoneLuan et al., 2002).versus syntide-2)was also observed to change the calciumCDPKs are unique owing to the presence of a C-terminalthreshold by more than ten-fold. Furthermore, there is evi-calmodulin-like regulatory domain that functions to coupledence that the calcium stimulation of some CDPKs is poten-the calcium sensor (i.e., calmodulin-like domain) directly totiated by putative lipid messengers (Harper et al., 1993; Binderits "responder"(i.e.,kinase).This group ofkinases was firstetal.,1994;FarmerandChoi,1999),interactionswith14-3-3discovered in plants (Harper et al., 1991; Suen and Choi,(Camoni et al.,1998),and phosphorylation byother kinases1991).While structurally analogous kinases have been(Romeisetal.,2000,2001).Theemergingthemeisthatdif-found in alveolate protists (e.g., Plasmodium; Zhao et al.,ferent CDPKs can be used to sense different calcium sig-1993;ZhangandChoi,2001),CDPKshavenotbeenfoundnals,andthespecificactivityofagivenCDPKwill dependin members of other phylogenetic branches, including fungi,on multiple factors, including its modification by other sig-insects,and humans.naling pathways and its interaction with different down-InArabidopsis,mostCDPKs havefour calcium binding EFstreamtargetshands, but there are several examples of degenerate orInsights into thephysiological roles of CDPKs have cometruncated EF hands (http://plantsP.sdsc.edu). At the ex-from three strategies: 1) identification of potential substrates, 2)treme end of the divergence spectrum, there are eightinplantaexpressionof recombinantCDPKs,and3)inplantaCDPK-relatedkinases(CRKs)that nolongerappeartobesuppression of CDPKactivity.regulated by calcium (Furumoto et al., 1996) due to the highThere is a growing list of potential CDPK substrates identi-degree of divergence in their calmodulin-like domains. Infied through approaches involving in vitro phosphorylation re-

S408 The Plant Cell can be divided into two types, sensor relays and sensor re￾sponders. Sensor relays, such as calmodulin, undergo a calcium-induced conformational change (sensing) that is re￾layed to an interacting partner. The interacting partner then responds with some change in its enzyme activity or struc￾ture (for example, calmodulin stimulation of an ACA calcium pump activity). This type of sensor includes calmodulin and calcineurin B–like proteins that are reviewed separately in this issue by Luan et al. (2002). In contrast, sensor responders undergo a calcium-induced conformational change (sensing) that alters the protein’s own activity or structure (e.g., intramolecular activation of a Ca2-dependent protein kinase [CDPK]). By definition, sensor relays function through bimolecular interactions, whereas sensor responders function through intramolecular interactions. These two different modes of decoding cal￾cium signals are used extensively in plants to provide many pathways by which calcium can trigger a diverse number of responses. Calcium-Regulated Kinases Kinases represent an important pathway by which calcium signals are decoded and propagated downstream into changes in structure and enzyme activity (Harmon et al., 2000). In Arabidopsis, the kinases shown to be involved in decoding calcium signals all belong to the CDPK/SNF1- related kinase family (http://plantsP.sdsc.edu). Of the 84 mem￾bers of this family, 59 are predicted to be regulated by calcium, based on structural similarities to biochemically character￾ized members, and of these 34 are CDPKs that bind calcium directly through their calmodulin-like regulatory domain (sensor-responders). The remaining 25 comprise the SNF1- related kinase 3 subgroup, members of which appear to in￾teract with a diverse collection of calcineurin B-like sensor relays and to play a role in a number of signaling processes, including those related to salt tolerance (Halfter et al., 2000; Luan et al., 2002). CDPKs are unique owing to the presence of a C-terminal calmodulin-like regulatory domain that functions to couple the calcium sensor (i.e., calmodulin-like domain) directly to its “responder” (i.e., kinase). This group of kinases was first discovered in plants (Harper et al., 1991; Suen and Choi, 1991). While structurally analogous kinases have been found in alveolate protists (e.g., Plasmodium; Zhao et al., 1993; Zhang and Choi, 2001), CDPKs have not been found in members of other phylogenetic branches, including fungi, insects, and humans. In Arabidopsis, most CDPKs have four calcium binding EF hands, but there are several examples of degenerate or truncated EF hands (http://plantsP.sdsc.edu). At the ex￾treme end of the divergence spectrum, there are eight CDPK-related kinases (CRKs) that no longer appear to be regulated by calcium (Furumoto et al., 1996) due to the high degree of divergence in their calmodulin-like domains. In addition, there are two closely related PPCKs (phospho￾enolpyruvate caboxylase kinases) and PPRKs (PPCK-related kinases) that have either no C-terminal domain or one with no significant similarity to EF hand–containing proteins. What appears to be missing from Arabidopsis are poten￾tial homologs of a CaMK (calmodulin-dependent protein ki￾nase) and protein kinase C, which represent the two major types of calcium-regulated kinases that function in animal systems (Satterlee and Sussman, 1998). In addition, an Ara￾bidopsis homolog has not been found for a calcium/cal￾modulin-dependent protein kinase, a kinase identified in lilly (Patil et al., 1995) and tobacco (Liu et al., 1998) that can be regulated by both calmodulin (analogous to a CaMK) and a visinin-like regulatory domain (analogous to a CDPK). There is evidence for cytosolic-, nuclear-, cytoskeletal-, and membrane-associated CDPKs (e.g., Schaller et al., 1992; Satterlee and Sussman, 1998; Martin and Busconi, 2000; Patharkar and Cushman, 2000; Romeis et al., 2000; Lu and Hrabak, 2002), raising the expectation that CDPKs could participate in regulating all aspects of cell physiology. While none of the CDPKs appear to be integral membrane proteins, 24 of the 34 Arabidopsis CDPKs have potential myristoylation sites at the beginning of their highly variable N-terminal domains. Myristoylation and palmitoylation at the N terminus have been implicated in CDPK–membrane asso￾ciations (Ellard-Ivey et al., 1999; Martin and Busconi, 2000; Lu and Hrabak, 2002). The potential for different CDPKs to sense and respond to different calcium spikes is provided by the functional diver￾gence observed in the EF hands of different calmodulin-like regulatory domains. Evidence that such differences can influence calcium activation thresholds was provided by biochemical characterization of three different soybean iso￾forms (Lee et al., 1998). Each of the three isoforms displayed a different calcium threshold for half-maximal activation, with the greatest difference between two isoforms being more than ten-fold (using the same syntide-2 as protein sub￾strate). Interestingly, the protein substrate itself (e.g., histone versus syntide-2) was also observed to change the calcium threshold by more than ten-fold. Furthermore, there is evi￾dence that the calcium stimulation of some CDPKs is poten￾tiated by putative lipid messengers (Harper et al., 1993; Binder et al., 1994; Farmer and Choi, 1999), interactions with 14-3-3 (Camoni et al., 1998), and phosphorylation by other kinases (Romeis et al., 2000, 2001). The emerging theme is that dif￾ferent CDPKs can be used to sense different calcium sig￾nals, and the specific activity of a given CDPK will depend on multiple factors, including its modification by other sig￾naling pathways and its interaction with different down￾stream targets. Insights into the physiological roles of CDPKs have come from three strategies: 1) identification of potential substrates, 2) in planta expression of recombinant CDPKs, and 3) in planta suppression of CDPK activity. There is a growing list of potential CDPK substrates identi￾fied through approaches involving in vitro phosphorylation re-

CalciumattheCrossroadsofSignalingS409actions (e.g., Weaver and Roberts, 1992), protein-proteinpling raises the problem of how a single messenger caninteractions (Patharkar and Cushman,2000),and consensusconvey information forspecific responsesto a wide rangeofsite predictions (e.g., Huang et al., 2001). Potential targets in-different stimuli (McAinsh and Hetherington, 1998). For ex-cludeimportantenzymes/proteinsinvolvedincarbonandni-ample,bothauxinandABAcanleadtoelevationofCa2+introgen metabolism, such as nitrate reductase and sucrosestomatal guard cells (Schroeder et al., 2001). However,synthase (Huber et al., 1996; Douglas et al., 1998); stress re-auxin leads to stomatal opening and ABA leads to closure.sponsepathways,includingphenylalanineammonia lyaseThequestionofspecificitybecomesmorecomplexwhen(Chengetal.,2001)andamino-1-cyclopropanecarboxylatefactorssuchasacclimationtopriorstimuliareconsidered.synthase(Huangetal.,2001);ionandwatertransport,suchasForexample,previoushistoryofdroughtorcoldstresscanaquaporins(Weaverand Roberts,1992;Johansson et al.,significantlyaffectthesubsequentCa2+responseofArabi-1996; Huang et al., 2001), calcium pump ACA2 (Hwang et al.,dopsis seedlings, which may involve changes in the relative2000),plasma membrane proton pump (Lino et al., 1998),chlo-contributions ofdifferentcellular Ca2+sources to the overallridechannel (Peietal.,1996),andtheKAT1potassiumchannelCa2+ signal (Knight et al., 1998).(Lietal.,1998;Berkowitzetal.,2000);cytoskeleton,suchasPlantcells also display signaling convergence,wherebyaactin depolymerizing factor (Allwood et al., 2001) and myosinwiderangeof inputscanbe integrated intoa smallernumberlight chain (McCurdy and Harmon, 1992); and transcription,ofoutputs.In contrasttospecificityconvergence involvessuch as a pseudoresponseregulator (Patharkar and Cushman,activation of similardownstream elements in response to dif-2000).ferent stimuli. A good example is stomatal closure in re-Evidencefora potential function of a CDPK came fromsponse to light, CO2, and drought, in which Ca2+ signalsthe expressionof a constitutivelyactiveCDPK in maizepro-appeartoplayakeyrole(Schroederet al.,2001).toplasts (Sheen, 1996). In this case, a constitutively activeversionof isoformAtCPK10, but not isoform CPK1or 11,triggered a signaling pathway,resulting in expression of re-CalciumSignaturesportersfordroughtandcoldstressunderthecontroloftheAlthoughthenumber ofreported stimulus-specific changesABA/stress regulated promoter HVA-1.Todate,therehavebeen no published reports on the stable expression of ain cellular Ca2+ (Ca2+ "signatures") in plants has steadily in-constitutivelyactiveCDPK in transgenic plants.creased (see Evans et al., 2001; Rudd and Franklin Tong,However,aplantphenotype that resulted from overex-2001 for recent reviews), relativelyfew of thesehavebeenpression of a wild-type CDPK has been reported. In thiscategoricallyassigned withspecific downstreamresponses.case,riceplants displayed improved drought, salt, and coldIndeed, certain changes in cellular Ca2+ may reflect pertur-stress tolerance through the overexpression of OsCDPK7bations of Ca2+homeostasis that do not have any specific(Saijo et al., 2000). There is also strong in vivo evidence forfunction (Plieth, 2001). Even where it has been shown thatthe potential role of CDPKs in controlling a pathogen re-Ca2+elevation is essentialfora specific process,asignalingsponse in tobacco (Romeis et al., 2001). In this case, sup-role maynot be clear.In pollen tubes,for example, BAPTApression of NtCDPK2 and NtCDPK3 by viral-induced genebuffer injections that abolish the apical Ca2+ gradient alsolead to growth inhibition (Miller et al., 1992). Moreover, artifi-silencing reduced and delayedthe Cf-9/Avr9-induced hy-persensitive response and blocked an expected wilting phe-cial elevation of Ca2+ at one side of the pollen tube apexleads to reorientation of growth (Malho et al., 1996). How-notype. Together, these in planta studies highlight thepotential to engineer plants with altered CDPKs to improveever, the direct role of Ca2+ as a specific signal in directinggrowthhasbeenquestionedbytheobservationthatoscilla-aplant'sresponsetobioticandabioticstresstions in apical Ca2+ lag behind corresponding growth oscil-lations (Messerli et al., 2000). Although there is compellingevidence for the widespread interaction between Ca2+-ASSIGNINGSPECIFICITYTOCA2+SIGNALSdependent and Ca2+-independent pathways in plant signal-ing (e.g., Jacob et al., 1999; Knight and Knight, 2001), it isConvergenceandSpecificityin Plant Ca2+Signalingalsobecoming clearthat specific patterns of Ca2+elevationalonecangiverisetospecificresponses.Specific responses to qualitatively orquantitatively differentHowever,assigning function to a particular Ca2+signal re-stimulicanbebroughtaboutby arange ofmechanismsquiresmonitoringtheresponsetoastimulusduringinhibi-tion of the Ca2+ signal and imposing the Ca2+ signal in theMostobviously,differentiationgivesrisetocelltypesthatare poised to respond differently by expressing differentabsence of a stimulus. In higher plant cells, this is often diffi-sensing or response elements. Indeed, cell type-specific recult, and specific manipulation of Ca2+ signals to directly af-sponses to drought, salt, and cold have been shown in Ara-fect a downstream response has been successful in onlyabidopsis roots bythe useof cell type-specific enhancer traplimited numberof cell types.In wholeArabidopsis seedlingsresponding to ozone treatment, a biphasic Ca2+ signaturetargetingofaequorin(Kiegleetal.,2000b).Atthesinglecelllevel, the involvement of Ca2+ in stimulus-response cou-was showntocorrelatewith increasedexpression-ofthe

Calcium at the Crossroads of Signaling S409 actions (e.g., Weaver and Roberts, 1992), protein–protein interactions (Patharkar and Cushman, 2000), and consensus site predictions (e.g., Huang et al., 2001). Potential targets in￾clude important enzymes/proteins involved in carbon and ni￾trogen metabolism, such as nitrate reductase and sucrose synthase (Huber et al., 1996; Douglas et al., 1998); stress re￾sponse pathways, including phenylalanine ammonia lyase (Cheng et al., 2001) and amino-1-cyclopropane carboxylate synthase (Huang et al., 2001); ion and water transport, such as aquaporins (Weaver and Roberts, 1992; Johansson et al., 1996; Huang et al., 2001), calcium pump ACA2 (Hwang et al., 2000), plasma membrane proton pump (Lino et al., 1998), chlo￾ride channel (Pei et al., 1996), and the KAT1 potassium channel (Li et al., 1998; Berkowitz et al., 2000); cytoskeleton, such as actin depolymerizing factor (Allwood et al., 2001) and myosin light chain (McCurdy and Harmon, 1992); and transcription, such as a pseudoresponse regulator (Patharkar and Cushman, 2000). Evidence for a potential function of a CDPK came from the expression of a constitutively active CDPK in maize pro￾toplasts (Sheen, 1996). In this case, a constitutively active version of isoform AtCPK10, but not isoform CPK1 or 11, triggered a signaling pathway, resulting in expression of re￾porters for drought and cold stress under the control of the ABA/stress regulated promoter HVA-1. To date, there have been no published reports on the stable expression of a constitutively active CDPK in transgenic plants. However, a plant phenotype that resulted from overex￾pression of a wild-type CDPK has been reported. In this case, rice plants displayed improved drought, salt, and cold stress tolerance through the overexpression of OsCDPK7 (Saijo et al., 2000). There is also strong in vivo evidence for the potential role of CDPKs in controlling a pathogen re￾sponse in tobacco (Romeis et al., 2001). In this case, sup￾pression of NtCDPK2 and NtCDPK3 by viral-induced gene silencing reduced and delayed the Cf-9/Avr9–induced hy￾persensitive response and blocked an expected wilting phe￾notype. Together, these in planta studies highlight the potential to engineer plants with altered CDPKs to improve a plant’s response to biotic and abiotic stress. ASSIGNING SPECIFICITY TO CA2 SIGNALS Convergence and Specificity in Plant Ca2 Signaling Specific responses to qualitatively or quantitatively different stimuli can be brought about by a range of mechanisms. Most obviously, differentiation gives rise to cell types that are poised to respond differently by expressing different sensing or response elements. Indeed, cell type–specific re￾sponses to drought, salt, and cold have been shown in Ara￾bidopsis roots by the use of cell type–specific enhancer trap targeting of aequorin (Kiegle et al., 2000b). At the single cell level, the involvement of Ca2 in stimulus–response cou￾pling raises the problem of how a single messenger can convey information for specific responses to a wide range of different stimuli (McAinsh and Hetherington, 1998). For ex￾ample, both auxin and ABA can lead to elevation of Ca2 in stomatal guard cells (Schroeder et al., 2001). However, auxin leads to stomatal opening and ABA leads to closure. The question of specificity becomes more complex when factors such as acclimation to prior stimuli are considered. For example, previous history of drought or cold stress can significantly affect the subsequent Ca2 response of Arabi￾dopsis seedlings, which may involve changes in the relative contributions of different cellular Ca2 sources to the overall Ca2 signal (Knight et al., 1998). Plant cells also display signaling convergence, whereby a wide range of inputs can be integrated into a smaller number of outputs. In contrast to specificity, convergence involves activation of similar downstream elements in response to dif￾ferent stimuli. A good example is stomatal closure in re￾sponse to light, CO2, and drought, in which Ca2 signals appear to play a key role (Schroeder et al., 2001). Calcium Signatures Although the number of reported stimulus-specific changes in cellular Ca2 (Ca2 “signatures”) in plants has steadily in￾creased (see Evans et al., 2001; Rudd and Franklin Tong, 2001 for recent reviews), relatively few of these have been categorically assigned with specific downstream responses. Indeed, certain changes in cellular Ca2 may reflect pertur￾bations of Ca2 homeostasis that do not have any specific function (Plieth, 2001). Even where it has been shown that Ca2 elevation is essential for a specific process, a signaling role may not be clear. In pollen tubes, for example, BAPTA buffer injections that abolish the apical Ca2 gradient also lead to growth inhibition (Miller et al., 1992). Moreover, artifi￾cial elevation of Ca2 at one side of the pollen tube apex leads to reorientation of growth (Malho et al., 1996). How￾ever, the direct role of Ca2 as a specific signal in directing growth has been questioned by the observation that oscilla￾tions in apical Ca2 lag behind corresponding growth oscil￾lations (Messerli et al., 2000). Although there is compelling evidence for the widespread interaction between Ca2- dependent and Ca2-independent pathways in plant signal￾ing (e.g., Jacob et al., 1999; Knight and Knight, 2001), it is also becoming clear that specific patterns of Ca2 elevation alone can give rise to specific responses. However, assigning function to a particular Ca2 signal re￾quires monitoring the response to a stimulus during inhibi￾tion of the Ca2 signal and imposing the Ca2 signal in the absence of a stimulus. In higher plant cells, this is often diffi￾cult, and specific manipulation of Ca2 signals to directly af￾fect a downstream response has been successful in only a limited number of cell types. In whole Arabidopsis seedlings responding to ozone treatment, a biphasic Ca2 signature was shown to correlate with increased expression of the

S410ThePlant Cellantioxidant enzyme glutathione S-transferase (GST; ClaytonACa2*ImagingCameleon-basedet al., 1999). La3+ pretreatment resulted in abolition of theratiosecond, but not the first, component of the Ca2+ elevation4.0andalso inhibitedGsTgene expression.Althoughthein-volvement of different cell types with different responsekinetics to ozone cannot be ruled out in this response,theresults point to a different function of each phase of theCa2+Ca?+"signature."1.5Sincethe demonstration that stomatal guard cells couldexhibit oscillations or repetitive increases in cytosolic Ca2+BWildtype(McAinsh et al., 1995), significant progress has been madein dissecting their functional significance. A key to the gen-eration of specific cytosolic Ca2+ signals may lie in the exist-StomataAence of multiple Ca2+ entry and efflux pathways,whichClosethemselvesmay be regulated bycytosolic Ca2+ (Harper,2001).In stomatal guardcells,several ofthese componentshavebeen manipulated with consequent effectson theformofstimulus-inducedchangesin[Ca2+lc.InCommelinacom-munis stomatal guard cells, inhibition of Ca2+ elevation bydet3BAPTAbuffer microinjection produced almost complete in-→noahibition of ABA-induced stomatal closure (Webb et al.,2001).Transient Ca2+ elevations can be brought about inguard cells followingactivation of plasmamembraneCa2+channels by hyperpolarization (Grabov and Blatt, 1998)orH,O,treatment(Peietal.,2000).Theeffectsonstomatalgca2aperture of manipulating Ca2+ oscillations by stimulation orinhibition of Ca2+ release by cADPR or InsP (Leckie et al.,1998; Staxen et al., 1999) suggest a clear involvement ofthese messengers but also indicate a degree of redundancyin the signaling pathways. Redundancy in guard cell Ca2+signaling is also evident from work with mutants defective inthe FIERY gene that encodes an inositol phosphatase. Fiery5minTimemutants have elevated levels of InsP3yet show apparentlynormal stomatal regulation,based on measurements ofFigure 5. The Use of Arabidopsis Mutants and New Imaging Tech-transpirational water loss (Xiong et al., 2001).The roles inniques Leads to a Better Understanding of Calcium Signaling.regulationofstomatalapertureofothermolecules,suchas(A) Cameleon indicators expressed in guard cells allowa ratiometricNAADP (Navazio et al.,2000) and inositol hexakisphosphatemeasurement of cytosolic Ca2+ changes (low at left and high at(InsPs) (Lemitri-Chlieh et al., 2000), likely to be involved inright).eliciting [Ca?+lcy elevations remain to be determined(B) Mutants display abnormal Ca2+ signals that fail to trigger sto-Furtherevidenceforthe response-dependenceofspecificmatal closure (Allen et al., 2000, 2001). In wild type (top),a variety ofhormone or stress treatments trigger a series of Ca2+ oscillationspatterns of Ca2+elevationhas comefromtherecentdiscov-that are followed by a prolonged closure of the stomata. In contrast,ery that sphingosine-1-phosphate (S-1-P) induces Ca2+ os-a steady state rise in calcium was observed indet3 mutants (middle)cillations in guard cells (Ng et al., 2001). The frequency andin response to treatments such as high external Ca2+ or oxidativeamplitude of these were dependent on the concentration ofstress (butnotABAand cold).Treatments thatfailedtoproduce nor-applied S-1-P,with correspondingeffectson thekinetics ofmal oscillations also failed to triggeraprolonged stomatal closure.stomatal closure.The stomatal closure response was also disrupted in the mutantOver the past three years, further significant advancesgca2 (bottom). In this case, calcium signals occurred as an abnor-have been made in assigning specificity to Ca2+ "signa-mal"high frequency"patterm of calcium oscillations.These resultstures" in guard cells by the use of ratiometic fluorescentprovide genetic evidence to support the hypothesis that a specificprotein Ca2+ indicators (cameleons)transformed into Ara-frequency of Ca2+ oscillations is required for a normal stomatal clo-sureresponsebidopsis guard cells (Allen et al., 1999). This work hascombined cameleon technology with the use of Ca2+ ho-meostasis or signaling mutants to provide some excitingnew insights.Thus, as shown in Figure 5, det3 mutants thatare defective in a V-type H+-ATPase respond to elevatedexternal Ca2+oroxidative stress withprolonged Ca2+ eleva-

S410 The Plant Cell antioxidant enzyme glutathione S-transferase (GST; Clayton et al., 1999). La3 pretreatment resulted in abolition of the second, but not the first, component of the Ca2 elevation and also inhibited GST gene expression. Although the in￾volvement of different cell types with different response kinetics to ozone cannot be ruled out in this response, the results point to a different function of each phase of the Ca2 “signature.” Since the demonstration that stomatal guard cells could exhibit oscillations or repetitive increases in cytosolic Ca2 (McAinsh et al., 1995), significant progress has been made in dissecting their functional significance. A key to the gen￾eration of specific cytosolic Ca2 signals may lie in the exist￾ence of multiple Ca2 entry and efflux pathways, which themselves may be regulated by cytosolic Ca2 (Harper, 2001). In stomatal guard cells, several of these components have been manipulated with consequent effects on the form of stimulus-induced changes in [Ca2]c. In Commelina com￾munis stomatal guard cells, inhibition of Ca2 elevation by BAPTA buffer microinjection produced almost complete in￾hibition of ABA-induced stomatal closure (Webb et al., 2001). Transient Ca2 elevations can be brought about in guard cells following activation of plasma membrane Ca2 channels by hyperpolarization (Grabov and Blatt, 1998) or H2O2 treatment (Pei et al., 2000). The effects on stomatal aperture of manipulating Ca2 oscillations by stimulation or inhibition of Ca2 release by cADPR or InsP3 (Leckie et al., 1998; Staxen et al., 1999) suggest a clear involvement of these messengers but also indicate a degree of redundancy in the signaling pathways. Redundancy in guard cell Ca2 signaling is also evident from work with mutants defective in the FIERY gene that encodes an inositol phosphatase. Fiery mutants have elevated levels of InsP3 yet show apparently normal stomatal regulation, based on measurements of transpirational water loss (Xiong et al., 2001). The roles in regulation of stomatal aperture of other molecules, such as NAADP (Navazio et al., 2000) and inositol hexakisphosphate (InsP6) (Lemitri-Chlieh et al., 2000), likely to be involved in eliciting [Ca2]cyt elevations remain to be determined. Further evidence for the response-dependence of specific patterns of Ca2 elevation has come from the recent discov￾ery that sphingosine-1-phosphate (S-1-P) induces Ca2 os￾cillations in guard cells (Ng et al., 2001). The frequency and amplitude of these were dependent on the concentration of applied S-1-P, with corresponding effects on the kinetics of stomatal closure. Over the past three years, further significant advances have been made in assigning specificity to Ca2 “signa￾tures” in guard cells by the use of ratiometic fluorescent protein Ca2 indicators (cameleons) transformed into Ara￾bidopsis guard cells (Allen et al., 1999). This work has combined cameleon technology with the use of Ca2 ho￾meostasis or signaling mutants to provide some exciting new insights. Thus, as shown in Figure 5, det3 mutants that are defective in a V-type H-ATPase respond to elevated external Ca2 or oxidative stress with prolonged Ca2 eleva￾Figure 5. The Use of Arabidopsis Mutants and New Imaging Tech￾niques Leads to a Better Understanding of Calcium Signaling. (A) Cameleon indicators expressed in guard cells allow a ratiometric measurement of cytosolic Ca2 changes (low at left and high at right). (B) Mutants display abnormal Ca2 signals that fail to trigger sto￾matal closure (Allen et al., 2000, 2001). In wild type (top), a variety of hormone or stress treatments trigger a series of Ca2 oscillations that are followed by a prolonged closure of the stomata. In contrast, a steady state rise in calcium was observed in det3 mutants (middle) in response to treatments such as high external Ca2 or oxidative stress (but not ABA and cold). Treatments that failed to produce nor￾mal oscillations also failed to trigger a prolonged stomatal closure. The stomatal closure response was also disrupted in the mutant gca2 (bottom). In this case, calcium signals occurred as an abnor￾mal “high frequency” pattern of calcium oscillations. These results provide genetic evidence to support the hypothesis that a specific frequency of Ca2 oscillations is required for a normal stomatal clo￾sure response.