Calcium. Expo Sic Ion Libia.

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Calcium: A Central Regulator of Plant Growth and Development

Author(s): Peter K. HeplerSource: The Plant Cell, Vol. 17, No. 8 (Aug., 2005), pp. 2142-2155Published by: American Society of Plant BiologistsStable URL: http://www.jstor.org/stable/4130945

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The Plant Cell, Vol. 17, 2142-2155, August 2005, www.plantcell.org ? 2005 American Society of Plant Biologists

HISTORICAL ERSPECTIVEESSAY

Calcium: A Central Regulator of Plant Growth and Development

Today no one questions the assertion thatCa2+ is a crucial regulatorof growth anddevelopment in plants. The myriad pro-cesses in which this ionparticipatesis largeand growingand involvesnearlyallaspectsof plant development (recent reviews inHarper et al., 2004; Hetherington andBrownlee, 2004; Hirschi,2004; Reddy andReddy, 2004; Bothwell and Ng, 2005).Despite this wealth of research, the con-cept of Ca2+ as an intracellular egulator srelativelyrecent and within he professionallifespan of manypeople who are stillactiveand workingon this topic today. The aim ofthis essay is to identify those lines ofthought and research that led to the ideathat Ca2+ is a second messenger in plantcell growth and development. This essaythus focuses primarily n work starting inthe mid sixties and extending to the mideighties. I do not provide an exhaustivereview of the historyof Ca2+ research, nordo I attempt to treat modern aspects ofCa2+ research. However, I do strive toidentify he roots of modern Ca2+ researchand to chart the origin of the currentrevolution.

EARLYSTUDIES ON PLANTCALCIUMCa2+ is an essential element; however, itsrole is elusive. When examiningtotal Ca2+in plants, the concentration is quite large(mM),but its requirement s thatof a micro-nutrient(pLM). a2+ is not usually limitingin field conditions, still there are severaldefects that can be associated with lowlevels of this ion, including poor rootdevelopment, leaf necrosis and curling,blossom end rot, bitter pit, fruitcracking,poor fruit storage, and water soaking(Simon, 1978; White and Broadley, 2003).The underlyingcauses for these effects arenot entirely clear; nevertheless, two areaswithin the cell have been recognized asbeing important targets. First is the cellwall,where Ca2+ plays a key role incross-linkingacidic pectin residues. The second

is the cellular membrane system, wherelow [Ca2 ]e increases the permeabilityofthe plasma membrane. These are brieflydiscussed below.

Ca2+ and the Cell WallSince the 19th century, it has been appre-ciated that Ca2+ plays a crucial role indetermining he structuralrigidityof the cellwall (reviewed in Wyn Jones and Lunt,1967; Burstrom, 1968). During cell wallformation, the acidic pectin residues (e.g.,galacturonic acid) are secreted as methylesters, and only laterdeesterified by pectinmethylesterase, liberating arboxylgroups,which bindCa2+. It ollows that low [Ca2+]eshould make the cell wall more pliableandeasily ruptured, whereas high concentra-tions should rigidify he wall and make itless plastic. Ithad become apparent inthemid to late fifties that modifying he [Ca2+]eproduced a pronounced effect on cellgrowth. Thus, elevating the [Ca2 ]e led toan inhibition n shoot or coleoptile growth,whereas reducing its concentration pro-moted cell and tissue elongation (Bennet-Clark, 1956; Tagawa and Bonner, 1957).Strong support for the Ca2+/pectate in-teraction came from a quantitativeexami-nation of the cation exchange capacity ofthe coleoptile cell wall, which was shownto be due to the number of free pecticcarboxyl groups (Jansen et al., 1960). Stillfurthersupport came from studies usingthe cation chelator EDTA,which had beenemployed to macerate plant tissues with-out destroying the cell structure (Letham,1958). The explanation centered aroundthe idea that EDTA,by chelating Ca2 , ledto a markedweakening or loss of pectatesin the middle lamella, thus removing theagent that cemented cells together.The importance of the Ca2+/pectateinteraction as a regulator of growth en-couraged researchers to include a role forauxin inthisscheme, particularly ecause itwas becoming evident that Ca2+and auxin

had antagonistic actions. Thus, auxin pro-moted shoot growth and inhibited rootgrowth, whereas Ca2+ inhibited shootgrowth and promotedroot growth.Workingwith oat coleoptiles, Bennet-Clark(1956)proposed that there might be a directantagonism between indoleacetic acid(IAA) nd Ca2+. Noting that Ca2+, and thelanthanide praseodymium, inhibited IAAinduced elongation, whereas EDTA re-versed the inhibitoryactivityof Ca2+, andeven promoted growth, Bennet-Clark(1956) suggested that IAAacts as a Ca2+/Mg2+ chelator. This model proposed thatIAAremoves Ca2+ and leads to a loss ofCa2+ pectates, which are replaced bypectate free acids or methyl esters. Thelatter, because they are not cross-linked,would render the wall plastic and able toelongate (Bennet-Clark, 1956). This ideawas challenged by Cleland (1960), whodemonstrated that IAAdoes not enhancethe loss of Ca2+ from the cell wall, nordoesit cause a redistributionof Ca2+ betweenpectin and proto-pectin. Somewhat later,Burling and Jackson (1965) used atomicabsorption spectroscopy to show thatCa2+ accumulated in the cell walls ofelongating coleoptiles and that this accu-mulationwas unaffected by auxin. Furthestudies by Baker and Ray (1965) and Rayand Baker (1965) established the separa-tion in action between Ca2+ and IAAprovidingclear evidence that the inhibitionof cell elongationby Ca2+ does not preventIAA rom stimulating cell wall synthesis. Inthe presence of Ca2+, and thus the inhi-bition of cell enlargement, they demon-strated a general promotion of synthesis ofmatrixpolysaccharides in the presence ofIAA Ray and Baker, 1965).

A compelling interactionbetween Ca2+,the call wall, and cell growth was alsofound in pollen tubes. It was shown in1963 that Ca2+ must be present in themedium to support pollen tube growthin vitro (Brewbaker and Kwack, 1963).Using 45Ca2+,Kwack (1967) showed that


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incorporation occurred exclusively in thepollen tube wall; some of the autoradio-graphic images indicated an enhancedaccumulation of Ca2+ in the apical region.Because the pollen tube cell wall, espe-ciallyat the tip, is composed almost entirelyof pectin, it is reasonable to assume that aCa2+/pectate interaction dominates therequirement or this ion.

Despite the attractiveness of the ideathat cell wall Ca2+ achieves its effectsthrough an interaction with pectates, itmust be recognized that not all resultscan be easily accounted for by this expla-nation (Cleland and Rayle, 1977; Tepferand Taylor, 1981). The failure to show aclose correspondence between the abilityof divalent cations to form a pectic gel withtheir ability to inhibit growth has led toa consideration of other ideas, forexample,a direct affect of Ca2+ on cell wall modify-ingenzymes (Clelandand Rayle, 1977). It simportantto keep in mind that within thecomplex frameworkof carbohydrates andproteins of the cell wall, there could beinteractions between Ca2+ and moleculesother than pectins that could contribute tocell wall structureand extensibility.Never-theless, a Ca2+/pectate interaction cannotbe ignored and deserves attentiontoday asa factor involved in the control of cellgrowth.Ca2+ and Membrane PermeabilityIthas also been known for many years thatCa2+ plays an importantrole in controllingmembrane structure and function (WynJones and Lunt, 1967; Burstrom, 1968). Ageneral idea is that Ca2+, by binding tophospholipids, stabilizes lipid bilayers andthus provides structural ntegrity o cellularmembranes. From a physiological pointof view, a frequent observation has beenthat Ca2+e controls membrane permeabil-ity (Epstein, 1972; Hanson, 1984). Thus,when cells are cultured in solutions oflow [Ca2l]e, especially in the presence ofEDTA, here is leakage of ions and metab-olites (Hanson, 1984). Using roots of soy-bean and maize, Hanson (1960) showedthata low [Ca2+]e aused a marked declinein the ability of these tissues to absorband retain solutes. A [Ca2+]e between

0.1 to 1.0 mM was found to be neces-sary to maintain the integrityand selec-tive iontransportof the plasma membrane.Epstein (1961) examined the competitionbetween different monovalent cations andreported that Ca2+ (0.1 to 1.0 mM), but notMg2+, promoted the uptake of potassiuminthe presence of sodium. Thus, Ca2+e bysome mechanism, impartsselectivity to theion transportprocess. In anotherexample,Van Steveninck (1965) found that low[Ca2+]e promoted a release of potassiumin cultured beet root tissues, which wascompletely reversed by adding back Ca2 ,but not Mg2+. Pollen tubes also showedchanges inpermeability n response to low[Ca2+]e,including a significant release ofcarbohydratesinto the medium(Dickinson,1967).

In a series of studies on leaf abscissionand tissue senescence, Poovaiah andLeopold (1973a, 1973b, 1976) reportedthat Ca2+ inhibited or slowed these pro-cesses. Recognizing that Ca2 , throughcross-linking pectates and cementing cellwalls, will directly retard abscission, theynoted that several other processes werealso affected. Duringsenescence in maizeand rumex leaf disks, they showed thatCa2+ retarded the loss of chlorophyll,theloss of protein,and the loss of free space,suggesting that the ion plays a regulatoryrole in maintaining and controlling mem-branestructureand function(Poovaiah andLeopold, 1973b).

Earlyultrastructuraltudies echoed thisrefrain. Thus, marked differences weredetected at the electron microscope levelin the membranes of barley shoot apicescultured in low [Ca2+le relative to thecontrols (Marinos, 1962). The low Ca2+-induced effect was apparent as relativelygross discontinuities in the nuclear enve-lope, plasma membrane, and tonoplast,and later nthe mitochondria.It s difficult oimagine that such lesions occur in theintact cell because they would immediatelylead to cell death. However, they may in-dicate reduced membrane stability,whichleads to breakage and discontinuitiesduringthe permanganate fixationprocess.For that reason, the details of this reportmust be treated with caution; neverthe-less, the differences observed suggest

that membranes cultured in low Ca2+e be-come structurallyweakened.If low Ca2+e makes the membrane morepermeable, it should follow that elevated

concentrations make the membrane lesspermeable. Using Ca2+ itselfas the probe,Robinson (1977) showed this to be true inzygotes of the alga Pelvetia. Thus, anincrease in the [Ca2+Ie from only 1 to3 mM reduced the influx of this ion by>10-fold. These results seem counterintuitive and are not well appreciated. Ex-amples certainlyexist inwhich it is evidentthat an increase in the [Ca2+]e causesa corresponding increase in the [Ca2+],(Gilroy et al., 1986), and an extracellulaCa2+sensor recentlyhas been identified nguard cells (Han et al., 2003). However,this situation does not automatically ex-tend to all cell types, as the study byRobinson (1977) shows. Inagreement withthe studies on Pelvetia, we find that in-creasing the [Ca2+le to 10 mM inhibitslilypollen tube elongation and causes thetip-focused gradient to drop to basallevels (D.A. Callaham and P.K. Hepler,unpublisheddata). Thus, in experiments inwhichthe [Ca2+]es modulated, he assump-tion cannot be made that similarchangesoccur on the cytosol. Rather,an increase in[Ca2+]e may generate a decrease in [Ca2+],.Briefly ummarizing,earlystudies on therole of Ca2+ in plants focused on the cellwalland on membranepermeability.At thattime, there was no widespread apprecia-tion that the [Ca2+], might be very low andthat this ion might be acting as a regulatorof cytoplasmic processes. Botanists ex-ploring Ca2+ effects in the concentrationrange between 0.1 and 100 mM wereunlikelyto see changes at the submicro-molarlevel. The concept of Ca2+as a reg-ulator nitially erives fromstudies of animalcells and only later in studies of plantcells. To see how this concept arose, I willfocus briefly on Ca2+ in animal cell phys-iology, giving attention to the process ofmuscle contraction.CALCIUMAND MUSCLECONTRACTIONMore than 120 years ago, Ringer (1883)showed that the repetitive beating of an


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isolated frog heartwas sensitive to different[Ca2+]for eview, ee Carafolit al., 2001).When cultured in distilled water, the heartsfailed to exhibit the proper contraction;however, when cultured in Londoncity tapwater, they exhibited repetitive contrac-tions. Using sequential ion addition to thedistilled water, Ringer (1883) discoveredthatCa2+ was the key factorthatsupportedcontraction.Despite these earlystudies, theidea that Ca2+ was a regulatorof musclecontraction did not expand at this point.Only considerably laterthroughthe effortsof Heilbrunn1940)was the emphasis againfocused on Ca2+. Heilbrunn1940)showedthat muscle contraction could be stimu-lated through the injection of Ca2+ into thefrog muscle fiber. Of note, the contractioncould take place even when the Ca2+solution was highlydiluted. Equally mpor-tant was the observation that musclecontraction was not supported by injec-tion of other importantphysiological ions,includingsodium, potassium, or Mg2+.Be-cause potassium at that time was consid-ered crucial,the additionalobservationthatmassive doses of potassium were ineffec-tive furtheremphasized the primaryrole ofCa2+ in stimulating contraction (Heilbrunnand Wiercinski,1947).

As insightful and penetrating as thesestudies were, Heilbrunn and Wiercinski(1947) were not able to establish the actual[Ca2+]i in the resting muscle fiber. Indeed,determining the [Ca2+]ihas been difficultfor any cell type. Hodgkin and Keynes(1957), using 45Ca2+ to examine the mobil-ityof this ion insquid axoplasm, made twoimportantobservations: first, that the mo-bilityof Ca2+ is extremely low; second, thatthe bulk of the Ca2+ is bound, with only10 FM or less being free and ionized.Further work that established the true[Ca2+]idepended on two technical devel-opments. The first was the application ofcation chelators EDTAand EGTA n phys-iological studies to carefully control the[Ca2+] (Bozler, 1954). Before the availabilityof effective chelators, it was nearly impos-sible to construct solutions in the submi-cromolarrange because of the presence ofCa2+ as a contaminant, or leaching fromglassware. Whereas EDTA has a highaffinityfor Ca2 , it also has a substantial

affinity or Mg2+. WithEGTA,he affinity orCa2+ is not as high as with EDTA,but therelative insensitivity of EGTA to Mg2+means that it is a more efficacious chelatorfor constructing solutions that are specifi-cally buffered for Ca2+. The second impor-tant development was the isolation andcharacterization of the photoprotein ae-quorin, a Ca2+ sensitive, bioluminescentprotein from the jelly fish Aequoria,whichprovideda means fordetecting changes inthe [Ca2+] in the submicromolar range(Shimomura et al., 1963). At resting[Ca2+]1,he protein generates only a faintglow; Shimomura et al. (1963) initiallyde-termined hatthe resting concentration wasbetween 0.1 and 1.0 ,uM.However, as the[Ca2+]i ncreases, there is an exponential(2.3 power) increase in the amount of lightgenerated, making aequorin a suitable re-agent for detecting regions of elevated ionconcentration or amplitudemodulation.

Despite the favorable properties of ae-quorinas an indicatorof the [Ca2+], in livingcells, there were substantialproblems in itsuse. First was the need to introduce theprotein into cells, and second was thedifficultyof detecting and imaging a ratherweak signal. The first problem was solvedusing large cells, which are easy to inject.Of course, more recently, using modernmolecularbiological methods, it is possibleto transfect cells with the aequorin geneand express the proteininvirtually ny cell(Knight et al., 1991), and even withinorganelles (Rizzutoet al., 1994). The prob-lems associated with the detection andimagingof the aequorin signal remain withus today. Although detection of a signalwithout imaging can be done effectivelywith photomultiplier ubes, imaging, espe-cially from single small cells is difficultdue to a low number of Ca2+-dependentphotons. Progress has been made in thedevelopment of extremelysensitive photonimaging equipment, which has permittedthe visualization of these weak signals(Gilkeyet al., 1978; Knightet al., 1993).The determination of the [Ca2+], in livingmuscle cells was performed by studies thatinvolveboth of these technologies. In1964,Portzehl and coworkers used EGTA toproduce carefully buffered Ca2+ solutionsand showed that contractionin an isolated

muscle fiber of the crab Maia squindooccurred between 0.3 and 1.5 FLM. fewyears later in 1967, Ridgway and Ashleyinjected the giant muscle of the acornbarnacle with aequorin. Within1 ms afterelectricalstimulation, hey recorded a sharpincrease in light, indicatingthat the [Ca2+],had risen (Figure 1). This was followed in5 ms by an increase in muscle tension.Although the results were not strictlyquantitative, Ridgway and Ashley (1967)argued, based on the work of Shimomuraet al. (1963), that at rest the [Ca2+], wouldbe between 0.1 and 1.0 1iM;therefore,upon stimulationit would be substantiallyhigher.These studies are dramaticand com-pelling; they clearly demonstrate that thestimulated depolarizationof the membranepotential is followed almost immediatelyby an abrupt increase in bioluminescence(i.e., [Ca2+]i) nd with only a furtherslightlag by the generation of tension (Ridgwayand Ashley, 1967). These studies were thefirst direct demonstration of Ca2+ ampli-tude modulation.Ca2+ AMPLITUDEMODULATION NNONMUSCLECELLSDuring the next decade, in studies ofseveral differentnonmuscle systems, bothEGTA nd aequorinwere used to show thatthe basal [Ca2+], was submicromolarandthat through stimulation elevations of the[Ca2+], could be elicited. For example,activation of the freshwater protozoans,Spirostomum (Ettienne, 1970), cell cleav-age in Xenopus (Bakerand Warner,1972),response of the photoreceptor of Limuluto light (Brown and Blinks, 1974), oscilla-tions in cytoplasmic streaming in the plas-modial slime mold, Physarum (Ridgwayand Durham, 1976), and egg activation inthe medaka fish, Oryzias atipes (Ridgwayet al., 1977), and sea urchin, Lytechinuspictus (Steinhardtet al., 1977) were shownto be anticipated by an increase in the[Ca2+]i.The examples of egg activation areespecially efficacious in establishing a pri-maryrole forCa2+ amplitudemodulation ndevelopment. Whereas Ridgway et al.(1977) employed eggs from medaka, afresh water fish, Steinhardt and coworkers(1977) used eggs froma marine sea urchin


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HISTORICAL PERSPECTIVE ESSA

Figure 1. A [Ca2+], Increase Precedes Muscle Contraction.After an electrical stimulus, the giant muscle of the acorn barnacle, which had been injected withaeqourin,exhibits an abruptrise in the [Ca2+]i (bottom trace). Soon thereafter,an increase in muscletension begins (top trace), which continues even though the Ca2+jquickly returns to basal level. TheCa2+-dependent lightemission from aequorinis measured witha photomultiplierube. Bar= 20 ms.(Figurecourtesy of Ridgway and Ashley, 1967, Figure la, withpermission of Elsevier.)In both instances, the eggs had beeninjected with aequorin, and in both exam-ples, clear documentation of a [Ca2+], in-crease was noted after fertilization.In anextension of the studies on medaka eggs,Gilkeyet al. (1978), using sensitive imag-ing equipment, were able to observe thespatial and temporal dynamics of the Ca2+-dependent light emission. Their resultsreveal that the [Ca2+]irises at the pointof sperm entry (the micropyle), reaching-30 ,uIIM,nd propagates as a wave thattravels at the rate of 12 ,u,m/s hrough thecortex of the egg. By the late seventies,therefore, ithad been established inseveralcell types that the basal [Ca2+], is -0.1 ,uMand, importantly, hat a varietyof differentevents can be activated through a changeor amplitude modulation of the [Ca2+], upto 1 pM or higher.

Ca2+ AMPLITUDEMODULATIONIN PLANTSAlthough plants do not possess musclesas such, it can be viewed as an interestingexample of parallelism that our under-standing of Ca2+ regulation in plant cellsin part originated from studies on thecontrol actomyosin incytoplasmic stream-

ing. In the sixties, it had been recognizedthat the action potential in large internodecells of the Characean algae would inducea very rapid but reversible inhibitionof cytoplasmic streaming (Barry, 1968;Tazawa and Kishimoto, 1968). Tazawaand Kishimoto (1968) showed that it wasnot the formationof a gel or the coagulationof the cytoplasm that led to streamingcessation but rather an inhibition of thedriving force. Realizing that there weresubstantial ion changes during the actionpotential, they focused primarilyon chlo-ride and potassium but nevertheless sug-gested that Ca2+ mightalso be involved.Atthe same time, Barry (1968), workingwithNitella and using ion replacements, pro-vided clear evidence that the presence ofCa2+, but not Mg2+, in the extracellularmedium caused the cessation of streamingduring the action potential. These studiesfurther emphasized that it was not theaction potential per se that led to streaminginhibitionbut rather he presumed influxofCa2+. Barry (1968) also directed attentionto the actomyosin system as the focus forCa2+ activity. Furtherwork, involving theperfusion of the large internode cells ofNitella and Chara, produced a system thatcould be readily manipulated experimen-tally. Williamson (1975) established that

streaming, in addition to requiring Awas dependent on a very low [Ca(0.1 ,uM). f he concentration was elevato 1.0 ,uM, there was a decreasecytoplasmic streaming by 20%, and if[Ca2+], was increased to 10 ,uM, streaming would be inhibitedby a >80Similar results reported by Tazawa et(1976) further emphasized the conclusthat elevated [Ca2+1]inhibited cytoplasmstreaming. At the time these studies wpublished, they may not have enjoywidespread acknowledgment becauthere were questions whether findifrom the Characean algae were relevto equivalent processes in higher planThe subsequent studies on Vallisndispelled this concern, showing that cyplasmic streaming, as in Nitellaand Chawas regulated by the [Ca2+] (Yamaguand Nagai, 1981; Takagiand Nagai, 198Today, it is widely recognized for nflowering and flowering plants alike tlow [Ca2+], (0.1 ,uM) permits streamiwhereas elevated [Ca2+], (1.0 ,uM) nhithe process.The major breakthrough hat establishthe relationshipbetween the action pottial, Ca2+, and the inhibition of streaing came from the pioneering studies Williamson and Ashley (1982). Usingternode cells of Nitella and Chara, iwhich the photoprotein aequorin had bemicroinjected, they showed that the actpotential elicited an abrupt rise in[Ca2+], (Figure 2) together with a paradecrease in cytoplasmic streaming. Tsystem also showed impressive recovwith a relatively rapid return to ba[Ca2+],, followed by a resumption in cyplasmic streaming. Williamson and Ash(1982) further established that the ba[Ca2+], in Chara was -0.1 ,uM,whereasNitella, twas 0.4 ,uM.Whenstimulated,[Ca2+], inChararose to 6.7 ,uIM, hereasNitella, it rose to 43 ,uM.A closely folloing study by Kikuyamaand Tazawa (19provided results in agreement wWilliamsonand Ashley (1982), firmlyestalishing the change of [Ca2+]iduringaction potential in Nitellaand Chara. Thstudies were the first and for several yeremained the most convincing exampleCa2+ amplitudemodulation in plants.


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-200 a(mV)

-100)

0*

O1

~'8

"~4

0L2s

Figure 2. The Action Potentialin CharaElicits a [Ca2+], Increase.A Charainternodecell, which had been injectedwithaequorin,is stimulated electrically o induce anaction potential (top trace). Following closely is a sharp increase in the photomultipliercurrentindicatingCa2+-dependent lightemission fromaequorin(bottom trace). Bar= 2 s. (Figurecourtesy ofWilliamson nd Ashley, 1982, Figure 2a, with permission of NaturePublishing Group.)

After these pioneering studies on Nitellaand Chara, there have been additionalstudies in plants showing that the basal[Ca2+], is low and that increases can occurfollowing different stimuli. Gilroy et al.(1986) used the permeant acetoxy methyl-ester of quin2 to show that the [Ca2+], inmung bean root protoplasts was 171 nM.This study is importantbecause it was thefirst o use a fluorescent indicator.Althoughquin2 is no longer used, the second-generation fluorescent dyes developed byR.Y. Tsien and colleagues, for examplefura-2and indo-1 (Grynkiewicz t al., 1985),and especially in their dextranated forms,have proved extremely effective in allowingus to assay [Ca2+], in plants. Other meth-ods have also provided compelling results.For example, Miller and Sanders (1987),using a Ca2+ selective intracellularmicro-electrode, found that the alga Nitellopsishad a basal [Ca2+], of 400 nM in the dark.However,when culturedinlight,the [Ca2+],dropped to 150 nM. The interpretationput

forthwas that the process of photosynthe-sis, together with ion uptake by chloro-plasts, caused the reduction of the [Ca21],.Also using Ca2+ selective microelectrodes,Felle (1988) showed that auxin inducedCa2+ oscillations in maize coleoptiles.Here, the basal [Ca2+], was 119 nM, whichin the presence of auxin rose in an os-cillatory fashion to 300 nM. Yet anotherexample was the induction of stomatalclosure by ABA, which was shown to beaccompanied by an increase in the [Ca2+],to 600 nM in Commelina guard cells thathad been injected with the fluorescentindicator dye fura-2 (McAinshet al., 1990).Note is also made of the dramatic tip-focused Ca2+ gradient observed in pollentubes (Obermeyer and Weisenseel, 1991;Rathore et al., 1991; Milleret al., 1992), aresult that was anticipatedgiven the earlierdemonstration of 45Ca2+ influx in thesecells (Jaffe et al., 1975). However, the fluo-rescent dyes allowed direct visualizationoffree Ca2+. Also, the use of fura-2 covalently

linked o a 10-kDdextranprovideda meansfor avoiding dye sequestration (e.g., intovacuoles) and for permitting long termrecording of the [Ca2+], (Miller et al.,1992). Finally, n a dramatic developmentthat fused molecularmethods to Ca2+ cellbiology, Knightet al. (1991) introducedtheaequorin gene into tobacco plants andwere able to show that different agents,including touch, cold shock, and fungalelicitors, induced Ca2+ stimulated lumines-cence. Suffice it to say that by the lateeighties and early nineties several studies,using different techniques, had docu-mented a low basal [Ca2+]iand demon-strated amplitude modulation nplantcells.CONCEPTOF Ca2+ AS A REGULATORThe studies discussed above make itabundantly clear that the [Ca2+], in plantcells, as in animal cells, is low and thatplantsare able to respond to various stimuliby eliciting a change in the [Ca2+]i.How-ever, just as a professional orchestra doesnot need the oboist to sound them theappropriate A, neither did the plant biolo-gists need these data to suggest that Ca2+was a potential signal transducer. By theearlyto midseventies, the ideas were in theair, and thus well before the actual docu-mentation of the [Ca2+]1,many scientistsworking on different aspects of plantgrowth and development were coming torecognize the potential importance of Ca2+as an intracellularignaling agent. Althoughthere were probably several paths thatwere responsible for focusing attentionon the regulatoryfunction of Ca2+, I willmention a few lines of thought and researchthat I thinkwere importantin shaping theideas of plant biologists.Ca2+ and Cyclic AMP:The Discoveryof Calmodulin and Calcium-DependentProtein KinasesIn he late fifties,Sutherlandand Rall 1958)discovered that adenosine 3',5'-mono-phosphate (cyclic AMP) levels increasedin liver tissues in response to epinephrineand furthermore hat this small nucleotidewas implicatedas a second messenger in awide varietyof cellular reactions frequently


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involved in the phosphorylationof proteins(reviewed in Rasmussen, 1970). It soonbecame apparent that Ca2+ was also in-volved in many of these reactions, whereit was recognized that a stimulus thatcaused an increase in cyclic AMP alsogenerated an increase in Ca2+ ion uptake.The confluence of the activities of thesetwo agents led Rasmussen (1970) tospeculate that, "The basic elements inthis widespread biochemical control mech-anism are: calcium ions, adenosine 3',5'-monophosphate (cyclic AMP), intracel-lular microtubules, microfilaments, secre-tory vesicles, and a class of enzymesknownas protein kinases which phosphor-ylate specific proteins with adenosine tri-phosphate (ATP) as substrate." I canclearly remember reading this article(Rasmussen, 1970) and being struck byits bold vision. At the time it was notpossible to make a case for the participa-tion of cyclic AMP in plant development(reviewed in Trewavas, 1976), but all theother components could be recognized aspossible contributors, including notablyCa2 , the cytoskeleton, directed secretion,and proteinkinases.

The continuingstudies indifferentanimalsystems on the regulationof cyclic AMP edto discovery of calmodulin. In1970, it wasreported that 3',5' nucleotide phosphodi-esterase (PDE), he enzyme that degradescyclic AMP to 5'-AMP, was in part regu-lated by a heat stable protein (Cheung,1970; Kakiuchi nd Yamazaki, 1970), whichitself did not show enzymatic activity. Animportant urtherobservation revealed thatPDE was controlled by Ca2+ with basalrates under 1 kLMnd maximalactivation at20 ,uM (Kakiuchi and Yamazaki, 1970).Subsequent biochemical investigationsestablished that the heat stable factorwas a protein that was dependent onCa2+, with half-maximal activation at2.3 pLM Teo and Wang, 1973). Initially,this was called the "calcium-dependentregulator," a name that was changed to"calmodulin"(Cheung, 1980). It was alsosoon realized that calmodulin was ex-tremely common, being found in virtuallyall tissues that were tested, and that itwasvery similar o troponinC, the Ca2+ switchfor striated muscle (Cheung, 1980).

The discovery of calmodulin did notescape the attention of the plant biolo-gists. Muto and Miyachi (1977) firstshowed that NAD kinase isolated frompea seedlings required an activator pro-tein, which was sensitive to acid and alkaliconditions, and also heat stable. Theseproperties, together with its relativelylowmolecular mass (28 kD), led Muto andMiyachi (1977) to draw a tentative con-nection of the protein they had identifiedwith the PDE activator in animalsystems.However, it was Anderson and Cormier(1978), using appropriatemetal chelators,who firstdiscovered the Ca2+ requirement fthe NADkinase activator protein in plants.Their results firmly established the closerelationship between it and the Ca2+-dependent regulatorprotein or calmodulin.It soon became apparent that calmodulinwas widelypresent nplants Watterson t al.,1980).These resultstrulymade the case forCa2+ as an intracellularegulatorof plantprocesses.

Continued investigations by many re-searchers revealed the existence of differ-ent kinases, inaddition to NADkinase, thatwere Ca2+/calmodulindependent. For ex-ample, Ca2+/calmodulin regulation wasdemonstrated for an unspecified proteinkinase (Polya and Davies, 1982) and forplant quinate:NAD+ 3-oxidoreductase(Ranjevaet al., 1983). Of particularnterestand excitement was the discovery at thistime of kinases that were Ca2+dependentbut calmodulin independent (Hetheringtonand Trewavas, 1982). This line of researchled eventuallyto the discovery of calcium-dependent protein kinases (CDPKs)(Putnam-Evanset al., 1986; Harmonet al.,1987), which are now recognized as mem-bers of a large familyof kinases that doesnot exist inanimals. Inanimals, there are nokinases that are directlyregulated by Ca2+;rather,theirCa2+-stimulated kinases workthrough a relay system, often involvingcalmodulin (forreview, see Carafoliet al.,2001). By contrast, plants have the cal-modulinpathway(forreview, see Sneddenand Fromm, 2001; Zhang and Lu, 2003)and a separate system involving theCDPKs (for review, see Harmon et al.,2001; Cheng et al., 2002; Harper et al.,2004); this marks a significantand unique

difference in the mechanism of Ca2+regulationbetween plantand animalcells.Ca2+ and Cell DivisionFor me, there was a distinct awakeningin the early seventies about Ca2+ as anintracellularregulator. First, the article byRasmussen (1970), mentioned above, wasenormously stimulating and provideda broad sweep about a role for Ca2+ inmany different signal transduction pro-cesses, perhaps especially including arelationshipbetween Ca2+ and the cyto-skeleton. But the trulydefiningmoment oc-curredwiththe publicationby Weisenberg(1972), which showed that a low [Ca2+](


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and itwas well known that contraction wasexquisitely attuned to the [Ca2+], fromresting (0.1 FLM)o those that activatedcontraction (0.3 to 1.5 jIM). In addition,studies on the sarcoplasmic reticulum romstriated muscle had led to the isolationandpartial characterizationof the Ca2+ pump(MacLennanand Wong, 1971).

Putting these lines of inquiry togetherswitched on the proverbial ight nmy mind.Itseemed plausible that microtubules n themitotic apparatus might be controlled bythe local [Ca2+],, which in turn would beregulated by the nearby endoplasmic re-ticulum (Figure3) (Hepleret al., 1981). Thisgeneral idea, which Barry Palevitz and Iarticulatedin our review on the cytoskele-ton (Hepler and Palevitz, 1974), guidedresearch in my laboratory or several yearsthereafter. Spindle-associated endoplas-mic reticulumwas found to contain depos-its of Ca2+ (Wickand Hepler, 1980; Wolniaket al., 1983), and spindle microtubuleswereshown to be sensitive to elevations in[Ca2+], with depolymerization occurringwhen the concentration was raised to1.0 FLMor more (Zhang et al., 1992).Restrictionof the [Ca2+],was seen to affectprogress through mitosis (Hepler, 1985),whereas stimulation of Ca2+ entry wasfound to promote bud initialformation inmosses (Saunders and Hepler, 1982) and

red light-stimulated spore development inferns (Wayneand Hepler, 1984). Neverthe-less, evidence for the occurrence of Ca2+amplitude modulationduringdivision wasand still is decidedly mixed (Hepler,1989);indeed, a compelling example of Ca2+amplitude modulationin plant cell divisionhas not been established.

Ca2+ and Polarized Cell GrowthAnarea of endeavor that drew early interesttoward Ca2+ concerned the regulation ofpolarized plant cell development. Throughthe pioneeringeffortsof Jaffe and coworkers(Jaffeet al., 1974; Weisenseel et al., 1975), itwas discovered that polarized cells (e.g.,Fucus zygotes, pollen tubes, and root hairs)drove substantial on currents hrough hem-selves. The bulkof the ioncurrentappearedto consist of a polarized nfluxof potassium,focused at the growingpoint (i.e., the rhizoidof Fucus or the growing tip of the pollentube). However, t was soon appreciated hatCa2+ influxconstituted a small but potenti-ally mportant omponent of the totalcurrent(Jaffeet al., 1975;RobinsonandJaffe, 1975;Weisenseel and Jaffe, 1976). Robinson andJaffe (1975), using Pelvetia eggs that werepolarized through unilateral illumination,showed that approximately ive times more45Ca2+ entered the shaded rhizoidpole than

entered the illuminated halluspole. A sub-sequent study in which the rhizoids grewtowardan experimentally mposed gradienof the Ca2+ionophore, A-23187, addedstrong support for the idea of localizedCa2+ influx as a regulator of polarizeddevelopment(Robinsonand Cone, 1980).Studies on growing pollen tubes alsoprovided persuasive support for localizedion fluxes and for a specific role forCa2+ inthe regulationof polarizedgrowth.Applica-tion of the vibrating electrode revealedstrong currentsinwhich an influxof potas-sium at the apex appeared to be balancedby an outward fluxof protons inthe regionof the grain (Weisenseel et al., 1975;Weisenseel and Jaffe, 1976). ImportantlyCa2+, as noted earlier (Brewbaker andKwack,1963),was essential fortube growth(Weisenseel and Jaffe, 1976). Additionallyautoradiographyrevealed an accumulationof 45Ca2+ n the apical domain, providingsupport forthe idea that ion flow into the tipcreated an intracellularradient(Jaffeet al.,1975). A certain amount of the autoradio-graphic signal observed by Jaffe et al.(1975) may be due to Ca2+ binding in thecell wallas noted by Kwack(1967). Never-theless, when considered with the otherstudies on current flow, these data areconsistent with a small but significant nfluxof Ca2+ across the plasma membrane.

Ca2+ in pollen tubes was subsequentlyexamined for gradients in membrane-associated Ca2+ (chlortetracycline) Reissand Herth, 1979) and total Ca2+ (proton-inducedx-ray emission) (Reiss et al., 1983),both of which provide results consistentwith the apex being elevated in the ion.However, the demonstration of the steep,tip-focused gradient, using fluorescentdyes (Obermeyer and Weisenseel, 1991;Rathore et al., 1991; Miller et al., 1992),finallyprovided the necessary proof forthepostulated asymmetric distribution of in-tracellular ree Ca2+. Based on the studieson polarized ion currents, the suggestionwas made thatCa2+ gradients might createan electricalfieldacross the cytoplasm thatwould be sufficientto segregate cytoplas-mic components by electrophoresis (Jaffeet al., 1974; Robinson and Jaffe, 1975).Robinson and Jaffe (1975) also suggestedthat Ca2+ might affect intracellularmotility

Figure 3. Diagramof a DividingPlantCell in Late Metaphase.Thisfigure depicts a system of Ca2+-containingendoplasmic reticulum hat extends from the spindlepoles to the chromosomes along kinetochoremicrotubules.It was suggested that duringanaphase,Ca2+ release from the endoplasmic reticulum activates motile processes (e.g., microtubuledepolymerization)and thus facilitatesmovement of the chromosomes to the spindle poles. Insupportof this model, Ca2+-stimulated depolymerization of microtubules and facilitation of chromosomemotion have been observed (Zhanget al., 1992). Although an endogenous increase in [Ca2+]iduringanaphase has been reportedusingthe absorbance indicatorarsenazo IIIHeplerand Callaham,1987),this has not been repeated with a more efficacious fluorescent dye. (Figurecourtesy of Hepleret al.,1981, Figure 7, withkindpermission of SpringerScience and Business Media.)


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and inparticularhe formationand functionof microtubules.Inbroadterms,these ideascan be appreciated as antecedents for theview that the Ca2+ gradient npolarizedcellscontributes to the control of secretion(Holdaway-Clarke nd Hepler, 2003).Ca2+ and SecretionThe substantial body of work showing thatCa2+ affected the permeability propertiesof the cell membrane, while interesting initself, did not provide a very compelling orcomplete understandingof the mechanismof action of the ion. Although usually notexplicitlystated, there are differentstudieswhere you can almost hear the authorsstruggling with this rathervague conceptand where they are attemptingto formulatea more specific model. An example is theinduction of ac-amylase release in barleyaleurone cells by gibberellic acid. Studieson this model system had gained attentionbecause itseemed that the molecular basisfor gibberellic acid mightemerge. An earlyand influential observation was that ofChrispeels and Varner 1967),who showedthat the presence of Ca2+e (mM)greatlyfacilitated the appearance of gibberellicacid-induced ot-amylase in the medium.The increase was not trivial;as shown byJones (1973), 20 mM CaC12 timulated an18-fold increase over the water control.If an increase in the [Ca2+]e renders themembrane less permeable, why thenwould there be an increased release ofx-amylase? It is my supposition that thisconundrum puzzled different researchersleading eventually to the realization thatCa2+specifically stimulated the process ofenzyme secretion (Jones and Jacobsen,1983), an idea that stands as a paradigmtoday in both plant and animal systems(Zorec and Tester, 1992).Ca2+ and Plant Growth RegulatorsIn addition to a potential interaction be-tween gibberellic acid and Ca2+, connec-tions between this ion and other plantgrowth regulators were emerging. Thus,Ca2+ was seen to enhance the ability ofcytokininto retard senescence (Poovaiahand Leopold, 1973b) and leaf abscission(Poovaiah and Leopold, 1973a) and to

promote cotyledon expansion (Leopoldet al., 1974). Ca2+ was also found to inhibitcytokininstimulationof anthocyanin(Elliott,1977) and betacyanin synthesis (Elliott,1979). Yet other studies identified a Ca2+/cytokinin/ethylene connection, althoughthere was disagreement between pub-lished reports on the nature of the in-teraction. Lauand Yang (1975), in studieson mung bean hypocotyl segments, re-ported that kinetin greatly stimulated up-take of 45Ca2+, and Ca2+ stimulated theuptake and metabolism of kinetin 14C. Ofparticular note, both Ca2+ and kinetincaused a striking ncrease in ethylene (Lauand Yang, 1975). By contrast, Poovaiahand Leopold (1973a), in studies of leafabscission in bean petiole explants, foundthat Ca2+ inhibited ethylene production.Quite apart from studies of higher plants,LeJohn and coworkers (1973) identified aCa2+ binding cell surface glycoprotein inthe oomycetes, Achlyaand Blastocladiella,which released Ca2+ when challenged withcytokinin. They postulated that cytokininstimulates the availabilityand uptake ofCa2 , thus promoting metabolism (LeJohnet al., 1973).

Although an early idea concerning aCa2+/auxin interaction has already beendiscussed and largely dismissed, espe-ciallyto the extent that these agents affectwallstructureand expansion, nevertheless,there were physiological processes sug-gesting a possible coordination in theiractivity. For example, auxin transport wasreduced by low [Ca2 ]e (EDTA) (DeLaFuenteand Leopold, 1973), whereas auxin-induced proton secretion was stimulatedby Ca2+ (Rubinsteinet al., 1977). The per-vasiveness of Ca2+ activity led Leopold(1977) to open a summary article with thestatement that, "Actions of each of theplant growth hormones can be alteredby calcium salts...." Despite these manyencouraging leads, the idea of Ca2+ asa signaling agent does not directlyemergefromthese studies. Withstimulatory ffectsbeing caused by millimolar hanges in the[Ca2 ]e, it would be nearly impossible toderive a sense about the Ca2+ status inthecytosol. Nevertheless, an awakening wastaking place, and one that emphasized theimportance of Ca2+ over Mg2+, and the

monovalent cations, as a regulatorof plantgrowthand development.Ca2+ and LightWell before the demonstration that photo-synthesis lowers the [Ca2+], in Nitellopsi(Millerand Sanders, 1987), a Ca2+/lighinteraction had been noted in differenorganisms.Thered/far-red eversiblephyto-chrome pigment system was a commonfocus of attention.Amongthe early studies,note is made of phytochrome stimulatednyctinastic eaf movement inAlbizzia,whichwas shown to be inhibitedby low [Ca2+]asgenerated by culture in EDTA McEvoyandKoukkari,1972). Ca2+ was found to mar-kedly enhance the photoreversible ed light-induced depolarization of the membranepotential nNitella Weisenseeland Ruppert1977). A photoreversible red light-inducedincrease in Ca2+ efflux was noted in oatcoleoptiles (Haleand Roux,1980).Red lightprovided as a microbeam to filaments ofMougeotia, was also shown to stimulatea2- to 10-fold increase inthe uptake of 45Ca2+(Dreyerand Weisenseel, 1979). Finally,redlight-stimulated hloroplastrotation nMou-geotia was reduced simplythrougheliminationofCa2+ rom he culturemedium Wagneand Klein, 1978). Based on these earlystudies and also influencedby Rasmussen(1970), Haupt and Weisenseel (1976) sug-gested that phytochrome molecules lo-cated inthe plasma membrane functionasCa2+ carriers and facilitatean increase inthe [Ca2+]1when irradiatedwith red light.Their results supported the idea that Ca2+wouldcontrolcontractile proteins within hecell, andthus the movement of chloroplasts,as demonstrated in Mougeotia(HauptandWeisenseel, 1976). Althoughmost currenresearch on phytochrome is not directedtowardCa2+, a potentialrole remainslikelygiven the identificationof SUB1, a Ca2+bindingprotein involved with both crypto-chromeand phytochrome Guo et al., 2001).Ca2+ HOMEOSTASIS:PUMPS ANDCHANNELSIN PLANTSCa2+ and MitochondriaGiven the enormous disparity in [Ca2+]between the cytosol (0.1 FM) and the


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outside medium or storage compartments(0.1 to 10 mM),it became obvious that thecell must exert extremelyclose control overthe movement of the ion. However, evenbefore the disparity in [Ca2+] was fullyappreciated, early studies revealed thatcertain organelles, in particularmitochon-dria, exhibited the abilityto take up largequantities of Ca2 . Initial studies con-ducted in the late fifties and early sixtieson mitochondria derived from differentanimalcells (e.g., liverand kidney)revealedthat Ca2+ sequestration depended onrespiration, was sensitive to uncouplers,and required inorganic phosphate (forre-view, see Carafoliet al., 2001). Also in thesixties, Hodges and Hanson (1965) dem-onstrated that maize mitochondria, liketheir animal cell counterparts, activelyparticipateinthe uptake and accumulationof Ca2+. These pioneeringstudies revealedthat Ca2+uptake requiresrespirationor theaddition of ATPand is blocked by uncou-pling agents. That the process of Ca2+uptake by plant mitochondriawas broadlyexpressed was shown by Chen andLehninger 1973), who examined and com-pared the activityof these organelles from14 different species of higher plants andfungi. Although all mitochondria activelysequestered Ca2 , those from sweet po-tato and white potato tubers were particu-larlyactive and comparable to those fromrat liver. Additional work also establishedthat plant mitochondria, like those fromanimal sources, exhibited greater uptakecapacity but lower affinity han the similarprocess inmicrosomes (Dieterand Marm6,1980, 1983). These observations led to theconclusion that mitochondria,while capa-ble of takingup largeamounts of ion, wouldnot be the organelle involved in establish-ing the low basal [Ca2+],.A further ntriguingobservation was thesensitivityof mitochondrialCa2+ uptake tolight. Roux et al. (1981) noted that the[Ca2+] in the culture medium surroundingisolated oat mitochondria increased whenthe preparation was irradiated with redlight. Because the change in the [Ca2+]was prevented by subsequent exposure tofar-red light, phytochrome was implicated.Additional studies with ruthenium red,which blocks sequestration, led them to

favor the idea that red light, rather thenblocking influx, stimulated efflux of Ca2+(Roux et al., 1981). Although further studiesat this time (Dieter and Marme, 1983;Yamaya et al., 1984) failed to agree withthe details provided by Roux et al. (1981),they nevertheless supported the basictenet that Ca2+ uptake by plant mitochon-dria is in part regulated by light.

An important finding concerning basicmitochondrial metabolism was the discov-ery that at least one associated enzyme,namely NADH dehydrogenase, was regu-lated by Ca2+. The early studies of Milleret al. (1970) and Coleman and Palmer(1971), using EDTA, directed attention to-ward Ca2+, but the later study of M0lleret al. (1981), using both EDTA and EGTA,definitively established a role for Ca2+ andnot for Mg2+. It appeared from thesestudies that plant mitochondria not onlywere able to sequester Ca2+, but that theion, through the control of NADH oxidation,participated in regulation of mitochondrialfunction. Inlight of these various issues, themitochondrial/Ca2+ connection in plantsdeserves renewed attention as empha-sized recently by Hetherington and Brownlee(2004). The idea of both plant and animalmitochondria being viewed only as a safetyvalve capable of responding to a vast over-load of Ca2+ is being challenged instudies ofanimal cell mitochondria. Evidence is emerg-ing that mitochondria respond to relativelysmall changes in the [Ca2+]j through spatialjuxtaposition with the endoplasmic reticulum(Rizzuto et al., 1994; Carafoli et al., 2001).Similarly, plant mitochondria may be playinga more central role in cytosolic Ca2+regulation than has been appreciated here-tofore.

Ca2+ and ChloroplastsChloroplasts also were seen to participatein the uptake of Ca2+. In 1964, Nobel andPacker, drawing parallels with studies onmitochondria, demonstrated that isolatedspinach chloroplasts sequestered Ca2+when irradiated with light and supple-mented with ATP. Several years later,when it was apparent that plant cellsmaintained very low [Ca2+], the role of thechloroplast in the regulation of this ion

received further attention. Different labora-tories using chloroplasts from wheat (Mutoet al., 1982) and spinach (Kreimer et al.,1985a, 1985b) confirmed the dramaticlight-dependent uptake of Ca2+. Kreimeret al. (1 985a, 1985b) further concludedthat photosynthetic electron transport wasessential and that it was the membranepotential rather than pH that drove Ca2+uptake.

A particular interest in the role of Ca2+ inthe chloroplast centered on the regulationof NAD kinase. Previous discussion hasemphasized the role these studies playedin the discovery of calmodulin in plants(Muto and Miyachi, 1977; Anderson andCormier, 1978). Considerable complexitysurrounded this problem because reportsarose showing both calmodulin-dependentand -independent forms of the enzyme andboth cytoplasmic and organellar location(Moore and Akerman, 1984). Nevertheless,it seemed apparent that at least some ofthe NAD kinase, which was associated withchloroplasts, was Ca2+ dependent andlight activated (Muto et al., 1982). With thefurther finding that calmodulin was local-ized in chloroplast stroma, Jarrett et al.(1982) suggested that light stimulated theuptake of Ca2+ and that the Ca2+/calmod-ulin complex activated NAD kinase. Theproduct, NADP, then served its importantfunction as the terminal electron acceptorfor photosystem 1.

Ca2+ and MicrosomesFrom a historical point of view, our appre-ciation of a microsomal Ca2+ sequestrationsystem is the most recent, although inmany ways it emerges as the most impor-tant in regulating the basal [Ca2+], (Evans,1998; Sze et al., 2000). Researchers ex-amining animal cells, in particular thoseworking on muscle, made early and impor-tant progress on the identification of anATPase on the sacroplasmic reticulum thatdrove the uptake of Ca2+ against a con-centration gradient (MacLennan and Wong,1971). Plants may not have such a hyper-trophied system for Ca2+ uptake, neverthe-less early studies provided evidence thatmicrosomal vesicles were capable of Ca2+uptake. Already in their landmark study on


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mitochondria, Hodges and Hanson (1965)made reference to preliminarywork show-ing Ca2+ uptake in a microsome pre-paration from etiolated maize seedlings.However, it was Gross and Marm6(1978)who demonstrated the presence of Mg2+/ATP-dependent Ca2+ uptake in micro-somes of maize, squash, oats, and mus-tard, suspension cells of parsley, and thealga Cryptomonas. Although the mem-brane fraction was not fully identified,thestudies were consistent with it being de-rived from the plasma membrane. A moredefinitive identification of plasma mem-brane activity derives from studies ofNeurospora, in which Ca2+ accumulationin invertedvesicles depended on a Ca2+/H+ antiporter Stroobantand Scarborough,1979).A major problem in these early studiesconcerned the identificationof the sourceof the membrane vesicles. For example,Dieter and Marm6 (1981) provided evi-dence for a calmodulin-dependent Ca2+-ATPase; however, they were not able toidentify the membrane. Rasi-Caldognoet al. (1982) isolated two nonmitochondrialmembrane fractions, the heavier of whichappeared to be a Ca2+-ATPase and thelighterone a Ca2+/H+-antiporter. tudies inwhich the source of the membrane systemwas defined included those of Kubowiczet al. (1982), who isolated a plasma mem-brane enriched fraction that was active inthe sequestration of Ca2+. Of furthernotehere was the observation that auxin pro-moted Ca2+ uptake, whereas cytokininwas inhibitory.Endoplasmic reticulumvesi-cles were isolated by Buckhout (1984),whodemonstrated high affinityCa2+ accumu-lationthat was not dependent on calmod-ulin.Finally,attention is directed toward thestudy of Schumaker and Sze (1985), whocarefullyisolated and identified two mem-brane fractions from oat roots. The moreprominent one derived from the vacuoleand the less prominent one from theendoplasmic reticulum. Both fractions se-questered Ca2 , withvacuolar membranesappearingto use a Ca2+/H+-antiporter ndthe endoplasmic reticulum membranesemploying a Ca2+-ATPase. In conclusion,it can be seen that studies conductedbetween the mid sixties and the mid

eighties and to the present day (Sze et al.,2000) established that plants possess a richand multifaceted mechanism for Ca2+sequestration.Ca2+ InfluxIn parallelwith the concept of uptake andsequestration, which lowers [Ca2+],, is theequally important matter of entry or re-lease, which raises the [Ca2+]i.Given thehuge concentration gradient, which maybe in the order of 1000- to 10,000-fold,together witha substantialcharge gradient,which may be -100 mVor more, there is anenormous combined force that will driveCa2+ into the cell. Itfollows that only a fewCa2+ channels may be requiredto imparta rapid increase in the [Ca2+]j.The activityof Ca2+ channels at the whole cell level isshown directlyin the previouslycited workby Williamson and Ashley (1982), whorecorded a sharp [Ca2+]irise immediatelyafter the action potential in Nitella andChara (Figure 2). The Characean algaewere also used to demonstrate the activ-ity of single Ca2+ channels (Berestovskyet al., 1976); however, this is a topic thathas exploded in more recent years (forreview, see Tester, 1990; HetheringtonandBrownlee, 2004).

CONCLUSIONSBeginning in the sixties and extendingthrough the seventies to the early eighties,several lines of investigation were beingfollowed by plant biologists that wereconsistent with the notion that Ca2+ isa crucial cellularregulator.The stage wasset, and as a result of these pioneeringefforts there was a virtual xplosion of workin the eighties (Hepler and Wayne, 1985;Trewavas, 1986; Kauss, 1987) (Figure 4)and to the present day that continues todefine and characterize the enormous rolethat Ca2+ plays in the regulation of plantgrowth and development. To some degree,studies on plants were impeded by thepresence of a cell wall, which provides anenormous reservoir for Ca2+. With its ownrequirements being very high (10 FLMo10 mM), he [Ca2 ]e in the wallwould easilyswamp the relatively rivial mounts seen inthe cytosol (0.1 to 10 FLM). ut as small asthe concentration is, Ca2+, clearly hasa powerful impact on a host of growthand developmental processes.

The extensive involvement of Ca2+ fre-quently leads to the vexing question: howcan one ion control so many events? Theanswer is not entirelyknown,but the broadframework or its solution can be drawn. In

Polarity

Figure 4. Diagram Depicting the Role of Ca2+ as a Signaling Agent in Plant Cells Published in 1985/86(Trewavas,1986).Althoughsome importanteatures of Ca2+ signalingwere not knownat this time(e.g., the existence ofCDPKs), he figure nevertheless represents and anticipates the central role that Ca2+ plays in manyaspects of plantgrowthand development. (Figure ourtesy of Trewavas, 1986, frontispiece figure,withpermission of SpringerScience and Business Media.)


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brief, Ca2+ regulation in plants is richlyendowed with many components that candefine and adjust responses in both timeand space (Reddyand Reddy, 2004). Influxchannels on the plasma membrane andrelease channels from internal stores (en-doplasmic reticulum, vacuole, and mito-chondria)provideseveral ways to generaterapid ion elevations or create local gra-dients. Once the [Ca2+]i as risen,there arethen a wide variety of response factors,including both calmodulin-dependentkinases and notably the CDPKs, whichwillphosphorylate a response element andthus stimulate or inhibit an event or pro-cess. Finally, plants exhibit frequency aswellas amplitudemodulation,providingyetanother means of generating signals thathave unique properties (Evanset al., 2001;Holdaway-Clarkeand Hepler, 2003). Be-cause these different pathways can in-teract, the numberof individual ignaturesmultiplies.As noted by Reddy and Reddy(2004) fromtheiranalysis of the Arabidop-sis proteome, there are -700 known pro-tein components that function at variousstages of Ca2+ signaling. Plants thuspossess myriad ways in which Ca2+ canoperate as the intermediary n transducingthe stimulus intothe appropriateresponse.The challenge for the future lies in identify-ing and characterizing hese many differentevents and processes.

Peter K. HeplerDepartment of Biology

Plant Biology Graduate ProgramUniversity of Massachusetts

Amherst, [email protected]

ACKNOWLEDGMENTSI thank Brian Gunning (Australian NationalUniversity, Canberra, Australia),Russell Jones(University of California, Berkeley, CA), StanRoux (Universityof Texas, Austin, TX), HevenSze (Universityof Maryland,College Park, MD),Tony Trewavas (University of Edinburgh,Edinburgh, Scotland), and my colleagues atthe University of Massachusetts for helpfuldiscussion duringthe preparationof this article.I also thank E. Ridgway (Universityof Virginia,Charlottesville, VA),A. Trewavas (University of

Edinburgh), and R. Williamson (AustralianNational University) for permission to usefigures from their publications. Researchfrom my laboratory has been suppor-ted by the National Science Foundation(MCB-0077599).

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