《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.