-
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].
-
CMLS, Cell. Mol. Life Sci. Vol. 55, 1999 217Multi-Author Review
Article
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
-
J. J. Rudd and V. E. Franklin-Tong Calcium signaling in plant
cells218
.
-
CMLS, Cell. Mol. Life Sci. Vol. 55, 1999 219Multi-Author Review
Article
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.
-
J. J. Rudd and V. E. Franklin-Tong Calcium signaling in plant
cells220
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
-
CMLS, Cell. Mol. Life Sci. Vol. 55, 1999 221Multi-Author Review
Article
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
-
J. J. Rudd and V. E. Franklin-Tong Calcium signaling in plant
cells222
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-
-
CMLS, Cell. Mol. Life Sci. Vol. 55, 1999 223Multi-Author Review
Article
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
-
J. J. Rudd and V. E. Franklin-Tong Calcium signaling in plant
cells224
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
-
CMLS, Cell. Mol. Life Sci. Vol. 55, 1999 225Multi-Author Review
Article
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
-
J. J. Rudd and V. E. Franklin-Tong Calcium signaling in plant
cells226
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
-
CMLS, Cell. Mol. Life Sci. Vol. 55, 1999 227Multi-Author Review
Article
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.
-
J. J. Rudd and V. E. Franklin-Tong Calcium signaling in plant
cells228
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
-
J. J. Rudd and V. E. Franklin-Tong Calcium signaling in plant
cells230
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.,