Download Calcium homeostasis in plants

Journal of Cell Science 106, 453-462 (1993)
Printed in Great Britain © The Company of Biologists Limited 1993
453
COMMENTARY
Calcium homeostasis in plants
Simon Gilroy, Paul C. Bethke and Russell L. Jones*
Department of Plant Biology, University of California, Berkeley, CA 94720, USA
*Author for correspondence
INTRODUCTION
Many aspects of Ca2+ homeostasis in plants are similar to
those in animals and fungi (Poovaiah and Reddy, 1989),
but an understanding of how Ca2+ transport and function
are integrated from the level of the whole plant to the subcellular level remains elusive. At the whole-plant level, a
constant supply of Ca2+ in the range 1-10 mM is required
to maintain normal growth and development (Epstein, 1972;
Clarkson and Hanson, 1980). Calcium uptake by roots leads
to millimolar concentrations of Ca2+ in plant tissues, and
in most plants Ca is the second most abundant metal and
the fifth most abundant element, after C, H, O and K
(Epstein, 1972). At the subcellular level, certain organelles,
such as the large central vacuole, may have similarly high
Ca concentrations, but cytoplasmic Ca2+ levels are three to
four orders of magnitude lower. Despite the abundance of
Ca in plant tissues and the small amounts required for most
cellular processes, the supply of Ca to the plant must be
uninterrupted. Removal of Ca from the nutrient supply
results in rapid death of cells in the apical meristem and a
cessation of growth (Epstein, 1972). Just why this calcium
starvation occurs when Ca levels in the plant are so high
is not fully understood, but the low mobility of Ca within
the plant body must be at least partially responsible.
The extremely low mobility of Ca in plants can best be
understood by examining solute transport through the plant.
Localized transport from cell to cell occurs by diffusion
through cytoplasmic connections called plasmodesmata
(Lucas and Robards, 1990), whereas long-distance transport occurs in a specialized vascular system through phloem
and xylem elements (Turgeon, 1989). Transport of solutes
from leaves or other potential Ca2+ stores occurs by a pressure-flow mechanism whereby solutes are loaded into the
cytosol of the phloem sieve elements, and an osmotically
driven pressure gradient drives solute flow at rates that can
average one meter per hour. Although phloem cells are efficient sugar transporters because they contain high sugar
concentrations, 100 mg/ml or more, they are inefficient
Ca2+ transporters because Ca2+ is unlikely to exceed 100200 nM in their cyotsol. Thus, whether Ca2+ is transported
by diffusion from cell to cell through plasmodesmata or by
pressure flow through the phloem, its transport is limited
by its concentration in the cytosol of the transporting cells.
Since plants cannot redistribute Ca2+ efficiently over long
distances, Ca absorbed by roots and transported through the
xylem is largely utilized or sequestered locally. Most of the
Ca absorbed by plants is found in the cell wall (Bush and
McColl, 1987; Cleland et al., 1990) and the vacuole (Clarkson and Hanson, 1980). The nature of the interaction of
Ca2+ with the cell wall is poorly understood, although a
role for Ca2+ in strengthening the wall by cross-linking the
carboxyl groups of the pectic polymers has been proposed
(Cleland et al., 1990). There is also almost universal agreement among plant physiologists that high Ca concentrations
are required at the outer surface of the plasma membrane
(PM) to maintain the structural and functional integrity of
the PM (Clarkson and Hanson, 1980). The precise roles of
these high Ca concentrations at the PM are not known. The
Ca that is sequestered in the vacuole is found mostly as
salts of oxalic, phosphoric and phytic acids. Although these
are often thought of as insoluble, they may be mobilizable
(Borchert, 1990).
Although internal Ca stores and the extracellular environment both contain millimolar Ca concentrations, cytoplasmic Ca 2+ homeostasis follows the same rules in plants
as it does in animals. The concentration of free Ca2+ in the
cytosol is maintained near pCa 7 by pumps and channels
located in the PM and in the membranes of cellular
organelles. These transporters are regulated by environmental and hormonal signals that induce changes in cytoplasmic Ca2+ concentrations. Recent progress in understanding the many roles played by Ca2+ in plant cells has
relied on improved methods for measuring intracellular
Ca2+ and monitoring its changes as cells respond to various stimuli. Further progress, especially with regard to Ca2+
transport, has been driven by electrophysiological measurements coupled with traditional biochemistry. This
review will emphasize these areas and will suggest directions in which we believe the field will advance.
MEASURING CALCIUM IN PLANTS
Fluorescent dyes
Over the last five years studies of Ca2+-based signal transduction and homeostasis have been advanced by the development of techniques to directly measure cytosolic Ca2+
levels in living, functioning plant cells. Calcium-selective
microelectrodes have been used extensively to monitor
Key words: aequorin, calcium, signal transduction
454
S. Gilroy, P. C. Bethke and R. L. Jones
changes in cytosolic Ca2+ and continue to provide insight
into Ca2+-based signal transduction and Ca2+ homeostasis
in plant cells. However, microelectrodes are difficult to construct and use (Miller and Sanders, 1987; Felle,
1988a,b,1989) and can only measure Ca2+ at one point in
the cell. These limitations have lead to the development of
fluorescent dyes that allow quantification of the temporal
and spatial dynamics of cytosolic Ca2+ (Grynkiewicz et al.,
1985; Tsien and Poenie, 1986; Haugland, 1992). The Ca2+indicating dyes fall into two broad categories, single-wavelength and ratio dyes. The range of fluorochromes available
allows plant biologists to select indicators that are compatible with windows in the autofluorescence spectrum of their
cells.
With single-wavelength dyes, such as Fluo-3 or Calcium
Green, the intensity of fluorescence increases with the concentration of free Ca2+. Calibration of these dyes is complex, as errors arise if the dye is not evenly distributed
within the cytoplasm. They have been successfully used to
visualize Ca2+ dynamics (Gilroy and Jones, 1992), however, and they are especially useful in confocal microscopy,
where rejection of out-of-focus blur allows much clearer
visualization of cytosolic ion levels (Fig. 1A; Gehring et
al., 1990 a,b; Williams et al., 1990; Fricker and White,
1992; Gilroy and Jones, 1992; Irving et al., 1992; Shacklock et al., 1992).
Ratio dyes, such as fura-2 or Indo-1, show a shift in their
excitation or emission spectra upon binding Ca2+. By monitoring the ratio of fluorescence intensity at two wavelengths, cytosolic Ca2+ can be measured (Grynkiewicz et
al., 1985; Tsien and Poenie, 1986). The advantage of ratio
dyes is that, although the ratio of fluorescence intensities is
Ca2+-dependent, it is independent of dye concentration or
localization. Ratio analysis of Ca2+ has found wide application in animal cells and has recently been adopted by
plant biologists (reviewed by Read et al., 1992, 1993). A
major advance in this technology is the combination of ratio
analysis with the superior spatial resolution and threedimensional reconstruction capability of the confocal
microscope. This approach is now being applied to plant
cells and promises to yield further insights into the intricacies of Ca2+ signalling (Fricker and White, 1992).
The primary obstacle to using fluorescent indicators in
plant cells has been dye uptake. Ca2+-sensitive dyes are
highly charged and do not readily cross biological membranes.The ester loading technique developed by Tsien
(1981) and used by animal cell biologists to load cells with
Ca2+ indicators has had limited applicability to plant cells
because of extracellular hydrolysis (e.g. see Cork, 1986;
Gilroy et al., 1986, 1991; Hahm and Saunders, 1991),
incomplete removal of ester groups in the cytosol (Bush
and Jones, 1987), or sequestration of the dyes by organelles
(e.g. see Read et al., 1992). To overcome these limitations,
plant biologists have developed or adapted other loading
techniques such as acid loading, electroporation, detergent
permeabilization and microinjection.
Acid loading
Acid loading of dyes into the cytoplasm utilizes the proton
binding properties of dyes to produce an uncharged molecule at low pH (approximately pH 5) that is lipid-soluble.
Once in the cytosol, where the pH is about 7, the dye is
deprotonated and trapped as the charged anion, the fluorescence of which indicates cytoplasmic Ca2+ concentration. This technique, developed by Bush and Jones (1987),
has been succesfully applied to plant protoplasts (Bush and
Jones, 1987, 1988; Lynch et al., 1989; Kiss et al., 1991;
Russ et al., 1991; Gilroy and Jones, 1992) and walled cells
(Hahm and Saunders, 1991; Russ et al., 1991).
Microinjection
Of the other dye-loading techniques applied to plant cells,
which include reversible permeabilization of the PM by
electroporation (Gilroy et al., 1986, 1987, 1989; Scheuerlein et al., 1991; Shacklock et al., 1992) and detergent treatment (Timmers et al., 1991), the most widely applicable
has been microinjection. Although technically demanding,
dyes have been loaded by both iontophoretic and pressure
injection into plant cells ranging in size from small stomatal guard cells (10 µm × 30 µm) (Gilroy et al., 1990, 1991;
McAinsh et al., 1990, 1992) to pollen tubes (15 µm × >100
µm) (Miller et al., 1992; Read et al., 1992) and even isolated protoplasts (40 µm diameter) (Gilroy and Jones,
1992). The advantage of microinjection is that it allows
simultaneous addition of other effector molecules, such as
caged Ca2+ or caged IP3, and observation of their effects
on cytosolic Ca2+ levels (Gilroy et al., 1990). The disadvantage is that analysis is limited to single cells, whereas
groups of cells and even tissues can be loaded by the esteror acid-loading techniques (Fig. 1C; Gehring et al., 1990a,b;
Irving et al., 1992; Williams et al., 1990).
Sequestration of fluorescent dyes
Serious problems associated with fluorescence imaging of
plant cells have been the sequestration of fluorescent dyes
by organelles such as endoplasmic reticulum (ER, Bush et
al., 1989), vacuole (Bush and Jones, 1987; Gilroy et al.,
1989; Callaham and Hepler, 1991; Gilroy and Jones, 1992)
and nucleus (McAinsh et al., 1990; Gilroy et al., 1990,
1991), and/or rapid leakage across the PM. Sequestration
can reduce the window for measurement of cytosolic Ca2+
from hours to a few minutes. For indicators that are
microinjected, the problem has been largely overcome by
using membrane-impermeant dyes linked to dextrans of >10
kDa (e.g. see Gilroy and Jones, 1992; Miller et al., 1992).
Such an approach has been used to distinguish hot spots of
cytosolic Ca2+ from accumulation of dye into organelles
(Gilroy and Jones, 1992). However, subcellular localizations of even dextran-coupled dyes have been reported in
fungal hyphae and pollen tubes (Read et al., 1992). The cell
wall can also be a significant binding site for dyes (Lynch
et al., 1989; Gilroy et al., 1991; Hahm and Saunders, 1991),
but confocal imaging can resolve cytosolic fluorescence
from that of the cell wall, and using protoplasts or microinjection circumvents problems with the cell wall.
Transformation with aequorin
A recently developed alternative to the dye technology is
the transformation of cells with the gene for aequorin. This
technique for measuring Ca2+ has great potential for
expanding our knowledge of the intricacies of Ca2+ dynamics, particularly in multicellular organisms, and has been
Calcium homeostasis in plants
applied to tobacco (Knight et al., 1991b, 1992), yeast (Shimada et al., 1991) and Escherichia coli (Knight et al.,
1991a). Aequorin is a Ca2+-dependent, luminescent protein
isolated from the coelenterate Aequoria victoriae sp. It consists of a 22 kDa apoprotein and a small prosthetic group
(coelentrazine). Upon binding Ca2+ the aequorin molecule
emits a photon of blue light and the coelentrazine is oxidized and inactivated. The molecule is responsive to Ca2+
in the range 10−7 to 10−5 M (Blinks, 1986, 1989). This wide
dynamic range and the high selectivity of aequorin for Ca2+
make it ideal for monitoring cytoplasmic Ca2+ levels. This
is especially true as the low autoluminescence from most
cell types makes detection of emitted light a particularly
sensitive indicator, immune to the photobleaching and photodamage often associated with fluorescent probes.
Aequorin, however, has multiple negative charges and so
does not readily cross membranes. Animal cells have been
loaded with aequorin by a range of treatments including
liposome fusion, hyperpermeabilization, scrape loading and
microinjection (reviewed by Blinks, 1986). Plant cells have
been loaded by electroporation (Gilroy et al., 1989) or, more
routinely, by microinjection (Williamson and Ashley,
1982). All of these approaches are traumatic to the cell and
technically demanding.
The aequorin apoprotein gene has been cloned in several
laboratories (Inouye et al., 1985; Prasher et al., 1985). The
availability of this cDNA, the ability to reconstitute active
aequorin in situ in the cytoplasm by simply adding coelentrazine, which readily crosses membranes, and the relative ease with which plants can be stably transformed
(Potrykus, 1991) have led to the use of plants transgenic
for aequorin for monitoring cytoplasmic Ca2+ levels. This
technology is especially attractive because it is non-invasive and aequorin is thought to remain in the cytosol and
be non-toxic (Blinks, 1989). Knight et al. (1991b) have
transformed tobacco plants with the aequorin gene under
control of the constitutive cauliflower mosaic virus 35 S
promoter and shown that an aequorin signal can be detected
in whole, intact seedlings. Aequorin is thought to be
expressed throughout the plant, although rigorous testing of
this assumption has yet to be reported. Using tobacco
seedlings transgenic for aequorin, stimuli such as touch,
cold shock and fungal elicitors have been shown to cause
rapid changes in cytoplasmic Ca2+ (Knight et al., 1991b,
1992). Imaging the transformed plants using a photoncounting camera has revealed waves of luminescence in
leaves responding to cold or mechanical shock (Fig. 2).
Such data would be extremely difficult to obtain by other
Ca2+ measurement approaches.
Further advances with this technology can be anticipated.
Tissue- or developmental-stage-specific promoters may
allow imaging of subpopulations of cells or tissue domains.
Synthetic aequorins are available with altered sensitivity to
Ca2+ and different emission spectra (Shimomura et al.,
1989,1990; Kendall et al., 1992a,b). A synthetic aequorin
that shows two peaks in its luminescence spectrum may be
especially useful and it is amenable to ratio analysis (Shimomura et al., 1989; Knight et al., 1993). Engineering
aequorins with reduced Ca2+ sensitivity is also possible
(Kendall et al., 1992b), and these novel aequorins should
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allow for measurement of relatively high (> 10−6 M)
organellar or apoplastic Ca2+, where native aequorin would
be rapidly discharged. The availability of signal sequences
that target proteins to mitochondria, chloroplasts, vacuoles,
ER or PM should allow Ca2+ concentrations at defined subcellular locations to be monitored. Indeed, measurements
of organellar Ca 2+ by targeting native aequorin, or aequorin
engineered to be less sensitive to Ca2+, to the mitochondria
and ER of animal cells have recently been reported (Kendall
et al., 1992a; Rizzuto et al., 1992).
Application of aequorin technology to plant cells is currently an area of intense activity. It provides plant biologists with a tool to monitor Ca2+ homeostasis and signalling
at the whole-tissue level in intact plants. Coupled with the
results from a range of studies using Ca2+-selective microelectrodes and fluorescent dyes, aequorin can be expected
to enrich our understanding of the multifaceted involvement
of Ca 2+ as a signalling molecule in plants.
We stress here the need for adequate controls to establish that cell function is not perturbed by the aequorin or
fluorescent dye methodology. Plant biologists have used
morphological criteria, such as maintenance of stomatal
response (Gilroy et al., 1990,1991; McAinsh et al.,
1990,1992), organellar morphology (Gilroy et al., 1990,
1991), cell turgor (Gilroy et al., 1990, 1991; McAinsh et
al., 1990, 1992), maintenance of growth (Miller et al., 1992)
and maintenance of cytoplasmic streaming (Hodick et al.,
1991; Miller et al., 1992), as indicators that the measurement protocol was non-perturbing. As an example of the
extent of trauma that can occur during dye loading, analysis of carrot protoplasts loaded with Quin-2 by electroporation revealed that cellular ATP levels were reduced by up
to 50% (Gilroy et al., 1986). We would also like to highlight the value of fluorescence measurements where the cell
under study is observed with a microscope during the experiment. Cuvette-based studies on populations of cells can be
fraught with difficulties. Artifacts can include the masking
of temporal changes due to averaging over an asynchronously responding population, the inclusion of optical artifacts arising from changes in cell shape or size and the
swamping of signals from live cells by the much larger fluorescence from dye bound to dead cells. For example, Chae
et al. (1990) and Shacklock et al. (1992) have both investigated changes in cytosolic Ca2+ in monocot protoplasts
irradiated with red light. Chae et al. (1990) observed sustained changes in Ca2+ whereas Shacklock et al. (1992)
observed transient increases in response to the light stimulus. Could these discrepencies be due to comparing population measurements (Chae et al., 1990) with results from
single-cell imaging (Shacklock et al., 1992)?
Ca2+ HOMEOSTASIS IN THE PLANT CYTOPLASM
Cytosolic Ca 2+ concentrations in plant cells at rest, approximately 100-200 nM, are typical of those found in all
eukaryotes. The cytoplasm is bounded on the outside by
the PM, which is tightly appressed to the cell wall. These
walls, especially in young tissue, are porous, allowing for
diffusion of water, nutrients and molecules as large as 40
kDa (Carpita, 1982). Within the matrix of the wall, and
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S. Gilroy, P. C. Bethke and R. L. Jones
often chelated by it, Ca2+ concentrations can be high, at the
least 10−4 to 10−5 M (Cleland et al., 1990; Evans et al.,
1991). The cytoplasm is bounded on the inside by the
endomembrane system, which delineates numerous
organelles. Of these, at least the vacuole and ER have Ca2+
concentrations higher than that of the cytoplasm. The vacuole may be particularly important as a repository for cell
Ca2+, as it often accounts for 90-95% of the cell’s volume
and contains millimolar concentrations of Ca2+ (Felle,
1988). Luminal ER Ca2+ has rarely been measured in plants,
but has been found to be above 3 µM in one case (Bush et
al., 1989). There is evidence that proteinaceous Ca2+ chelators analogous to BiP are also present within the ER (Jones
and Bush, 1991). Mitochondria and plastids also have
higher than cytoplasmic Ca2+ levels, but will not be considered further here.
Because the cytoplasm is surrounded by extensive
regions of high Ca2+ and cytoplasmic Ca2+ must be maintained at nanomolar levels to support ATP-based metabolism, a variety of Ca2+ transporters are employed by plant
cells to regulate cytoplasmic Ca2+. Recent reviews (Evans
et al., 1991; Johannes et al., 1992a,b; Hetherington et al.,
1992; Maathuis and Sanders, 1992) are suggested for the
interested reader. A brief discussion of this very active field
is given below.
Ca2+-ATPases
Ca2+-ATPases have been found in the PM, tonoplast and
ER of plant cells. The functions of the PM and vacuolar
Ca2+-ATPases seem to be long-term maintenance of steadystate Ca 2+ levels (Evans et al., 1991) and resetting of cytoplasmic Ca2+ concentrations (Felle et al., 1992) by transport of Ca 2+ from the cytoplasm to the apoplast or vacuole.
The ER Ca2+-ATPase may also serve this function (Hepler
and Wayne, 1985) and also provide regions of high Ca2+
within the ER, which may be necessary for protein folding
(Sambrook, 1990). Calcium cotransporters and antiporters
have not been widely studied in plants, although H+/Ca2+
antiporters have been reported in the vacuolar membranes
of oats (Schumaker and Sze, 1990) and red beet (Blackford
et al., 1990), presumably functioning in conjunction with
the H +-ATPases and H+-PPiases that acidify the vacuole.
Ca2+ channels
Within the last few years, several plant Ca2+ channels have
been identified by their electrophysical characteristics, but
identification of Ca2+ channel proteins has proved problematical (Harvey et al., 1989; Hetherington et al, 1992;
Thuleau et al., 1993). They have been reported to open in
response to voltage (Johannes et al., 1992b), stretch (Cosgrove and Hedrich, 1991), IP3 (Alexandre and Lasalles,
1990,1992; Alexandre et al., 1990) and abscisic acid (ABA)
(Schroeder and Hagiwara, 1990). As opposed to the Ca2+ATPases, Ca2+ channels in the PM, and perhaps in the tonoplast, seem to function in short-term modulation of cytoplasmic Ca2+ levels, often in response to extracellular
factors. An IP3-regulated channel in the tonoplast may play
a major role in the release of Ca2+ into the cytoplasm from
intracellular stores, although this has yet to be demonstrated
(Schroeder and Thuleau, 1991). The function of stretchactivated Ca 2+ channels is still to be established, but these
Table 1. Stimuli that cause changes in cytosolic Ca2+ in
plants
Stimulus*
Hormonal
ABA
Auxin
CK
GA
Cell type
Guard cell
Maize coleoptile &
root
Parsley hypocotyl &
root
Aleurone cell
Maize epidermal cell
Maize coleoptile &
root
Parsley hypocotyl &
root
Funaria
Aleurone cell
Physical/chemical
Cold shock Tobacco seedling
Gravity
Maize coleoptile
Light
Maize coleoptile
Oat protoplasts
Oat protoplasts
Nitellopsis
Mougeotia scalaris
Salinity
Maize root protoplast
Touch
Tobacco seedling
Wind
Tobacco seedling
Yeast
Tobacco seedling
elicitors
Change in Ca2+
Localised, transient
increase
Localised, transient
increase
Localised, transient
increase
Transient decrease
Sustained oscillations
Localized, sustained
increase
Localized, sustained
increase
Sustained increase
Localized, sustained
increase
Transient increase
Sustained increase
Sustained increase
Sustained decrease
Transient increase
Sustained decrease
Increase
Increase
Transient increase
Transient increase
Transient increase
Reference†
1-5
6
6
7
8
6
6
9
10-13
14
15
15
16
17
18
19
20
14, 21
21
14
*ABA, abscisic acid; CK, cytokinin; GA, gibberellic acid.
†1, Gilroy et al. (1991); 2, McAinsh et al. (1990); 3, McAinsh et al.
(1992); 4, Irving et al. (1992); 5, Schroeder and Hagiwara (1990); 6,
Gehring et al. (1990a); 7, Wang et al. (1991); 8, Felle (1988a); 9, Hahm
and Saunders (1991); 10, Bush and Jones (1987); 11, Bush and Jones
(1988); 12, Bush (1992); 13, Gilroy and Jones (1992); 14, Knight et al.
(1991b); 15, Gehring et al. (1990b); 16, Chae et al. (1990); 17 , Shacklock
et al. (1992); 18, Miller and Sanders (1987); 19, Russ et al. (1991); 20,
Lynch et al. (1989); 21, Knight et al. (1992).
may prove to be an integral part of the plants response to
water availability, externally applied physical forces and
growth.
PERTURBATION OF CYTOSOLIC Ca2+
Without the use of neurons, plants perceive and respond to
environmental stimuli and internally produced signaling
molecules. Changes in cytoplasmic Ca2+ are central to these
processes. Indeed, cytoplasmic Ca2+ levels have been
reported to rise following so many different stimuli (Table
1) that the true role of elevated Ca2+ may be to bring the
cell out of a metabolic state of rest and into one of graded
response. Of those modulators of cytoplasmic Ca2+, physical stimuli and plant growth regulators will be briefly discussed as areas where significant progress has been made
or where exciting new possibilities exist.
Physical stimuli
It has been known for centuries that when some plants are
exposed to physical stimuli, morphological changes occur.
Plants shaken by wind are often more compact than those
grown in still air; tendrils twine around objects, but only
Calcium homeostasis in plants
after coming into contact with them. With the production
of transgenic plants expressing aequorin, a new avenue has
been opened for probing these responses and the role of
cellular Ca2+ in them. Knight et al. (1992a,b) have produced transgenic tobacco plants that constituitively express
aequorin and have demonstrated that cytoplasmic Ca2+
concentration increases instantly in response to touch.
Intriguingly, touch also induces the expression of a set of
genes including that for calmodulin (Braam and Davis,
1990; Braam, 1992). Similar changes in cytosolic Ca2+
were seen in response to wind, cold-shock and fungal elicitors that induce plant defense genes. During wind stimulation, Ca2+ increased only while the plants were in motion.
Repetitive stimulation resulted in a loss of sensitivity,
which returned after 45-60 seconds. The source of
increased Ca2+ resulting from wind or cold-shock was
examined with the putative ion channel blockers La3+,
Gd3+ and ruthenium red. Both 10 mM La3+ and Gd3+ eliminated the cold shock response but did not affect the windinduced Ca 2+ increase. Ruthenium red at 50 µM prevented
the wind response but not the cold-shock response. It was
proposed that the origin of increased cytosolic Ca2+ is
extracellular in the case of cold-shock and organellar in
the case of wind induction.
Evidence for the involvement of Ca2+ in the gravitropic
response has been provided by Gehring et al. (1990b).
Using the dye Fluo-3 AM loaded into abraded maize stem
segments and confocal microscopy, they found that repositioning coleoptiles from vertical to horizontal resulted in an
increase in cytoplasmic Ca2+ from 255 to 370 nM in cells
on the lower side of the tissue. The plant growth regulator
auxin is widely thought to be an integral part of the gravitropic response. Gerhing et al. (1990a) have also shown
that epidermal and cortical cells of maize coleoptiles exhibited an increase in cytoplasmic Ca2+ after treatment with
the synthetic auxin 2,4-D, suggesting that the auxin
response is linked to Ca2+. Because of inherent difficulties
associated with dye loading and calibration with large tissue
pieces, further work in this area is eagerly awaited.
Hormonal responses
Where changes in cytosolic Ca2+ can be linked to an easily
measured response, progress in understanding how Ca2+
levels change and what different calcium concentrations
may do has been most rapid. Studies on the cereal aleurone
and the epidermal guard cell pair have benefited from the
availability of a functional assay and represent the bestunderstood examples of how higher plant cells use Ca2+ to
link signal with response.
The cereal aleurone
The cereal aleurone cell secretes a battery of hydrolytic
enzymes, predominantly the Ca2+-containing metaloprotein
α-amylase, upon stimulation by gibberellic acid (GA). Bush
(1992) has shown that when aleurone layers from wheat are
exposed to GA, cytosolic Ca2+ levels rise within minutes.
Incubation with the Ca2+ channel blocker nifedipine prevented the rise in cytosolic Ca2+. GA treatment also reduced
the ability of the PM Ca2+-ATPase to lower cytosolic Ca2+
following an external Ca2+ pulse. The suggestion is that
both increased activity of Ca2+ channels in the PM and
457
decreased activity of a PM Ca2+-ATPase are responsible for
the GA-stimulated increase in free Ca2+. Gilroy and Jones
(1992) have found a similar, though slower (4 hour) rise in
cytosolic Ca2+ from 100 to 300 nM upon incubation of wallless aleurone protoplasts with GA. This precedes the onset
of enzyme secretion. Subsequent incubation with ABA reset
cytosolic Ca 2+ to resting levels, and secretion then ceased.
Ratio and confocal imaging of individual protoplasts loaded
with Fluo-3 revealed heterogeneity of Ca 2+ within the cytoplasm, with the region just below the PM showing the highest concentration (Fig. 1A,B; Gilroy and Jones, 1992). This
further suggests that in the aleurone cell, the increase in
cytosolic free Ca 2+ results from import through the PM, not
from internal stores. Following GA stimulation, a
Ca2+/calmodulin-regulated Ca2+-ATPase in the ER also
becomes more active (Bush et al., 1989; Gilroy and Jones,
1993), providing the substrate for formation of the Ca2+requiring α-amylase holoenzyme.
The stomatal guard cell
The epidermal guard cell pair forms a pore, the stomate,
through which CO2 enters and water leaves the plant. The
aperture of the pore is controlled by the guard cells, which
when fully turgid produce maximal opening and when nonturgid close the pore. This provides the primary mechanism
by which plants regulate their rate of water loss. ABA has
long been known to cause stomatal closure. Several groups
have recently shown that application of ABA to guard cells
results in a rapid rise in cytosolic Ca2+ preceding stomatal
closure (McAinsh et al., 1990, 1992; Gilroy et al., 1991;
Irving et al., 1992). Fluorescence ratio imaging of Com melina communis guard cells using Indo-1 revealed hot
spots of Ca 2+ in the cytoplasm around the nucleus and vacuole (Fig. 1D,E; Gilroy et al., 1991). The rate of cytosolic
Ca2+ increase was reduced by 1 mM La3+, but an increase
still occured with external EGTA pretreatment. These
results suggested that Ca2+ release from internal stores
could play a role in triggering the stomatal response.
Microinjection of caged Ca2+ or caged IP3 into guard cells
and photolysis of the caged group also results in increased
Ca2+ levels and subsequent stomatal closure (Gilroy et al.,
1990), reinforcing the idea that an increase in cytosolic Ca2+
triggers stomatal closure. Gilroy et al. (1991), however,
observed that, although guard cells always closed in
response to ABA, Ca2+ levels were elevated in only 40%
of the guard cells tested. This led Gilroy et al. (1991) to
suggest that ABA-induced stomatal closure may occur
through both Ca2+-dependent and Ca2+-independent pathways, although this is controversial (McAinsh et al., 1992).
Schroeder and Hagiwara (1990) used simultaneous patch
clamping of the guard cell PM and fluorescent imaging of
cytosolic Ca2+ using fura-2 to demonstrate that ABA
induced repetitive increases in cytosolic Ca2+. Calcium
increase was coincident with nonspecific cation channel
openings, and reversal potential data indicated that Ca2+
enters the cell through these channels. Intriguingly,
Schroeder and Hagiwara (1990) found that only one third
of the guard cells in epidermal strips of Vicia faba closed
when perfused with ABA, and 37% of the protoplasts examined by patch clamping showed an ABA-induced rise in
cytosolic Ca 2+. Hedrich et al. (1990) have also shown that
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S. Gilroy, P. C. Bethke and R. L. Jones
Fig. 1. Imaging of cytosolic
Ca2+ levels in plant cells. Ca2+
concentrations have been colorcoded according to the inset
scale. (A,B) Barley aleurone
protoplast microinjected with
Calcium Green-dextran and
treated with 5 µM GA for 0 (A)
or 18 hours (B). Bar, 10 µm. (C)
Arabidopsis mesophyll cells in a
whole leaf, loaded with Calcium
Green. Lower epidermis was
peeled from a leaf and indicator
was loaded into the whole tissue
by incubation at pH 4.5 for 2
hours with 25 µM dye. Leaves
were then washed twice with
water and imaged using the
Phoibos 1000 confocal
microscope and the 488 nm line
of the argon ion laser, a 500 nm
dichroic mirror and a 530 nm,15
nm half-bandwidth emission
filter. Due to the difficulties in
calibrating dye responsiveness
in whole tissues the values
shown should be taken as
approximations only. Bar, 50
µm. (D,E) Guard cell of
Commelina communis
microinjected with Indo-1.
Lower epidermis was peeled
from leaves and mounted in a
perfusion chamber. Individual
guard cells were microinjected
with Indo-1 and cytosolic Ca2+
imaged before (D) or 15
minutes after (E) stimulating the
stomate to close by elevating
extracellular Ca2+ from 20 µM
to 1 mM, according to Gilroy et
al. (1991). Bar, 10 µm. v,
vacuole; p, phytic acid bodies;
c, chloroplast; m, mesophyll; a,
air space; n, nucleus. (A-B)
Gilroy and Jones, unpublished;
(C) Gilroy and Bent,
unpublished; (D-E) modified
from Gilroy et al. (1991).
elevated cytosolic free Ca2+ activates voltage-dependent
anion channels. This leads to a possible scheme for ABA-
induced stomatal closure, i.e. efflux of ions from the stomatal guard cell and resultant reduction in turgor (Macrobbie, 1992). In this scheme ABA causes opening of non-
Calcium homeostasis in plants
459
Fig. 2. The effects of coldshock on the luminescence of a
cotyledon from an aequorintransformed tobacco plant.
Images were accumulated for
25 seconds at the indicated time
after treatment with ice (star) at
the tip of the cotyledon. The
total photon count for each
image (± 10%) is shown, top
right. The images have been
coded according to: red, high
photon fluxes (i.e. elevated
Ca2+); blue, low; black,
background. Bar, 100 µm.
Modified from Knight et al.
(1993).
specific cation channels in the PM, allowing for entry of
Ca2+ and resulting in an increase in cytosolic free Ca 2+ concentration. Higher Ca2+ concentrations might lead to Ca2+induced Ca2+ release from internal stores or IP3 production, causing release of additional Ca2+ from an
IP3-mobilizable pool. The opening of Ca2+-permeable
channels in the PM could depolarize the membrane and
activate Ca2+-dependent, voltage-dependent anion channels.
Depolarization would also tend to close inward K+ channels. Activation of an outward K+ channel may subsequently occur in response to changes in cytosolic pH. The
net efflux of ions would then cause an increase in water
potential, a reduction in turgor as water leaves the cells,
and closure of the stomatal pore. For recent reviews of
guard cell function with respect to Ca2+ and ion channels
see Macrobbie (1992) and Schroeder (1992).
FUTURE PROSPECTS
We foresee rapid progress in the isolation and sequencing
of genes encoding Ca-transporters. A putative ER Ca2+ATPase from tomato has been cloned, and the availability
of this probe should lead to the isolation of other ER Ca2+ATPase genes (Wimmers et al., 1992). Rapid progress in
the molecular cloning of Ca2+ channels from plants can also
be expected now that it has been demonstrated that Xeno pus oocytes can be used for the functional expession of
plant ion channel genes (Boorer et al., 1992; Cao et al.,
1992). Elucidation of the primary amino acid sequence of
Ca2+-transporters should lead to new insights into their regulation and an enhanced understanding of how plant cells
control cytoplasmic and organellar Ca2+ concentrations.
Although recent work on Ca2+ in plants has emphasized
Ca2+ at the single cell level, we anticipate that the focus
will change, both by narrowing to the subcellular level, and
by broadening to the tissue level. At the subcellular level,
Ca2+ heterogeneity within the cytoplasm, perhaps resulting
from segregation of differentially regulated transporters,
may aid in disecting out spatially distinct Ca2+-mediated
events, as recently suggested for hippocampal neurons
(Bading et al., 1993). At the tissue level, there has been
little effort to understand how Ca2+ acts to transmit information over long distances in plants. The recent work from
Trewavas’ laboratory using tobacco plants transgenic for
aequorin suggests that stimuli such as cold shock or wounding are propagated by waves of Ca2+ (Fig. 2; and Knight
et al., 1991b, 1993). How these waves are propagated, how
far they extend, and the responses that they induce as they
pass through tissues are unknown.
Our knowledge of how Ca2+ homeostasis and signalling
are intertwined with other cellular signals, such as pH, is
also likely to expand exponentially in the next few years.
The evidence is overwhelming that fine control of cellular
processes is not accomplished by isolated signals but by a
web of interwoven signal transduction chains. Identification
of these chains and the interactions that tie them together
will pose a serious challenge to plant cell biologists in the
coming decade.
The authors thank Drs Marc Knight, Nick Read and Tony Trewavas for the images of the aequorin-transformed plants, Dr
Andrew Bent for the image of dye-loaded Arabidopsis leaves and
Eleanor Crump for help in preparing this manuscript. Russell
Jones acknowledges the support of the National Science Foundation and the US Department of Agriculture.
460
S. Gilroy, P. C. Bethke and R. L. Jones
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