calcium signaling in plants - usp€¦ · calcium signaling in plants j. j. rudd and v. e....

CMLS, Cell. Mol. Life Sci. 55 (1999) 214–2321420-682X/99/020214-19 $ 1.50+0.20/0© Birkhäuser Verlag, Basel, 1999

Calcium signaling in plantsJ. J. Rudd and V. E. Franklin-Tong*

Wolfson Laboratory for Plant Molecular Biology, School of Biological Sciences, University of Birmingham,Edgbaston, Birmingham, B15 2TT (UK), Fax +44 121 414 5925, e-mail: [email protected]

Abstract. Changes in the cytosolic concentration of cal- Characteristic changes in [Ca2+]i have been seen toprecede the responses of plant cells and whole plants tocium ions ([Ca2+]i) play a key second messenger role in

signal transduction. These changes are visualized by physiological stimuli. This has had a major impact onour understanding of cell signaling in plants. The nextmaking use of either Ca2+-sensitive fluorescent dyes or

the Ca2+-sensitive photoprotein, aequorin. Here we challenge will be to establish how the Ca2+ signals areencrypted and decoded in order to provide specificity,describe the advances made over the last 10 years or so,

which have conclusively demonstrated a second messen- and we discuss the current understanding of how thismay be achieved.ger role for [Ca2+]i in a few model plant systems.

Key words. Cytosolic calcium; signaling; Ca2+ imaging; angiosperms.

Signal transduction in plant cells has rapidly become amajor topic of research that has emerged from thedisciplines of genetics, molecular biology, physiologyand biochemistry and now largely combines these disci-plines in order to advance our knowledge of how plantssense, and respond to, the diversity of extracellularstimuli they are exposed to. These studies have oftendrawn comparisons with how a mammalian cell pro-cesses the information it receives in the form of extracel-lular signals, and how this information is then decodedto produce an appropriate response. The concept ofsignal perception followed by intracellular signal trans-duction, which precedes the cellular response, hasproven a consistent formula adapted by many eukary-otic cells, including those of plants.The exact mechanisms whereby this is achieved fromstart to finish is obviously beyond the scope of anysingle review article. We must, therefore, focus in onspecific mechanisms of particular interest. This articlefocuses on what is now regarded as an extremely impor-tant ‘second messenger’ in plant cells, the Ca2+ ion.The focus of the discussion will be on the characteristicsof changes in the cytoplasmic concentration of Ca2+

observed in intact, living cells that have been exposed toa physiological stimulus. We will briefly summarize how

cytosolic free Ca2+ ([Ca2+]i) is generally regulated inmammalian and plant cells. We will consider the majoradvances achieved in this field over the last 10 years orso, which have been made possible by the new genera-tion of dyes which accurately report [Ca2+]i, togetherwith the advent of use of aequorin in transformedplants. These new technologies, coupled with theavailability of a handful of model systems which haveclearly identifiable responses to defined physiologicallyrelevant stimuli, have had a major impact on our cur-rent understanding of cell signaling in plants. Althoughlagging behind the mammalian field with respect to thedetailed knowledge of calcium signaling, it has clearlybeen established that plants use similar methods ofsignaling to animal cells. Nevertheless, despite the ad-vances made, there is much more to be learned aboutcalcium signaling in plants.

Calcium dynamics in animal and plant cells

The cytosolic free Ca2+ ([Ca2+]i) in the cell is understrict biochemical and physiological control. Cells gen-erally operate to keep [Ca2+]i low (�100–200 nM) inthe ‘resting’ or quiescent state (see fig. 1). Increases in[Ca2+]i can be used as a ‘second messenger’ followingcell stimulation, and in this way [Ca2+]i can bring about* Corresponding author.

CMLS, Cell. Mol. Life Sci. Vol. 55, 1999 215Multi-Author Review Article

Figure 1. The localization and mobilization of Ca2+ during signal transduction in animal and plant cells. This cartoon of a generalizedcell uses a pseudocolor scale (as indicated in the scale bar), in order to indicate the calcium concentration within the cytoplasm andorganelles of this cell. It contains organelles that are known to store Ca2+ in the �M to mM range. These organelles are illustrated inred to indicate their high [Ca2+] (see scale bar). The intracellular concentration of Ca2+ ([Ca2+]i) of a quiescent, or unstimulated, cellis generally accepted to be �100–200 nM (shown as blue; see scale bar). This low [Ca2+]i is maintained, at least in part, by the actionof Ca2+-ATPases which act to pump Ca2+ into intracellular stores. The extracellular Ca2+ concentration ([Ca2+]e) is significantlyhigher than the [Ca2+]i of the cytosol of a quiescent cell. Interaction between signal molecules and receptors (shown at the cell surfacehere) may result in Ca2+ influx. This is achieved by ‘opening’ of Ca2+ channels that are present in the peripheral plasma membrane,and this leads to local increases in [Ca2+]i, as illustrated by the color coding. Ca

2+-ATPases act to return [Ca2+]i to basal levels. Signalmolecules can also interact with receptors to result in the mobilization of Ca2+ from intracellular stores. The organelles identified asthe predominant pool for mobilizable Ca2+ in animal and plant cells are thought to be different. Animal cells are thought to mainlyrelease Ca2+ from the ER and/or the SR. Plant cells, however, appear to use the vacuole as the major mobilizable Ca2+ store, althoughthere is some emerging evidence that they also use Ca2+ stored in the ER. Ca2+ in intracellular stores can be mobilized by thegeneration of IP3 and cADPR, as indicated. These second messengers interact with specific receptors (IP3R and RyR) located onintracellular Ca2+ stores. These receptors form ion channels through which Ca2+ is released into the cytosol, resulting in spatially (andtemporally) defined increases in [Ca2+]i which are indicated in the diagram in pseudocolor. The dotted arrows linking the plasmamembrane receptor to the IP3R indicates that the pathway(s) are not well characterized in plants. These increases in [Ca

2+]i mediatethe elicitation of specific cellular responses to the extracellular signals. The pseudocolors used in this figure may be broadly related tothe images in figure 2, and in this way one can begin to envisage how some of the alterations in [Ca2+]i may be brought about.

a cellular response to the stimulus. We attempt toexplain some of the dynamics and controls involved inregulating Ca2+ in a ‘generalized’ cell, shown in figure1, which encompasses features found in both animaland plant cells. The actual increases in [Ca2+]i varywith cell types and the stimulus applied, but it is gener-ally accepted that the [Ca2+]i can increase up to 1–2�M in stimulated cells. The nature of the downstreameffectors are quite diverse and will not be described inany detail here, but interested readers should refer to [1]for a good general discussion.The biochemical means by which [Ca2+]i increasesvaries with the source of the mobilizable Ca2+ store. Itmay arise from outside the cell or from an internal pool.

As indicated in figure 1, the [Ca2+]i in both these localesis generally accepted to be in the micromolar to mil-limolar range, which is much higher than in the cytosol(100–200 nM). This is due, at least in part, to theactivity of Ca2+-ATPases (as shown in fig. 1) whichactively pump the Ca2+ into these stores, thereby main-taining low [Ca2+]i. Figure 1 illustrates that followingstimulation by receptor binding, this stored Ca2+ ispermitted to move down its concentration gradient andinto the cell cytoplasm, increasing [Ca2+]i. This caninvolve extracellular or intracellular Ca2+, or both.Ca2+ that originates from outside the cell often entersvia selective ion channels located in the plasma mem-brane (as indicated in fig. 1). Intracellular Ca2+ can be

J. J. Rudd and V. E. Franklin-Tong Calcium signaling in plant cells216

released in a number of ways. The first mechanism isthe classical example of receptor-mediated increase in[Ca2+]i, resulting from the operation of the phospho-inositide-signaling pathway. This involves phospho-inositidase C (PIC)-mediated generation ofinositol(1,4,5)-trisphosphate (IP3) from the membranephospholipid phosphatidylinositol(4,5)bisphosphate(PIP2). IP3 interacts with specific IP3 receptors (IP3R)that form Ca2+ channels in the membranes of intracel-lular Ca2+ stores (see fig. 1). Once bound to its recep-tor, IP3 induces channel opening, allowing the efflux ofCa2+ into the cytosol, through IP3-induced Ca2+-re-lease (IICR) [2, 3]. A further mechanism for release ofCa2+ from intracellular stores involves Ca2+ itself.This process, Ca2+-induced Ca2+ release (CICR), in-volves a second distinct Ca2+ channel receptor, theryanodine receptor (RyR), which is located on internalstores, as indicated in figure 1. RyrR is also responsiveto cyclic adenosine diphosphate (ADP) ribose(cADPR), another second messenger [4].Examples of production of IP3 and cADPR in responseto physiological stimuli in plant cells remain sparse. Ofthe few reported examples, IP3 production has beenobserved for the response of Medicago sati�a to fungalelicitors [5] and for Brassica napus in response to freez-ing [6], and cADPR has been seen to be produced in theetiolated hypocotyls of tomato as a result of treatmentwith abscisic acid (ABA) [7]. The detailed operation ofthe phosphoinositide pathway has, consequently, notbeen conclusively demonstrated in plant cells.A major difference exists between plant and animal cellswith respect to the identity of the major intracellularmobilizable Ca2+ store. In mammals this is believed tobe the endoplasmic reticulum (ER) or sarcoplasmicreticulum (SR) (see fig. 1). However, the experimentalevidence acquired so far points to the vacuole being themajor mobilizable intracellular Ca2+ store in plant cells(as shown in fig. 1). Good evidence exists for the pres-ence of receptors for both IP3 and cADPR on plant cellintracellular Ca2+ stores [8, 9], as indicated in figure 1.There is little doubt that the second messengers, IP3 andcADPR, are able to mobilize Ca2+ sequestered in thevacuole of plant cells. In vitro studies using isolatedplant vacuoles demonstrated the ability of both IP3 andcADPR to induce the release of Ca2+ from this store[10, 11]. Moreover, it has become clear that both agentscan work independently to release Ca2+ from the samepopulation of vacuoles. This suggests the presence ofthe respective receptor/ion channels for both agents onthe plant vacuole [11]. Although studies of pollen tubeshave suggested that the ER is likely to be a potentialCa2+ store in plant cells [12, 13], it is only very recentlythat evidence that plant cells can mobilize Ca2+ storedin the ER has been obtained. By using cells from

cauliflower inflorescences that were not highly vacuo-lated, it has been demonstrated that IP3-induced Ca2+

release may take place from nonvacuolar membranes[14]. This suggests that plant cells also have the abilityto mobilize Ca2+ stored in the ER as well as thevacuole, for use in cell signaling.Figure 1 illustrates how these mechanisms may operatein a cell to produce spatially localized high [Ca2+]i as aresult of the activity of both intracellular Ca2+ ionchannels and those that are distributed on the periph-eral plasma membrane. It is thought that messagescarried by [Ca2+]i are decoded to give an appropriatecharacteristic response in the stimulated cell. This canonly be achieved if the signaling inputs that controlCa2+ dynamics via ion channels and second messengergeneration corroborate to give spatially and temporallydistinct Ca2+ signals. Changes in [Ca2+]i are thought toprovide signaling information that is ‘encoded’ by theirmagnitude, duration and spatial patterning. This infor-mation appears to be in the form of quarks, blips,sparks, puffs, spikes, repetitive spikes (oscillations) andwaves of [Ca2+]i [15]. The first four of these are re-garded as fundamental and elementary changes in[Ca2+]i and have been observed in small ‘microdo-mains’ of cells, where they result from the localizedrelease of Ca2+ from individual or small numbers ofintracellular ion channels [16]. Oscillations and waves of[Ca2+]i are believed to result from the recruitment ofthese elementary release events, which have been visual-ized in single cells [17, 18]. Since at least some of thesepatterns of Ca2+ increases have been observed in plantcells, this phenomenon also appears to be a feature ofCa2+ based signaling in plants and will be discussedlater. We will first consider how changes in [Ca2+]i maybe observed/measured in living cells, together with someexamples.

Measuring cytosolic free calcium in living plant cells

Measuring cytosolic calcium ([Ca2+]i) in living cellsrequires a nondestructive means of incorporating a cal-cium sensor into the cytoplasm. Assuming that thesensor does not affect the normal functioning of thecell, a stimulus may be applied, and both the physiolog-ical and calcium response may be monitored. In thisway, direct evidence of signal-response coupling is pos-sible. Broadly speaking, there are two major forms ofcalcium sensors that have been introduced into plantcells in order to report [Ca2+]i. These are calcium-sensi-tive fluorescent dyes and the calcium-binding photo-protein, aequorin. We will briefly describe some of themethodology below, but readers requiring more detailof the technology involved are referred to [19].


Measuring [Ca2+]i using calcium-sensitive fluorescentdyesThe use of Ca2+-sensitive fluorescent dyes is an approachwhich has been widely used to investigate the quantita-tive, spatial and temporal distribution of intracellularcalcium in living plant cells. Figure 2A–E illustratessome examples of plant cells where Ca2+i has beenimaged using these dyes. Techniques routinely used forintroducing these dyes into mammalian cells have not, ingeneral, been widely used in plants. This is primarily dueto the permeability problems posed by the plant cell wall.The most successful, and therefore most widely used,method of introducing dyes into plant cells is to microin-ject the dye, using either pressure injection or ion-tophoretic injection (using an electrical current), directlyinto the cytoplasm of plant cells. Protoplasts have alsobeen used to study plant cell calcium, and are moreamenable to noninvasive techniques such as ester andacid loading. However, as protoplasts have lost their cellwall and polarity, their responses may not always bestrictly physiological.The nature of the dye that is introduced, and the methodof detecting calcium-induced changes in fluorescence,dramatically affects the nature of the information ob-tained. Two major classes of fluorescent dyes are gener-ally used: the single-wavelength dyes such as Fluo-3 andCalcium Green, and the dual wavelength ‘ratiometric’dyes, such as Indo-1 and Fura-2. The single-wavelengthdyes have the advantage that, to date, they are compat-ible with laser confocal scanning microscopes equippedwith an argon laser. When calcium is bound, there is anincrease in fluorescence, but as the fluorescence is onlymeasured at one wavelength, calibration of the increasein [Ca2+]i is notoriously difficult, because fluorescence isproportional to the amount of dye present. Furthermore,there is a potential problem with possible artefacts, asredistribution of the dye could potentially give the im-pression of increases in [Ca2+]i. However, the evidencesuggests that single-wavelength dyes are generally reli-able, and are liable to be ‘missing’ detail, rather thancreating artefacts. For example, the apical gradient ofhigh [Ca2+]i known to be present in pollen tube tips isusually not detected using single-wavelength dyes (see fig.2B). Nevertheless, single-wavelength dyes in conjunctionwith detection mechanisms such as laser confocal scan-ning microscopy have provided significant informationwith respect to alterations in the spatial distribution ofcalcium in living plant cells following application ofstimuli (see later). One particular advantage these dyeshave over currently available ratiometric dyes is that theymay be used in conjunction with caged probes, whichrequire a flash of ultraviolet (UV) light to release theprobe (see later, and [20–23]).Single-wavelength calcium-sensitive dyes, however, suf-fer from the drawback that they are unable to provide

accurate information with respect to [Ca2+]i. This prob-lem is overcome by using dual-wavelength ratio dyes.These dyes have two characteristic excitation/emissionwavelengths, one of which is Ca2+-dependent and mon-itors changes in [Ca2+]i, the other Ca2+-independent andused as a correction factor for differences in dye distribu-tion. A ‘ratio’ may be calculated from fluorescent imagescollected in pairs at the Ca2+-dependent and Ca2+-inde-pendent wavelengths in order to obtain accurate, quanti-tative information with respect to spatial and temporalpatterns of [Ca2+]i. Figure 2A, C, D, E illustrates thespatial and temporal diversity of alterations in [Ca2+]iobserved in different cells following specific stimulation,which have been detected using ratiometric techniques;these examples are discussed later in detail. Readersinterested in further technical detail about these dyes arereferred to [24] and [19].

Measuring [Ca2+]i using aequorinIn contrast, the photoprotein aequorin is suited to thestudy of calcium-mediated responses to stimuli imposedupon an entire plant, rather than individual cells. In brief,aequorin is more correctly referred to as a calcium-sensi-tive luminescent protein. The biochemical mechanism ofsensing changes in [Ca2+]i by this method relies on thedissociation of the apo-aequorin polypeptide and theluminophore coelenterazine upon binding Ca2+. Theluminophore then emits blue light. Emitted light can bedetected by a luminometer, or can be imaged by using anintensified CCD photon-counting imaging camera. Theuse of luminometry provides an excellent method fordetermining the temporal characteristics of changes in[Ca2+]i, but it is unable to provide spatial information,for which imaging is necessary.Aequorin was first used to report [Ca2+]i in the algaChara [25], following microinjection directly into thecytosol. Use of transformation to introduce these probesinto whole plants has revolutionized this type of study[26]. The growth or form of transgenic Nicotianaplumbaginifolia plants that constitutively expressed apo-aequorin in the cytoplasm of each cell was not affectedwhen compared with wild type [26]. Furthermore, incu-bation with coelenterazine resulted in the production ofreconstituted aequorin that was able to report changes in[Ca2+]i not only in cells and tissues, but also in wholeseedlings.This technology has enabled the study of Ca2+-basedsignal-response coupling in whole plants and tissues,rather than generally being limited to studying Ca2+

responses in individual cells. Figure 2E demonstrates anexample of the use of imaging for visualization of theresponse of a leaf to a ‘cold shock’ stimulus. It is alsopossible to use this approach to observe ‘long-range’signaling responses whereby [Ca2+]i can be monitored in


.


cells and tissues distant to the site where a stimulus isapplied. Examples of this will be discussed later. Theaequorin technology has been developed to increasesensitivity, using aequorin in conjunction with asemisynthetic, h-form coelenterazine, which has in-creased sensitivity to Ca2+ [27–30]. This has allowedsmall, yet potentially significant, changes in [Ca2+]i tobe detected. It has been suggested for some time thatthe major drawback of the use of transgenic plantsexpressing aequorin as a means of reporting [Ca2+]i isnot knowing the proportion of the apo-aequorin that isreconstituted in each cell. This presents problems withrespect to determining the quantitative changes in[Ca2+]i. However, the development of the semisyntheticaequorins has overcome this problem as they now pos-sess the ability to report [Ca2+]i due to an ability toluminesce at distinct wavelengths. Therefore, as previ-

ously described for the use of ratiometric dyes, the ratioof luminescence can used to quantify [Ca2+]i.Another significant adaptation to the standard recombi-nant aequorin technology has arisen from the ability totarget the photoprotein to distinct subcellular locations,where it can act to report fluctuations in [Ca2+]i eitherwithin organelles or in the cytosol immediately adjacentto the membranes of these organelles, in regions re-ferred to as ‘microdomains’. For instance, the introduc-tion of an H+-pyrophosphatase-apo-aequorin constr-uct into Arabidopsis thaliana seedlings has enabled visu-alization of changes in [Ca2+]i in regions adjacent to thevacuole [31].All of the methods described for reporting plant cellcalcium have advantages and drawbacks, some of whichwe have touched on here. These will be highlighted inthe following discussion that will focus on the few key

Figure 2. Imaging dynamic changes in cytosolic Ca2+ [Ca2+]i in living plant cells. Figure 2A–E illustrates some examples of the useof Ca2+-sensitive fluorescent dyes in single cells. (F) Example of the use of the photoprotein aequorin to report changes in [Ca2+]i (inphotons of light emitted) for an entire leaf. The images are presented in pseudocolor, which indicates alterations in [Ca2+]i clearly, andeach has a color calibration scale indicating [Ca2+]i for that particular set of images. (A) The response of a Commelina guard cell totreatment with ABA. The Ca2+-sensitive dual-wavelength dye Indo-1 was microinjected into the cytosol of a guard cell on an epidermalstrip, and fluorescence ratio imaging was used to visualize [Ca2+]i. The left-hand image shows the resting [Ca

2+]i in an unstimulatedguard cell. The middle and right-hand images display [Ca2+]i in the same guard cell at 15 s and 2 min after the addition of 100 nMABA. Note that the response to ABA induces punctate, transient elevations in the [Ca2+]i, which are spatially localized spikes. ©American Society of Plant Physiologists. Reproduced, with permission, from McAinsh et al. (1992). (B) Reorientation of an Agapanthuspollen tube. The Ca2+-sensitive single-wavelength dye Calcium Green-1 together with caged Ca2+ (Nitr-5) was microinjected into thecytosol of a pollen tube growing in vitro, and Ca2+i was imaged using laser confocal scanning microscopy. The first two images (at 0and 30 s) show the pollen tube before treatment. At 100 s, the caged Ca2+ was photoactivated using a flash of UV light in a regionto the left of the pollen tube tip (as indicated by the arrow). The images at subsequent time points show further localized increases in[Ca2+]i in the tip region, and reorientation of the direction of growth of the pollen tube. © American Society of Plant Physiologists.Reproduced, with permission, from Malhó and Trewavas (1996). (C) The response of a Papa�er rhoeas pollen tube to stigmaticself-incompatibility (S) proteins. The Ca2+-sensitive dual-wavelength dye fura-2 dextran was microinjected into two pollen tubes inorder to visualize alterations in [Ca2+]i in response to addition of incompatible stigmatic S proteins, using fluorescent ratio imaging.The left-hand series illustrates alterations (at 10-s intervals) in the apical Ca2+ gradient in a pollen tube. This gradient of Ca2+i isclearly lost within 60 s of receiving the stimulus. The left-hand series shows changes in the [Ca2+]i in the shank of a pollen tube, in aregion �100 �m behind the tip. Alterations in [Ca2+]i (indicated in seconds after application of the stimulus) were visualized, andappear to originate in the shank of the tube. The spatiotemporal appearance of these changes appears to have the form of a wave, whichmoves forward, towards the tip of the pollen tube. © American Society of Plant Physiologists. Reproduced, with permission, fromFranklin-Tong et al. (1997). (D) The response of Medicago sati�a root hairs to treatment with Rhizobium meliloti Nod factors. TheCa2+-sensitive dual-wavelength dye fura-2-dextran was microinjected into a root hair, and [Ca2+]i was visualized using fluorescentratio imaging. The images shown are a series taken �50 min after the application of a Rhizobium Nod-factor, with 5-s intervalsbetween each image, reading from left to right. A transient increase in [Ca2+]i, that is localized to the nuclear region of the cell, isvisualized. These transient increases in [Ca2+]i appear to oscillate approximately every 60 s (data not shown). © Cell Press. Reproduced,with permission, from Ehrhardt et al. (1996). (E) The deformation of a Vicia sati�a root hair following application of a Rhizobiumlipochito-oligosaccharides (Nod factors) The Ca2+-sensitive dual-wavelength dye Indo-1 was introduced into root hairs using acidloading, and [Ca2+]i was determined using fluorescence ratio imaging. Image (i) illustrates the apical high [Ca

2+]i gradient of growingzone I root hairs. Images (ii) and (iii) show nongrowing zone II and III root hairs that lack the apical high [Ca2+]i gradient. Images(iv); (v) and (vi) show [Ca2+]i in a zone II root hair, 70, 100 and 130 min, respectively, after the addition of lipochitooligosaccharides.Reestablishment of a high apical [Ca2+]i gradient is accompanied by reinitiation of growth in (vi). © Blackwell Science Ltd.Reproduced, with permission, from de Ruijter et al. (1998). (F) The response of a Nicotiana leaf to cold-shock. The gene encoding theCa2+-sensitive photoprotein apo-aequorin was introduced into Nicotiana plumbaginifolia plants, using transformation. The activeluminophore was reconstituted by incubation with coelenterazine. Luminescence, which reports [Ca2+]i, was imaged using an intensifiedCCD photon-counting imaging camera (courtesy of Prof. Anthony Campbell, University of Wales College of Medicine). The images areat 10-s intervals (reading from the top left-hand corner to the bottom right-hand image) and illustrate the response of a leaf to chilling(a reduction in temperature from 25 °C to 2 °C). The images show that the cold shock stimulus induces transient, spatially localizedincreases in [Ca2+]i of the leaf. Note that although a small number of cells initially respond, the increases in [Ca

2+]i spread throughthe leaf before returning to basal levels. Reproduced with permission, © Marc Knight, University of Oxford.


Table 1. Examples of physiological stimuli that have been shown to induce changes in [Ca2+]i in plants.

Response ReferenceCell/tissue typePhysiological stimulus

stomatal closure [20, 32–34]Abscisic acid (ABA) stomatal guard cellsstomatal closure [19]stomatal guard cellsHigh CO2stomatal opening [32]Auxin (IAA) stomatal guard cellsreorientation [23, 52, 60]pollen tubesDirectional growth signals

pollen tubes growth inhibition [12, 13, 56]Self-incompatibility (S) proteinsfertilization [74]egg cellsSperm cell fusionreorientation [83]Physical obstruction root hairsdeformation, curling and nodule formation [84, 88, 89]root hairsNodulation (Nod) factors

seedlingsTouch morphological changes [26, 27, 92]morphological changes [26, 92, 95]seedlingsWind

seedlingsCold chill resistance [26, 27, 31, 92]Fungal elicitors seedlings defense response [26]

We list examples of plant systems in which responses to physiologically relevant stimuli have been studied and shown to involve Ca2+

as a second messenger. We indicate the stimulus, the cell tissue and type, and the response. In all of these examples, measurements of[Ca2+]i were made using either Ca

2+-sensitive fluorescent dyes or the Ca2+-sensitive photoprotein aequorin, and increases in [Ca2+]iwere shown to precede the response.

model experimental systems that have been used toinvestigate signal transduction via changes in [Ca2+]i inhigher plants. These studies (which are summarized intable 1) have collectively highlighted the different rolesCa2+ appears to play in the mediation of many differentbiological responses, and will now be described in somedetail.

Plant systems for studying stimulus-response couplingvia alterations in [Ca2+]i

Several contrasting model systems have been used tomeasure changes in [Ca2+]i in plant cells that have beenexposed to a physiological stimulus. By this we mean astimulus that a plant or a plant cell would expect toencounter in its natural environment. We will attempt toconcentrate our discussion mainly on responses to phys-iological stimuli in order to provide greater depth withrespect to the systems that have been studied in thismanner. Table 1 lists some of the best-characterizedsystems where responses to physiological stimuli havebeen studied with respect to Ca2+i acting as a secondmessenger. Figure 2 illustrates some examples of imaging[Ca2+]i in some of these systems.

Changes in [Ca2+]i are associated with stomatalopening/closureStomatal guard cells are the key cells in the epidermis ofthe leaf which allow it to control gaseous exchange: entryof CO2 and exit of H2O as vapor. Although some of thewater is used to cool the plant in high temperatures, mostwater is lost unnecessarily, and the plant generally has toattempt to conserve water, using the stomata. The stom-ata, therefore, have to balance maximizing photosynthe-

sis with minimizing water loss. Responsiveness toenvironmental factors enables this.Two major environmental factors, light and CO2, influ-ence the behavior of stomata. In addition, other factors,including air humidity, temperature and wind movementalso interact to modulate changes in stomatal aperture.Stomata generally operate under a circadian rhythm, andmost plants open their stomata in the day and close themat night. This enables them to take up CO2 during the dayfor photosynthesis in the tissues of the plant. In additionto light as a stimulus, stomata also respond to CO2.Generally stomata respond rapidly to changes in CO2,closing as CO2 rises and opening as CO2 decreases. Theguard cells play a key role in the regulation of the waterrelations of the plant, and act to control water loss byclosing under conditions of drought in order to preventloss of water through transpiration. Osmotic changes inthe environment, therefore, play a major role in regulat-ing guard cell aperture. In addition to environmentalfactors, stomata also respond to hormonal stimuli. Per-haps the best-characterized is the response to the hor-mone ABA. It should be remembered that the effects ofenvironmental factors on stomata may be mediated byhormones. For example, water stress and salt stress canresult in elevated ABA levels, resulting in subsequentstomatal closure.Arguably the best-characterized plant system with re-spect to signaling via changes in [Ca2+]i is that of thestomatal guard cell. As a model system, the stomatalguard cells are ideal, as many stimuli are known to elicitopening and closing, as described above. The movementsresulting from changes in guard cell turgor are easilyidentifiable and measurable. Furthermore, as the twoguard cells act independently, one of the pair can act asan integral control. A well-established assay using epider-mal strips containing stomata makes this a model system


which is amenable to both the perfusion of extracellularagents, and the imaging and photometry of individualguard cells microinjected with Ca2+-sensitive fluorescentdyes.Convincing evidence, using these approaches, has estab-lished that Ca2+ acts as a second messenger in the processof both stomatal closure and opening [32]. ABA-inducedincreases in [Ca2+]i were first reported in guard cellsprepared as epidermal strips that had been treated withexogenous ABA [33]. Figure 2A illustrates this responsefor guard cells of Commelina communis. Ca2+-imaging ofa guard cell prior to the addition of ABA is shown in theleft-hand cell of figure 2A, and indicates the [Ca2+]i ofthe resting cell, which is �200 nM (comparable withresting levels in cells in general, as indicated in fig. 1).However, �15 s following the addition of ABA, in-creases in the [Ca2+]i were observed (see the middle cellof fig. 2A). These peak levels of [Ca2+]i are �1 �M, andcomparable to concentrations in stimulated cells (again,indicated in fig. 1). The right-hand cell displays the[Ca2+]i �2 min after the addition of ABA, and shows[Ca2+]i decreasing. It is therefore clear that ABA inducestransient and localized increases in the [Ca2+]i of thestomatal guard cell. The distribution and duration ofthese increases will be discussed later when we considerspecificity in Ca2+-based signaling responses of plantcells.A curious observation that has arisen from studies of thistype is that ABA-induced increases in [Ca2+]i do notprecede stomatal closure in all of the test groups. Al-though elevations in [Ca2+]i were observed in some of thestomata exposed to ABA, all of them subsequentlyclosed, regardless of whether [Ca2+]i increased [20]. Thistype of response, whereby ABA-induced stomatal closureoccurs independent of a rise in [Ca2+]i, has prompted thesuggestion that there may exist another, Ca2+-indepen-dent, pathway for ABA-induced stomatal closure [34].Further data suggest that [Ca2+]i is also involved inmediating stomatal closure in response to high CO2 andoxidative stress [35, 36]. Again, a small proportion of thecells responded in the absence of any detectable increasein [Ca2+]i. These ‘anomalies’ are discussed later, when weconsider the way in which changes in [Ca2+]i can elicitspecific responses in plant cells.Research has focused on elucidating how the informationencrypted in these changes in [Ca2+]i is decoded by thecell. It is also important to establish the source of the[Ca2+]i, and how it affects other components. Guard cellturgor is dependent upon several important physiologicalprocesses, the most significant of which is the influx andefflux of K+, which is associated with stomatal openingand closure (see the chapter on ion channels). Increasesin [Ca2+]i have been shown to be involved in theregulation of stomatal aperture [32]. A key question,therefore, is whether increases in [Ca2+]i are also in-

volved in changes in K+ channel activity. Use of cagedIP3 in guard cells of Vicia faba suggested that IP3-inducedrelease of [Ca2+]i influenced K+ fluxes associated withstomatal closure [37], and direct evidence for a role for[Ca2+]i was provided by imaging the effect of release ofcaged IP3 and caged Ca2+ on [Ca2+]i [38]. Both of thesetreatments resulted in a reduction in stomatal aperture,thereby clearly demonstrating that guard cells possess theability to respond to extracellular stimuli that may induceincreases in IP3 and the subsequent mobilization of Ca2+

from internal stores. Exactly how these changes in [Ca2+

]i affect ion channels is suggested to involve changes inthe phosphorylation state of what are presumably regu-latory proteins [39].A further interesting feature of the study of [Ca2+]i inguard cells again relates to the mobilizable calcium pooland more specifically to the intracellular distribution ofthe elevated [Ca2+]i. Due to the immobility of Ca2+ withrespect to diffusion in the cytosol, the spatial identifica-tion of the source of the increase is of significant interest.For instance, a difference in the subcellular distributionof [Ca2+]i during oscillatory increases that were inducedby high extracellular Ca2+ ([Ca2+]e) has been observed[40]. Specifically, it was noted that the transient increasesin [Ca2+]i that preceded the closing response to 1.0 mMexternal Ca2+ was localized to the peripheral regions ofthe guard cell in proximity to the plasma membrane. Thiswas then followed by an increased [Ca2+]i surroundingthe vacuole of the cell. This illustrates the tight control,and the possible utilization of several Ca2+ stores, incalcium signaling in these systems. Increases in [Ca2+]iin regions surrounding the vacuole and nucleus of guardcells have also been reported to occur when the external[K+] is lowered [20].

The importance of Ca2+ in plant reproductionAnother model system that has been the subject ofextensive Ca2+ imaging is the pollen tube. An importantfeature of these studies has, again, been the availabilityof a simple experimental technique by which pollen canbe grown in vitro. This facilitates investigations into thebiochemical control of pollen tube growth in vitro whichwould otherwise be practically impossible in vivo usingthe technology presently available. To date, fluorescentdyes have been used to monitor [Ca2+]i. However,although technical problems preclude the current use ofaequorin in this system at present, in the near future itmay be possible to use this technique to study truly in vivoevents.How might the pollen tube, in its natural environment,be signaled? Although it is clear that pollen can growwithout the presence of a stigma or style, there isconsiderable evidence for communication between pollentubes and the pistil. Let us consider pollination.First, pollen grain hydration upon contact with the


stigma is a situation that may involve signaling. Estab-lishment of polarity and initiation of tube growth mayalso require signals.The pollen tube must require mechanism(s) for ensuringcorrect directional growth. This is a situation for whichsome sort of guidance system, which is likely to involvesignaling between the pollen tube and the stylar trans-mitting tract, is required. An extracellular matrix in thetract contains components which are thought to beimportant in adhesion, nutrition and directional guid-ance involved in pollen tube growth [41, 42]. Althoughthis interaction is poorly understood at present, there isevidence that the stylar exudate contains several hy-droxyproline-rich glycoproteins, including heavily gly-cosylated arabinogalactans (AGPs) [43], some of whichmay be taken up and incorporated into pollen tubesgrowing in vivo [44]. Some AGPs, such as tobacco-transmitting tissue glycoprotein (TTS), can stimulatepollen tube growth and may play a role in pollen tubeguidance [45–47], though this topic is controversial [48].By whatever guidance mechanism they use, pollen tubesreaching the bottom of the pistil must undergo furtherreorientation, because the pollen tube has to find themicropyle through which it enters the ovary. Ulti-mately, there is the meeting of the pollen sperm cellswith the egg cell, between which one would expectsignaling in order to effect fertilization.All this occurs when the original pollen grain is per-ceived as compatible by the recipient pistil, and isallowed to grow normally! A situation which frequentlyoccurs is an interaction between pistil and pollen that istermed ‘incompatibility’. The most obvious example ofthis is the interaction of a pollen grain and pistil fromdifferent species, where interspecific incompatibility ex-ists to prevent fertilization. Although the mechanismsinvolved have not yet been determined, it is reasonableto suggest that these involve signal perception andtransduction. Incompatibility also occurs within species.This intraspecific incompatibility is often known as self-incompatibility (SI). These are often genetically deter-mined mechanisms whereby an individual plantprevents self-fertilization by selectively inhibiting pollenof identical self-incompatibility genotype (S-genotype)to that of its pistil. This obviously includes its ownpollen. We will discuss some of the evidence for Ca2+

signaling in normal pollen tube growth before Ca2+

signaling in incompatible interactions.The role of Ca2+ in mediating pollen tube growth. Acrucial requirement for Ca2+ in pollen tube growth hasbeen appreciated for many years. However, it was notuntil the availability of reliable Ca2+-sensitive fluores-cent imaging techniques that information about thespatial and dynamic changes in [Ca2+]i in pollen tubesbecame available. It is therefore relatively recently thatwe have begun to obtain information about [Ca2+]i and

its distribution in the pollen tube. Over the last 10 yearsgood evidence for a positive correlation between highapical [Ca2+]i in pollen tubes and growth has accumu-lated. This tip-focused gradient in [Ca2+]i is a consistentfeature of growing pollen tubes, and is common toother tip-growing cells, including neurites of nerve cells,fungal hyphae and root hairs (see later discussion).A number of independent studies on pollen tubes, usingratiometric Ca2+ imaging, have established that tip-fo-cused apical Ca2+i gradients (see fig. 2C), estimated tobe between 3 and 10 �M at its maximum [49–57], arelikely to be a general phenomenon common to allgrowing pollen tubes. It is worth noting that the [Ca2+]iin the tip region is consistent with the generally ac-cepted values of [Ca2+]i in stimulated cells, whereas the[Ca2+]i in the ‘shank’ region behind this area is withinthe levels of [Ca2+]i in resting or quiescent cells (see fig.1). Evidence from a variety of sources supports the ideathat the apical Ca2+i gradient results from Ca2+ influxrestricted to the extreme apex of the pollen tube [52–55,57–60]. It is generally thought that when growth ceases,the apical gradient is dissipated due to the closing ofthese channels. The nature of these Ca2+ channels iscurrently not known.Good evidence that apical Ca2+ has an oscillatorybehavior in growing pollen tubes has recently beenobtained [54–57, 61]. Detailed measurements have es-tablished that oscillating [Ca2+]i and growth rate areclosely associated and have the same periodicity [54, 55,57]. This has resulted in the suggestion that there is adirect causal link. The exact relationship between influxand growth needs further clarification, as does the exactfunction of the Ca2+ gradient. As these processes arenot the result of any obvious, identifiable physiological‘stimulus’ in the strict sense of the word, we will notconsider [Ca2+]i with respect to ‘normal’ pollen tubegrowth in any further detail here. Several recent reviewshave discussed the evidence that Ca2+ plays a key rolein the regulation of the pollen tube growth in detail[62–65].A role for Ins(1,4,5)P3 in the regulation of pollen tubegrowth. Although there is good evidence for the in-volvement of extracellular Ca2+ in pollen tube growth,increases in intracellular Ca2+ and the calcium storesinvolved are largely uncharacterized in normally grow-ing pollen tubes. However, there are data which providegood evidence that a functional phosphoinositide sig-nal-transducing system, involving IP3-stimulated in-creases in [Ca2+]i, plays a role in the regulation ofpollen tube growth in Papa�er rhoeas [22]. Measurableincreases in [IP3]i could be stimulated in pollen tubes,and these increased levels of IP3 could result in increasesin [Ca2+]i in growing pollen tubes. Subsequent modula-tion of pollen tube growth (altered tip morphology,temporary or permanent inhibition of growth, and re-


orientation) correlated well with these events [22]. Datasuggest that low-level phosphoinositide turnover andIICR are also required for normal pollen tube growth[22]. This prompts one to envisage a two-tier level ofcontrol involving phosphoinositide signaling modulat-ing pollen tube growth. Evidence suggests that low-levelturnover of IP3 is required for normal pollen tubegrowth, whereas levels consistent with those in ‘stimu-lated’ cells result in modulation of growth, includinginhibition.Confocal imaging of Ins(1,4,5)P3-stimulated increases in[Ca2+]i in P. rhoeas pollen tubes has provided evidencefor Ca2+ waves which travel towards the pollen tubetip. We will discuss the possible physiological role ofthese waves later. Data suggest that heparin-sensitiveIP3 receptors and IICR play a role in Ca2+ wavepropagation in the pollen tube. A model has beensuggested whereby the wave is propagated by Ca2+-de-pendent positive feedback loop on pollen PIC, resultingin a cascade of increasing IP3 generation and Ca2+

mobilization [22]. The nature of these Ins(1,4,5)P3-sensi-tive Ca2+ stores remains to be established, but there isevidence, in contrast to plant systems characterized indetail to date, that suggests that rather than the vac-uole, the ER may be the major Ca2+ store implicated inthis response [12, 13, 22].Ca2+ is involved in the reorientation of pollen tubegrowth. How the perception of extracellular signalsleads to modulation of growth patterns and directional-ity of plant cells is currently a key question in theplant-signaling field. Alterations in [Ca2+]i clearly havethe potential to act on many cellular processes. Al-though changes in [Ca2+]i are clearly linked to pollentube growth, the mechanisms involved remain unclear.There remains much speculation as to what are the cuesand signals which initiate the necessary changes in di-rectional growth of pollen tubes as they grow throughthe pistil, and these were briefly mentioned earlier inthis review. However, these components have not, todate, been tested to see if they alter the [Ca2+]i of thepollen tube. However, studies aimed at elucidating thecontrols involved in reorientation of pollen tubes isbeginning to throw some light on some of the controlmechanisms. Below we discuss some of the evidence forthe involvement of Ca2+ signaling in modulating pollentube growth.Using Ca2+-imaging, a series of studies by Malhó et al.[23, 52, 60] have established a second messenger role forCa2+ in the control of directional growth in Agapanthuspollen tubes. Several treatments, all of which resulted intransient elevations of [Ca2+]i, perturb the polarity ofthe pollen tube, which results in the temporary inhibi-tion of growth (with loss of the apical Ca2+ gradient).This was followed by tip swelling and coincided withthe establishment of a new apical Ca2+ gradient as

growth resumed, usually in a different direction [52, 60].Figure 2B illustrates the rapid reorientation of pollentube growth, stimulated by localized release of cagedCa2+ to one side of the pollen tube [23]. The directionof reorientation could be predicted accurately usingfluorescence ratios of the left versus the right of thepollen tube [23]. Modification of external Ca2+ locally,to one side of the pollen tube, also resulted inreorientation.These data, therefore, provide strong evidence that re-orientation of pollen tubes can be determined by local-ized alterations in [Ca2+]i. Use of ‘manganesequenching’ has allowed the measurement of Ca2+ chan-nel activity more directly. Inhibited pollen tubes showgreatly reduced Ca2+ channel activity, but when theystart to swell, prior to reinitiation of growth, consider-able localized Ca2+ channel activity in the first 20 �mof the pollen tube was measured [60]. These data sug-gest that Ca2+ channel activity in the apical dome playsa critical role in determining pollen tube reorientation.Data suggest that slight alterations in the exact localiza-tion of high Ca2+ channel activity within the tip regionis likely to determine directional growth in pollen tubes.The nature of the type of Ca2+ channels is un-known.Together, these data suggest that spatial alterations in[Ca2+]i can control the direction of pollen tube growth.Further investigations into the factors involved are re-quired, and it should be borne in mind that there arelikely to be many other factors controlling directionalgrowth of the pollen through the pistil tissues towardsthe ovary. At this stage it would be naive to assume thatCa2+ is likely to be the sole means of regulating direc-tional growth.The role of Ca2+ in the SI response of Papa�errhoeas. SI is one of the most important geneticallycontrolled mechanisms used by plants to regulate theacceptance or rejection of pollen, in order to preventfertilization by unwanted pollen. This is, in many spe-cies, controlled by a single, multiallelic S-locus. Self-fer-tilization is prevented when pollen carrying an S-allelewhich is genetically identical to that carried by the pistilon which it lands is inhibited, whereas pollen carryingother S-alleles is not. SI is an example of a very precisecell-cell communication and signaling system, wherebyinhibition of pollen tube growth is a response to aprecise, defined signal. Several alleles of the stigmaticS-gene of Papa�er rhoeas, the field poppy, have beencloned and sequenced [66–69]. They encode small, basicglycoproteins (14–15 kDa), and both stigmatic extractsand recombinant S-proteins have been shown to haveS-specific biological activity (i.e. they inhibit incompat-ible pollen). It therefore provides a good model systemfor studying signal-response coupling. The availabledata suggest that the stigmatic S-proteins act as signal


molecules that, when perceived by the alighting pollenas ‘self’, initiate pollen tube inhibition.Calcium imaging of pollen tubes challenged with Sproteins has provided good evidence that Ca2+ acts asa second messenger mediating pollen inhibition in theSI response [12, 13, 56]. Transient increases in [Ca2+]iin pollen tubes were only triggered when biologicallyactive S proteins were used in combination with incom-patible pollen. The peak increases in [Ca2+]i coincidedwith the cessation of pollen tube growth, and release ofcaged Ca2+ was used to demonstrate a direct linkbetween increases in [Ca2+]i and the biological re-sponse. These data all point to increases in [Ca2+]ibeing involved in inhibition of pollen tube growth, andsuggest that the S proteins achieve their effect by stimu-lating changes in [Ca2+]i.Early imaging studies of pollen tubes responding to theSI reaction suggested the SI-stimulated Ca2+i increaseswere localized in the ‘shank’ of the pollen tube [12, 13,70]. Recent studies using ratiometric Ca2+ imagingconfirmed this and provided further evidence for thepropagation of Ca2+ waves in pollen elicited by the SIresponse [56]. Figure 2C illustrates a pollen tube [Ca2+]iresponse to addition of incompatible S proteins in boththe apical region and the ‘shank’ region. S-specific in-creases in [Ca2+]i were visualized in the subapical andshank regions of the pollen tube virtually immediatelyafter S proteins were added, and [Ca2+]i in this regionincreased over several minutes, increasing from �200nM to �1.5 �M (as shown in the right hand section offig. 2C). A coincident diminution of the apical Ca2+

gradient was observed at the pollen tube tip (as shownin the left-hand section of fig. 2C), which followingsome fluctuation was reduced to basal levels within �1min [56]. These changes appeared to involve not onlyincreases but also a redistribution of [Ca2+]i and sug-gested that some of these alterations in [Ca2+]i might beinterpreted as a calcium wave.We have previously discussed a possible mechanism forthe generation of Ca2+ waves in pollen tubes withrespect to the PI-signaling pathway. The source of theCa2+, the mechanisms involved in Ca2+-release and thenature of these Ca2+ waves with respect to this SI-spe-cific stimulus are unknown, and require further investi-gation. Clearly IP3-induced Ca2+ release could beinvolved, though other stores and pathways could alsobe implicated. Nevertheless, it seems clear that alter-ations in Ca2+i in regions of the pollen tube other thanthe apical region are crucial for some processes regulat-ing pollen tube growth.Important questions arising from these studies includethe question of how these changes in [Ca2+]i mightsignal the arrest of pollen tube growth. It is tempting tospeculate that it is merely the loss of the apical [Ca2+]igradient that mediates this, and based on the impor-

tance of this gradient for the growth of pollen tubes inall species investigated, there can be little doubt thatthis plays a role. However, SI-induced pollen tube inhi-bition becomes irreversible within �20 min, even whenthe inhibitory S proteins are removed [71]. Changes inthe phosphorylation of pollen proteins are also ob-served subsequent to the changes in [Ca2+]i [71, 72],and there is evidence for specific gene transcription inincompatibly challenged pollen [73]. This suggests thatthe elevated [Ca2+]i also serves to signal downstreamcomponents that probably play key roles in the irre-versible inhibition of pollen tube growth. Later discus-sion will highlight what some of these intracellulartargets might be.An involvement of Ca2+ signaling in the fertilizationevent. Recent work, using confocal imaging of in vitrofertilization in Zea mays has demonstrated changes inthe [Ca2+]i of egg cells upon fertilization by isolatedpollen sperm cells [74]. A single slow, but transient,elevation in [Ca2+]i occurred in the egg cell followingthe fusion of a sperm cell. The spatial and temporalcharacteristics were strikingly similar to those observedin animal cells during fertilization. It is clear that theinteraction between gametes during reproduction inhigher plants involves dynamic changes in [Ca2+]i, asituation that has been observed for algae [75, 76], seaurchins [77] and mammals [78]. The spatial and tempo-ral characteristics of these increases vary to some extent,though they all appear to take the form of a Ca2+ waveacross the cell, originating from the point of fertiliza-tion. It will be interesting in the future to see what thisinformation encodes, with respect to the subsequentfertilization processes.

A role for Ca2+ signaling in the growth of root hairsThe growth of root hairs closely resembles that ofpollen tubes, and appears to be typical of tip-growingcells in general. It has been demonstrated that apicalgradients of [Ca2+]i are also correlated with growth inroot hairs [79–84]. Analysis using Ca2+-selective vi-brating probes first revealed that Ca2+ influx was local-ized almost exclusively to the tips of growing root hairsof Sinapis [79]. More recently, apical [Ca2+]i gradientshave been imaged in root hairs from several species[81–84]. It is of interest that the levels of [Ca2+]i mea-sured [81, 82] are similar to those in pollen tubes, withtip [Ca2+]i being broadly comparable (up to �1 �M)to those in stimulated cells, and the [Ca2+]i in theregions behind (100–200 nM) being comparable to qui-escent cells.There is strong evidence from a variety of sources thatCa2+ influx is responsible for the apical gradient, and iscorrelated with growth. This suggests that these fluxesare essential for tip growth and may be involved in


directing growth in both root hairs and pollen tubes. Asexpected, inhibition of the apical [Ca2+]i gradients by avariety of treatments generally results in inhibition ofgrowth. Furthermore, [Ca2+]i distributions during roothair development in A. thaliana have recently beendescribed [82], and studies of the rhd-2 mutant, defec-tive in sustained root hair growth, have provided strongevidence that implicates [Ca2+]i in regulating the tipgrowth process. However, there are data, using Limno-bium root hairs, which suggest that the magnitude ofthe Ca2+ flux entering the root hair tip does not deter-mine growth rate [80].Regulation of root hair directional growth. The questionof whether this tip-based [Ca2+]i gradient orientatesapical growth has been investigated [83]. An asymmetri-cal calcium influx across the root hair tip, which wasartificially generated, stimulated a change in the direc-tion of tip growth towards the high point of the new[Ca2+]i gradient. However, this reorientation, unlikethose stimulated in pollen tubes, was only temporary,and the root hairs soon established growth in the origi-nal direction. Similarly, in root hairs which had theirpath of growth blocked by mechanical means, reorien-tation occurred, but as the root tip passed the obstacle,growth returned to the original direction. Ca2+ imagingrevealed that when the root hair changed direction, thegradient also reoriented, and when growth returned tothe original direction, so did the [Ca2+]i gradient. Thus,the tip-focused [Ca2+]i gradient was always centred atthe site of active growth. These data suggest that whilethe tip-focused [Ca2+]i gradient is an important factorin mediating apical growth, it is not the primary deter-minant of directional growth in root hairs. It suggeststhat root hairs either have a predetermined direction ofgrowth, or that the root hair growth process is lesssusceptible to directional signals than extending pollentubes. Additional cues, therefore, are likely to regulateroot hairs in a manner different from those regulatingpollen tubes. One suggestion is that they may havepredetermined polarity.Ca2+-signaling during the symbiosis of root hair cells andRhizobium. The ability to fix atmospheric nitrogen isfacilitated by the symbiotic relationship between legu-minous plants and Rhizobium bacteria. This provides uswith another example of a physiological processwhereby changes in [Ca2+]i appear to play key cell-sig-naling roles. In brief, this process involves the interac-tion between bacterial-derived Nod factors and roothairs at a specific developmental stage [85]. This is whenthey have just, or are about to, arrest polarized growth.The Nod factors themselves are race-specific lipochi-tooligosacharides. Although the initial stages of theinteraction with respect to the identification of Nodfactor receptors awaits elucidation, the interaction isknown to induce several distinct physiological responses

of the root hairs. First, the root hair undergoes ‘defor-mation’. Subsequent swelling of the root hair is fol-lowed by ‘curling’ which appears to be outgrowth fromthat swelling [86, 87].As mentioned above, root hair deformation is one ofthe earliest responses to Nod factors, and occurs some60 min or so after application. This is followed byreinitiation of root hair extension. The hypothesis thatroot hair deformation in V. sati�a (vetch) induced byNod factors is the reinitiation of growth directed byreestablishment of an apical Ca2+ gradient has beenrecently tested [84]. This showed that root hairs termi-nating growth were susceptible to deformation, anddemonstrated that these root hairs respond initially bytip swelling, followed by reestablishment of polarity,with a new tip emerging from the swelling. Using Ca2+

imaging, these hairs were shown to have characteristicstypical of tip-growing cells, including a tip-focused cal-cium gradient of 1–2 �M at its peak. As illustrated infigure 2E, the most recently formed ‘zone I’ root hairs,which still exhibit tip growth, had a high apical [Ca2+]i(as shown in panel i), whereas older root hairs whichhave ceased to extend did not (see panels ii and iii). Thisconfirms the idea that root hair growth is correlatedwith a tip-focused gradient, as in pollen tubes. Swollentips of root hairs treated with Nod factor exhibit a[Ca2+]i that is 6–10 times higher than in untreated roothairs. This high [Ca2+]i soon polarized, and tip growthwas reactivated, as illustrated in figure 2E (panels iv, vand vi). These data provide good evidence that reinitia-tion of growth, mediated by [Ca2+]i, is responsible forNod factor-induced deformation of root hairs [84].A clear role for [Ca2+]i in the mediation of thesecharacteristic responses of root hairs to Nod factors hasrecently been established. Ca2+ imaging in root hairs ofVigna unguiculata has enabled the identification of avery early Ca2+ response to R. meliloti Nod factors,within seconds of the stimulus being applied [88]. Thereappeared to be a sustained increase in [Ca2+]i, up to aplateau. Although a distinct subcellular locale for thisincrease could not be defined, it was possible to demon-strate a direct correlation between the increases in[Ca2+]i and the curling and deformation responses ofthe root hair. Ca2+ imaging has been used to study theresponses of root hairs of M. sati�a (alfalfa) followingthe application of Nod factors from R. meliloti [89].After a lag period of �9 min, repetitive transientelevations in [Ca2+]i in the form of asymmetric Ca2+

‘spikes’ were observed, in response to addition of Nodfactors. These oscillations, with a periodicity of 60 s,continued for several hours. Unexpectedly, the spatialpatterns of the changes in [Ca2+]i were distinctive (asillustrated in fig. 2D), and originated in the nuclearregion of the root hair [89]. The increases in [Ca2+]i inthe nuclear region were �700 nM and are comparable


to those in stimulated cells. It is of interest to note that,although most root hairs respond by producing Ca2+

spikes, the timing of the responses of adjacent root hairswere apparently not coordinated.These studies suggest that there are at least three dis-tinct phases or patterns to the Ca2+ increases producedin response to Nod factors in root hairs. These comprisean early plateaulike increase in [Ca2+]i, which is fol-lowed by transient repetitive increases, in the form ofCa2+ spikes. Finally, a reinitiation of tip growth isstimulated, which is accompanied by a reestablishmentof high apical [Ca2+]i. We will discuss the significanceof differences in the spatial and temporal patterns in[Ca2+]i later.

The study of changes in [Ca2+]i observed in wholeplants and tissues in response to environmental stimuliWe now move on to studies using the photoproteinaequorin. The use of this technology has enabled thestudy of Ca2+-based signal-response coupling in wholeplants or tissues, rather than generally being limited tostudying Ca2+ responses in individual cells (see earlier).This approach has so far been used to investigate moreenvironmental stimuli, such as variations in wind andtemperature and the response to touch. This contrastswith the systems previously described, which have con-centrated on more specific signal molecules as stimuli.These types of stimuli affect the final morphology of aplant by influencing aspects of ‘plant form’ [90]. It istherefore of interest to investigate how these stimulimay bring about morphological changes and to investi-gate a role for Ca2+ as a second messenger, which maymediate these responses.Demonstration of an involvement of [Ca2+]i in mediat-ing these types of environmental responses was ob-tained by subjecting Nicotiana plumbaginifolia seedlingsto touch stimuli. A consequent dramatic elevation in[Ca2+]i was observed in the seedlings [26]. Reductionsin external temperature from 20 °C to 0–5 °C alsoresulted in transient increases in [Ca2+]i [26]. Interest-ingly, changes in [Ca2+]i were not observed for transi-tion from ambient to higher temperatures, includingthose thought to represent a heat shock. This clearlysuggests that [Ca2+]i plays a signaling role in the chill-ing or freezing tolerance of the seedling, but not in heatshock responses. It is worth noting that the timing ofthe elevations in [Ca2+]i observed in response to thedifferent stimuli varied. For example, very short tran-sients were induced by touch, and longer transients wereobserved in response to cold shock.Subsequent studies, using synthetic aequorins withhigher luminescence signals [27], has enabled spatialinformation to be obtained by imaging [Ca2+]i. Forinstance cold shock was observed to have a dramatic

effect by increasing the [Ca2+]i in the cotyledons androots of seedlings, but had little effect on [Ca2+]i inhypocotyls [27]. The cells in the cotyledons displayed anincrease, and then subsequent decrease over time, thatsuggested a ‘wavelike’ response. A small localized num-ber of cells initially respond before other cells, dis-tributed in adjacent areas, respond by increasing[Ca2+]i in the same manner [27]. This type of response,for a tobacco leaf exposed to a cold shock, is shown infigure 2F. This shows sequential photometric images ofa leaf, taken 10 s apart, following cold-shock treatmentand illustrates the transient increase in the [Ca2+]i ofthe cells of the leaf. It is of interest to note that not allof the cells respond in the same way and at the sametime.Studies have shown that even if a whole seedling issubjected to the same stimulus, different tissues appearto have differential sensitivity. Cotyledons appear to besignificantly less sensitive to cooling than roots. Forexample, [Ca2+]i in root tissue of N. plumbagifoliaseedlings was observed to respond to controlled temper-ature reductions ahead of the cotyledons [91]. Thissuggests that the temperature-sensing mechanisms ofthese two tissues vary, presumably in line with theirunique physiological requirements. Application of astimulus to a defined region of the plant has establishedthat long-range signaling is also likely to play a role inthese types of responses. By subjecting the roots, butnot the shoots, of a plant to cold treatment, it wasobserved that increases in [Ca2+]i were stimulated in theleaves, which were some distance from the site of thestimulus [91]. This was the first suggestion of a coordi-nated response of a whole plant, using long-range Ca2+

signaling [91]. However, despite an apparently identicallevel of stimulation, not all the leaves responded, andthose that did respond did not respond in the same way.We will discuss the significance of this later.It is becoming apparent that [Ca2+]i from differentpools may be mobilized by different stimuli. Studies onthe response of seedlings to environmental stimuli [92]suggest that touch and wind stimulation mobilize thesame Ca2+ pools. This is thought to involve Ca2+

release from intracellular stores. The cold shock re-sponse, on the other hand, appears to largely utilizeextracellular Ca2+ ([Ca2+]e). Further technological ad-vances, which have allowed aequorin to be targeted todistinct intracellular, subcellular locations has enabledmore detailed analysis of subcellular localization ofchanges in [Ca2+]i. Apo-aequorin expressed both in thecytosol and on the cytoplasmic face of the vacuole wasused to report fluctuations in [Ca2+]i in distinct subcel-lular ‘microdomains’ of A. thaliana seedlings [31].Again, the effect of cold shock resulted in a largeincrease in [Ca2+]i. The largest increases in [Ca2+]i weremeasured in the cytosol. In contrast, [Ca2+]i adjacent to


the vacuolar membrane was much lower than this,but took longer to return to resting levels [31]. Thissignificant observation clearly indicates differences inspatiotemporal changes in [Ca2+]i, which suggests theutilization of different pools of Ca2+ which can besimultaneously mobilized by a single stimulus. Thereis some evidence that suggests that IICR from thevacuole is a feature of the cold or ‘chilling’ response[31].

The question of specificity for plant cell Ca2+ responses

There is obviously more information encrypted in thechanges in [Ca2+]i of a cell than would immediatelymeet the eye. If we consider the guard cell studiesdescribed earlier, it is clear that the responses to auxinand ABA, which have opposite effects on guard cellturgor and stomatal aperture, are both mediated byincreases in [Ca2+]i [30, 32]. How this may be accom-plished has led to discussion of the elements thatcould aid in the generation of specificity of responsevia changes in [Ca2+]i.Mechanisms whereby specificity can be encoded in-clude the concept of the ‘calcium signature’ [93, 94].Certain features of Ca2+ signals are thought to ‘en-crypt’ the information that is necessary for the gener-ation of a specific response to a stimulus. Informationmay be passed on, within and between cells, usingtemporal and spatial patterning. This could use digital‘encoding’, in the form of variations in amplitude ofthe increases in [Ca2+]i that are often referred to asCa2+ transients. The duration of the transient is alsolikely to contain information. In situations whererepetitive transients (oscillations) are observed, the fre-quency of these is likely to encode information.Spatial patterning is also regarded as important.There may be heterogeneity within the cell and ortissues of the plant. Localization of the increases in[Ca2+]i, and their origin and propagation through thecell and tissues may be important. Alterations in[Ca2+]i may be regionally confined, or they may takethe form of Ca2+ waves, and move across cells ortissues. Coordination of these intracellular responses islikely to be important.The strength of the signal, and whether [Ca2+]ireaches a threshold, may determine the resulting re-sponse. In animal cells this information appears to bein the form of quarks, blips, sparks, puffs [15] whichall occur in highly localized regions of the cell, andare all regarded as fundamental and elementarychanges in [Ca2+]i. Ca2+ spikes, oscillations andwaves [15] are believed to result from the recruitmentof these elementary release events, which have beenvisualized in single cells [17, 18].

Temporal patterning and ‘calcium signatures’We have described several model plant systems whichhave been demonstrated to use [Ca2+]i as a secondmessenger. These include stomatal guard cells respond-ing to ABA, pollen tubes responding to reorientationstimuli and SI proteins; root hairs responding to Nodfactors; and leaves responding to chilling (see fig. 2).From the patterns of changes in [Ca2+]i, it is clear thatthere are a variety of calcium signatures generated.Digital encoding of the calcium signals and the forma-tion of a calcium signature in the form of amplitude,duration and frequency of the increases in [Ca2+]i arethought to be one way in which specificity is encoded.Using the example of N. plumbaginifolia seedlings, dif-ferent stimuli, in the form of chilling, wind and touch,elicit transient increases in [Ca2+]i. However, the tem-poral nature of these transients varies [26]. As each ofthese stimuli elicits specific characteristic responses inthe plants, the duration of Ca2+ transients are inter-preted to be specific signals. Stomatal guard cells andtheir response to ABA provide another example of thegeneration of [Ca2+]i transients. Whereas some stimulitrigger a single transient, other stimuli may triggermultiple increases in the form of oscillations. Further-more, the [Ca2+]i responses in both guard cells and inN. plumbaginifolia protoplasts have been found to bedirectly proportional to the strength of the stimulus,and this affects the pattern of the oscillations [40, 95].This is an important observation that is consistent withthe responses of mammalian cells to changes in theconcentrations of agonists [17], suggesting that the sameunderlying mechanisms for controlling and relaying in-formation through changes in [Ca2+]i may apply.The same stimulus can also trigger different responseswith respect to [Ca2+]i. One example which we havedescribed is that of the root hair to Nod factors. In roothairs of Vigna unguiculata, a very rapid increase in[Ca2+]i, within seconds of R. meliloti Nod factor beingapplied, was observed; this was sustained in the form ofa plateau [88]. A later response has been observed in M.sati�a [89]. Following the application of R. meliloti Nodfactors, there was a lag period of �9 min, after whichrepetitive asymmetric Ca2+ ‘spikes’ were observed, asillustrated in figure 2D. These oscillations, with a peri-odicity of 60 s, continued for several hours [89]. Evenlater than these responses, increases in [Ca2+]i in swol-len root hairs of Vicia sati�a, which precede reinitiationof tip growth are observed in response to R. legumi-nosarum bv �iciae Nod factors [84]. Although these areall responses to Nod factors in root hairs, the calciumsignatures are clearly different, and they may be respon-sible for different aspects of the response. Although it isnot yet clear, the amplitude, duration and frequency ofthese increases in [Ca2+]i may all contribute to thespecific responses within the plant cell.


Spatial patterning and ‘calcium signatures’Although the amplitude, duration and frequency ofthese increases in [Ca2+]i are important in encodingspecificity of response, the spatial patterning of theseincreases is considered to be equally important. Figure 2illustrates some of the diversity in the spatial patterningof alterations in [Ca2+]i. There is clearly heterogeneitywith respect to the [Ca2+]i response to stimuli, and thelocalization of increases in [Ca2+]i are generally notuniformly distributed within the cell/tissues. For exam-ple, within the cytoplasm of the guard cell, there arelocalized ‘hot spots’ of high [Ca2+]i triggered in re-sponse to ABA, as illustrated in figure 2A. In somesituations, this localization has been correlated withdistinct regions of the cell. For example, in root hairsstimulated by Nod factor, increases in [Ca2+]i areclearly highly localized (as shown in fig. 2D) and associ-ated with the region of the cell containing the nucleus[89]. Similar localization of transient increases in[Ca2+]i in the ‘nuclear region’ have been observed inpollen tubes stimulated by S proteins [12, 13]. This maypoint to the specific use of particular Ca2+ stores incertain responses. The specific distribution and posi-tioning of increases in [Ca2+]i may also be important insending messages to particular intracellular targets withdistinct localities within the cell.The ability to elicit the formation and propagation ofCa2+ waves has been observed in plant cells [22, 56, 74],as illustrated for a pollen tube in figure 2C, is anothermeans whereby specificity may be encoded by spatialpatterning of [Ca2+]i within the cell. In this way, rela-tively small increases in [Ca2+]i may be elicited in adefined region of the cell, and then this message may bepropagated to other parts of the cell. This propagationis likely to involve generation of further increases in[Ca2+]i which spread across or through the cell in aparticular pattern. This has been likened to a ‘flood’ ora ‘tide’ of [Ca2+]i. This wavelike movement of [Ca2+]imay propagate the signal within the cell. In this way,the response of a cell may be integrated using informa-tion from a single message (the original point source ofCa2+). Ca2+ waves appear to be a distinctive feature offertilization, in animal cells [96], algal cells [76] andplant cells [74]. This may be a characteristic ‘Ca2+

signature’ evoked by the fertilization event, with in-creases in Ca2+ propagating from the point of initialfertilization across the cell. In contrast, the Ca2+ wavesobserved in pollen tubes clearly have a different modeof action (see fig. 2C), and the result is inhibition ofgrowth.

The concept of ‘physiological address’In order to explain the considerable variability of re-sponses of cells to specific stimuli, the concept of ‘phys-

iological address’ in plant cells has been suggested [93,94]. During development, individual cell types, tissuesand, indeed, whole plants, are exposed to a variety ofstimuli. Depending on the combination of these stimuli,it is thought that they acquire a unique complement ofsignaling components. In this way, it is thought thatdifferent cells, even if they are adjacent to each other,may respond differently to the same stimulus. We havealready previously described some of the variability inresponses to the same stimuli, for example for guardcells [20, 33, 34], for root hairs [89] and for seedlingsexposed to environmental stimuli [91]. It has been estab-lished that, with respect to the stomatal guard cellresponse to ABA, the temperature and water stress thatthe plants were previously exposed to dictated whetherincreases in [Ca2+]i were observed to precede stomatalclosure [34]. This illustrates the importance of the con-cept of ‘physiological address’. This unique program-ming, therefore, currently provides a good explanationfor the unique responses of cells, tissues and plants toparticular stimuli.While specificity is likely to involve both calcium signa-ture and physiological address, there is obviously fur-ther complexity within the system, which may allow‘fine tuning’. The concept of ‘cross-talk’ between signal-ing pathways is likely to play a role in this. By this wemean that pathways are unlikely to be exclusive andlinear. Completely different pathways are likely to oper-ate at the same time, and it is the communicationbetween these ‘independent’ pathways that is likely tobe important in generation of specificity. A furtherconsideration for the generation of a specific responsevia changes in [Ca2+]i therefore probably also includesthe distribution of ‘downstream’ signaling componentsthat exist as targets for the increase in [Ca2+]i.

The intracellular targets of elevated [Ca2+]iThe nature of some of the intracellular targets forelevated [Ca2+]i are addressed in other articles withinthis issue, and we will therefore only highlight a selectedfew of these. Among the elements which may be trig-gered by a Ca2+-signaling pathway are protein kinasesand phosphatases, which may influence gene expression.Other components affected by [Ca2+]i may be morestructural, such as the cytoskeleton and exocytosis.A signaling role for [Ca2+]i in the activation of genetranscription has been identified for the response ofplants to red/far-red light. This stimulus is detected by asoluble receptor referred to as phytochrome A (PhyA)and results in the development of chloroplasts andanthocyanin synthesis, which involves gene transcrip-tion [97]. As in the absence of red light increases in[Ca2+]i-stimulated gene expression and chloroplast de-velopment [97, 98], this provides evidence that Ca2+

signals can be decoded to influence gene transcription.

CMLS, Cell. Mol. Life Sci. Vol. 55, 1999 229

One way in which [Ca2+]i may pass on informationwithin the cell is through Ca2+-binding proteins such ascalmodulin (CaM) and calcium-dependent CaM-inde-pendent protein kinases, which are generally nowknown as calmodulin-domain-like protein kinases(CDPKs) [99, 100]. CaM and CDPKs have been iden-tified in a variety of subcellular locations in plants [101].This suggests that they are likely to play a key role indecoding the information encrypted in spatiotemporalchanges in [Ca2+]i. As the name suggests, CDPKs con-tain a CaM domain as part of the structure of thenative enzyme and are therefore directly activated byCa2+ [100, 101].Both animal and plant cells are known to possess CaM,which mediates a wide variety of the cellular responsesto elevated [Ca2+]i. Activated CaM (i.e. Ca2+-bound)has been shown to induce gene expression in plant cells[97], which suggests a key role for Ca2+-CaM in medi-ating developmental responses. This also provides evi-dence that CaM is an important downstream target forelevated [Ca2+]i in plants. We have already discussedthe role of elevated [Ca2+]i in plants in response totouch stimulation [26, 92]. There is good evidence thatthis stimulus can result in the increased expression of afamily of CaM-related genes, referred to as the TCHgenes [102, 103]. These examples imply that increases in[Ca2+]i may transmit messages to the nucleus, therebyactivating gene transcription, through the activities ofCa2+-binding proteins.Other targets thought to be mediated by increases in[Ca2+]i are more structural in function. One examplewhich we draw attention to is the cytoskeleton. Asmicrofilament and microtubule assembly and disassem-bly are thought to be influenced by [Ca2+]i [104], it isalso considered likely that the cytoskeleton is directly orindirectly influenced by the changes in [Ca2+]i. There isincreasing evidence that the cytoskeleton, as well ashaving a structural role, may also have a signaling role.In many eukaryotic cells, actin-binding proteins func-tion as stimulus-response modulators, translating sig-nals into alterations in the cytoarchitecture [105]. Thereis evidence emerging that this is likely to be true forplant systems, and that this may be modulated byalterations in [Ca2+]i. For example, a kinesin-like CaM-binding protein (KCBP) has been identified in Ara-bidopsis, and Ca2+-CaM has been shown to regulatethe interaction between KCBP and tubulin. This sug-gests that this interaction may be regulated by [Ca2+]i[106]. This clearly indicates ‘cross-talk’ between signal-ing pathways and ‘structural’ elements, whereby the cellarchitecture may be influenced by these signals. Severalof the responses mediated by [Ca2+]i, which we de-scribed earlier, such as the root hair deformation re-sponse to Nod factor [84], the responses of seedlings towind and touch [26, 92], pollen tube reorientation [23,

52, 60] and inhibition stimulated by S proteins [12, 22,56] are likely to involve alterations in the cytoskeletonas an end product of the stimulus. Many other targetsfor Ca2+ probably remain to be identified. Their futureidentification will, no doubt, provide much data to-wards the elucidation of how the information containedwithin Ca2+ signals are processed in plant cells.

Future perspectives

Ten years ago we had little idea about the levels anddistribution of [Ca2+]i in plant cells. We also had noidea that [Ca2+]i was involved as a second messenger inthese cells. Great leaps forward have been achieved overthis time period, and we now have indisputable evidencethat [Ca2+]i has this role in plant systems. We havedescribed some of the well-characterized systems, andwe expect more examples of different stimulus-re-sponse-coupled systems to emerge in the future. Al-though there is no doubt that increases in [Ca2+]i arestimulated in response to physiological stimuli, themechanisms whereby this is achieved are largely un-known. Future studies will, no doubt, elucidate in detailthe pathways involved in the mobilization of [Ca2+]iunder physiological conditions. Combined with furtheradvances in technology, we should expect to look for-ward to considerable advances in our understanding ofCa2+ signaling in plant cells in the future. We havediscussed the perceived importance of the Ca2+ ‘signa-ture’. The spatiotemporal ‘encoding’ of the signals is arelatively new concept in the mammalian field, and weexpect that in the near future the plant field will developin a similar direction. More detailed information aboutthe downstream elements that are triggered by Ca2+

signaling pathways will enable further understanding ofstimulus-response coupling mediated by [Ca2+]i.

Acknowledgments. Work in the authors’ laboratory is funded bythe Biotechnology and Biological Sciences Research Council(BBSRC). We would like to thank Dominic Manu for help withthe reference list. We also thank Chris Franklin for criticalreading of the manuscript.

1 Clapham D. E. (1995) Calcium signalling. Cell 80: 259–2682 Berridge M. J. and Irvine R. F. (1989) Inositol phosphates

and cell signalling. Nature 341: 197–2053 Berridge M. J. (1993) Inositol trisphospate and calcium

signalling. Nature 361: 315–3254 Galione A. (1993) Cyclic-ADP-Ribose: a new way to control

calcium. Science 259: 325–3265 Walton T. J., Cooke C. J., Newton R. P. and Smith C. J.

(1993) Evidence for the generation of inositol trisphosphateand hydrolysis of phosphatidylinositol 4,5 bisphosphate arerapid responses following addition of a fungal elicitor whichinduces phytoalexin synthesis in lucerne suspension cells.Cell Signal 5: 345–356


6 Smolenska-Sym G. and Kacperska A. (1996) Inositol 1,4,5-trisphosphate formation in leaves of winter oilseed rapeplants in response to freezing, tissue water potential andabscisic acid. Physiol. Plantarum 96: 692–698

7 Wu Y., Kuzma J., Maréchal E., Graeff R., Lee H.-C., FosterR. et al. (1997) Abscisic acid signalling through cyclic ADP-ribose in plants. Science 278: 2126–2130

8 Muir S. R. and Sanders D. (1996) Pharmacology of Ca2+

release from red beet microsomes suggests the presence ofryanodine receptor homologs in higher plants. FEBS Lett.395: 39–42

9 Muir S. R., Bewell M. A., Sanders D. and Allen G. J. (1997)Ligand-gated Ca2+ channels and Ca2+ signalling in higherplants. J. Exp. Bot. 48: 589–597

10 Schumaker K. S. and Sze H. (1987) Inositol triphosphatereleases calcium from vacuolar membrane vesicles of oatroots. J. Biol. Chem. 262: 3944–3946

11 Allen G. J., Muir S. R. and Sanders D. (1995) Release ofCa2+ from individual plant vauoles by both InsP3 and cyclicADP-ribose. Science 268: 735–737

12 Franklin-Tong V. E., Ride J. P., Read N. D., Trewavas A. J.and Franklin F. C. H. (1993) The self-incompatibility re-sponse in Papa�er rhoeas is mediated by cytosolic free cal-cium. Plant J. 4: 163–177

13 Franklin-Tong V. E., Ride J. P. and Franklin F. C. H.(1995) Recombinant stigmatic self-incompatibility (S-)protein elicits a Ca2+ transient in pollen of Papa�er rhoeas.Plant J. 8: 299–307

14 Muir S. R. and Sanders D. (1997) Inositol 1,4,5-trisphos-phate-sensitive Ca2+ release across nonvacuolar membranesin cauliflower. Plant Physiol. 114: 1511–1521

15 Berridge M. J. (1997) Elementary and global aspects ofcalcium signalling. J. Physiol. 499: 291–306

16 Parker I. and Yao Y. (1992) Regenerative release of calciumfrom functionally discrete subcellular stores by inositoltrisphosphate. Proc. R. Soc. Lond. B 246: 269–274

17 Bootman M. D. and Berridge M. J. (1996) Subcellular Ca2+

signals underlying waves and graded responses in HeLa cells.Curr. Biol. 6: 855–865

18 Bootman M., Niggli E., Berridge M. and Lipp P. (1997)Imaging the hierarchical Ca2+ signalling system in HeLacells. J. Physiol. 499: 307–314

19 Webb A. A. R., McAinsh M. R., Taylor J. E. and Hether-ington A. M. (1996) Calcium ions as intracellular secondmessengers in higher plants. Adv. Bot. Res. 22: 45–97

20 Gilroy S., Fricker M. D., Read N. D. and Trewavas A. J.(1991) Role of calcium in signal transduction of Commelinaguard cells. Plant Cell 3: 333–344

21 Shacklock P. S., Read N. D. and Trewavas A. J. (1992)Cytosolic free calcium mediates red light-induced photo-morphogenesis. Nature 358: 753–755

22 Franklin-Tong V. E., Drobak B. K., Allan A. C., Watkins P.A. C. and Trewavas A. J. (1996) Growth of pollen tubes ofPapa�er rhoeas is regulated by a slow-moving calcium wavepropagated by inositol 1,4,5-trisphosphate. Plant Cell 8:1305–1321

23 Malhó R. and Trewavas A. J. (1996) Localised apical in-creases of cytosolic free calcium control pollen tube orienta-tion. Plant Cell 8: 1935–1949

24 Haughland R. P. (1996) Handbook of Fluorescent Probes,6th Ed., Spence M. T. Z. (ed.), Molecular Probes, Eugene

25 Williamson R. E. and Ashley C. C. (1982) Free Ca2+ andcytoplasmic streaming in the alga Chara. Nature 296: 647–651

26 Knight M. R., Campbell A. K., Smith S. M. and TrewavasA. J. (1991) Transgenic plant aequorin reports the effects oftouch and cold-shock and elicitors on cytoplasmic calcium.Nature 352: 524–526

27 Knight M. R., Read N. D., Campbell A. K. and TrewavasA. J. (1993) Imaging calcium dynamics in living plants usingsemi-synthetic recombinant aequorins. J. Cell Biol. 121: 83–90

28 Shinomura O., Musicki B. and Kishi Y. (1988) Semi-syn-thetic aequorin. Biochem. J. 251: 405–410

29 Shinomura O., Musicki B. and Kishi Y. (1989) Semi-syn-thetic aequorins with improved sensitivity to Ca2+ ions.Biochem. J. 261: 913–920

30 Shinomura O., Inouye S., Musicki B. and Kishi Y. (1990)Recombinant aequorin and recombinant semi-synthetic ae-quorins. Biochem. J. 270: 309–312

31 Knight H., Trewavas A. J. and Knight M. R. (1996) Coldcalcium signaling in Arabidopsis involves two cellular poolsand a change in calcium signature after acclimation. PlantCell 8: 489–503

32 Irving H. R., Gehring C. A. and Parish R. W. (1992)Changes in cytosolic pH and calcium of guard cells precedestomatal movements. Proc. Natl. Acad. Sci. USA 89: 1790–1794

33 McAinsh M. R., Brownlee C. and Hetherington A. M.(1990) Abscisic acid-induced elevation of guard cell cytosolicCa2+ precedes stomatal closure. Nature 343: 186–188

34 Allan A. C., Fricker M. D., Ward J. L., Beale M. H. andTrewavas A. J. (1994) Two transduction pathways mediaterapid effects of abscisic acid in Commelina guard cells. PlantCell 6: 1319–1328

35 Webb A. A. R., McAinsh M. R., Mansfield T. A. andHetherington A. M. (1996) Carbon dioxide induces increasesin guard cell cytosolic free calcium. Plant J. 9: 297–304

36 McAinsh M. R., Clayton H.,

calcium signaling in plants - usp€¦ · calcium signaling in plants j. j. rudd and v. e....

Documents