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Development 100, 181-184 (1987)
Printed in Great Britain © The Company of Biologists Limited 1987
Review Article
Calcium: a regulation system emerges in plant cells
Botany Department, University of Edinburgh, Edinburgh EH9 3JH, UK
Key words: calcium, regulation, plant cells, calmodulin, Chara, Fucus, NAD kinase, ATPases, protein kinases, cytoplasmic streaming,
polarized growth
Calcium occupies a pre-eminent place in the cellular
control systems of animals (Campbell, 1983). Because of the cytotoxic effects of calcium, cells pay
very particular attention to keeping cytoplasmic calcium levels very much lower than the normal extracellular 10~ 3 M level; usually it is in the range
1CT 8 -10~ 6 M. This is accomplished using a variety of
calcium-pumping systems located both in the plasma
membrane and organelles and together these operate
a very efficient calcium-stat system. But, in addition,
cells use the temporary elevation of cytoplasmic
calcium to between 10~6 and 10~5 M that may follow
plasma membrane perturbation and alteration of
calcium channel activity, as signals, eliciting a variety
of predetermined responses. The concentration of
cytoplasmic calcium is sensed by calcium-binding
proteins, most notably calmodulin, and the calcium/
calmodulin complex in turn modulates the activity of
numerous enzymes and proteins. Calcium is also
associated with other signalling systems such as IP3
and cyclic AMP.
Calcium injected into cytoplasm has very limited
mobility. This may be the consequence of efficient
internal sequestration systems which may include
calcium-binding proteins bound to the cytoskeleton.
A localized stimulus to the plasma membrane can
elicit an equally localized response in the cytoplasm.
This may explain why calcium figures so strongly
in the control systems involving cytoskeleton rearrangement, secretion and endocytosis, cell division,
membrane electrical activity, motor and contractile
activity and cell-to-cell communication. All of these
processes at some stage involve a differential response between the different parts of the cytoplasm
of the same cell.
Calcium research in plants has only been seriously
undertaken in.the last 7-8 years and this short article
outlines our current understanding; greater details
can be found elsewhere (Hepler & Wayne, 1985;
Trewavas, 1986). Even with the limited knowledge
available, however, it is evident that the control
exerted in plant cells is as profound as that in animals.
Is Cytoplasmic calcium regulated in plant
In order that changes in cytosolic calcium levels can
regulate cellular processes the level of the ion itself
must be closely regulated. Indirectly we know that
cells must efficiently hold cytosolic calcium well
below the cytotoxic concentration found in the extracellular fluid, 10~ 3 M (Hepler & Wayne, 1985), but
technical problems have dogged attempts to directly
measure this level in plants. However, the giant algal
cells of Chara and Fucus have proved uniquely suited
to microinjection of Ca2+ indicators (Williamson &
Ashley, 1982) or insertion of Ca2+-sensitive microelectrodes, backed by the use of a permeant Ca 2+ indicating dye (Brownlee & Wood, 1986). These
investigations have shown that lower plants maintain
free, cytosolic calcium at about 10~ 7 M. Recently the
development of a technique to trap Ca2+-indicating
dyes in the cytoplasm of plant cell protoplasts has
confirmed that a similar concentration is found in
higher plant cells (Gilroy, Hughes & Trewavas,
1986). Measurements have also demonstrated the
stability of the Ca2+-regulatory system that maintains
this 'resting' Ca2+ level despite changes in the extracellular ionic environment, including changes in Ca 2+
concentration over the range 10~ 7 M to 10~ 3 M (Gilroy, Hughes & Trewavas, 1987).
Superimposed on this stable low background, increases in Ca2+ concentration to 10~ 6 M have been
observed in several plant systems. From these studies
two classes of modulation have emerged: a sustained
but localized ion gradient as seen in the tip-growing
cells of Fucus (Brownlee & Wood, 1986) or pollen
tubes (Reiss & Nobiling, 1986) and a transient increase in level as shown by lily cells undergoing
mitotic progression (Keith, Raten, Maxfield, Bajer &
5. Gilroy, D. P. Blowers and A. J. Trewavas
Selanski, 1985) or Chara cells responding to an action
potential (Williamson & Ashley, 1982).
Thus it has been demonstrated that plant cells
precisely regulate their cytosolic Ca2+ levels. However, the molecular nature of the regulatory 'machinery' that brings this about is now only slowly
being revealed.
How is cytoplasmlc calcium regulated in plant
The low 'resting' submicromolar calcium level of the
plant cell has to be maintained against a very unfavourable 10~3 M concentration of calcium found in
the cell wall compartment or vacuoles/organelles.
Plant cells possess a range of active transport systems
which either sequester Ca 2+ into organelles or pump
it back to the extracellular space. Mitochondria,
chloroplasts and vacuoles accumulate Ca 2+ , often to
10~ 3 M levels, and the transporting systems of these
organelles or membrane vesicle preparations generally have an affinity for Ca 2+ of less than 10~ 6 M
(Moore & Akerman, 1984). In these cases, then, the
organelles have relatively low affinity, but probably
high capacity Ca 2+ storage sites. In contrast the Ca 2+ ATPase efflux pump at the plasma membrane and the
calcium-sequestering pump of the endoplasmic reticulum have a tenfold higher affinity. It is most likely
that these represent the cellular sites at which a low
'resting' Ca 2+ level is set.
The plasma and vacuolar membranes may also
possess Ca2+-sensitive ion channels allowing controlled influx since these membranes specifically bind
Ca2+-channel antagonizing drugs in vitro (Hetherington & Trewavas, 1984; Andrejauskas, Hertel &
Marme, 1986), such as verapamil and nifedipine.
These same drugs are known to inhibit physiological
processes in plants (Hepler & Wayne, 1985). Attempts to detect and detail the properties of calcium
channels using patch/clamp technology are only at
a very early stage. However clear detection of channels will require sound electrophysiological evidence;
drug binding is insufficient on its own.
The activity of these components of the Ca 2+ regulatory system have also been shown to be modulated by developmentally important stimuli. Active
phytochrome increases Ca 2+ influx at the plasmamembrane (Roux, Wayne & Datta, 1986) whilst
modulating Ca 2+ uptake by mitochondria and chloroplasts in several systems (Moore & Akerman, 1984).
The activity of the plasma membrane and endoplasmic reticulum Ca2+-pumps seem to be regulated
by changes in Ca 2+ level via the calcium-dependent
regulatory protein, calmodulin (Moore & Akerman,
Detection of a change in Intracellular free
calcium by calcium-binding proteins
The calcium receptor, calmodulin, has been detected
and isolated from a number of different plants (Allan
& Trewavas, 1985). Spinach calmodulin has been
sequenced and differs by only 13 residues from
bovine calmodulin (Roberts, Lukas, Harrington &
Watterson, 1986). Calmodulin has been detected in
both membrane and soluble plant cell fractions.
Bovine and plant calmodulin are often interchangeable in activating capability. Other calcium-binding
proteins have been detected in plants most notably in
phloem (Sabnis & McEuen, 1986) and carrot cells
(Ranjeva, Graziana, Dillenschneider, Charpentean
& Boudet, 1986) and there is circumstantial evidence
to support the presence of others elsewhere.
Calcium- and calmodulln-dependent enzymes
In plants
After binding three or four calcium ions the calmodulin molecule undergoes a conformational change and
then forms a ternary complex with various target
proteins transducing the original calcium signal. Only
a few calcium- and calmodulin-dependent enzymes
have been studied in any detail in plants. The
following examples are those best characterized and
indicate the range of processes under control.
NAD kinase (E.C. catalyses the phosphorylation of NAD to produce NADP and was the
first calcium- and calmodulin-dependent enzyme to
be discovered in plants (Anderson & Cormier, 1978).
The capability for manipulating NAD and NADP
levels argues for a pivotal role of this enzyme in the
regulation of anabolic and catabolic processes. Significantly phytochrome activation by red light increases the NADP/NAD ratio suggesting a direct tie
up between light, Ca 2+ , NAD kinase and metabolic
Quinate:NAD oxidoreductase (QORase, E.C.I.1.1.24) catalyses the oxidation of quinate to produce
dehydroquinate, an intermediate in the shikimate
pathway involved in aromatic amino acid synthesis.
In dark-grown carrot cells the enzyme appears to
contain its own calcium-binding protein (Graziana,
Dillenschneider & Ranjeva, 1984). The 'light' form of
the enzyme is activated by a protein kinase and is
discussed further below.
Numerous adenosine triphosphatases (ATPases)
have been described in plant tissues and the calciumand calmodulin-activated membrane associated types
may represent active calcium pumps. Calcium- and
calmodulin-activated calcium transport has been
found in the plasma membrane (Dieter & Marme,
Calcium regulation in plant cells
1980), the endoplasmic reticulum (Gross, 1982) and
the tonoplast (Fukumoto & Venis, 1986).
Finally, there are numerous calcium- and calmodulin-regulated protein kinases. These enzymes have
the potential to amplify a weak signal and thus figure
prominently in control concepts. Such enzymes have
been detected in both membrane and soluble fractions and in numerous plants. However in only one or
two cases have the substrates been identified. The
activity of the 'light' grown form of QORase is
controlled by a calcium- and calmodulin-dependent
protein kinase (Graziana, Ranjeva & Boudet, 1983).
In addition a plasma-membrane-associated protein
kinase which autophosphorylates has also been described (Blowers, Hetherington & Trewavas, 1985).
Autophosphorylation of this protein kinase alters its
catalytic activity, possibly releasing it from calcium
and calmodulin dependence, and thus giving prolonged activity after the transiently elevated Ca2+
concentration has returned to normal.
The binding characteristics of calmodulin to enzymes have been investigated in only a few cases but
presently determined binding constants span a range
of 1 0 ~ 8 - 1 0 ~ 6 M as has been found for animal cells.
The /CD for activation of NAD kinase by calmodulin
can be 10~ 8 M although this is dependent on the
source of calmodulin whereas for the plasma-membrane-bound protein kinase it is nearly 10~ 6 M. In this
latter case control of enzyme activity by calmodulin
availability is a real possibility.
modulated (Jaffe's second rule) with Ca 2+ ionophores and Ca 2+ buffers whilst Ca2+ influxes have
been blocked (Jaffe's third rule) with Ca2+-channel
antagonists or by reduction of extracellular Ca 2+ .
These treatments affect diverse plant processes including: cytokinin-induced bud formation in mosses;
desmid morphogenesis; protoplast fusion; a multitude of phytochrome-triggered responses (notably
chloroplast motility and fern spore germination);
gravitropism; secretion; leaf movements; guard cell
swelling and, of course, polar growth, mitosis and
cytoplasmic streaming (Hepler & Wayne, 1985;
Trewavas, 1986). Coupled to these studies have been
observations of changes in 'membrane bound' calcium; calcium isotope fluxes and total calcium level
which are thought to indicate changes in the cytoplasmic level of the ion (direct measurements have rarely
proved feasible).
Further evidence of Ca2+ regulation has come from
the inhibitory action of anticalmodulin drugs on many
of these processes (Hepler & Wayne, 1985). Though
inhibition often requires high drug concentrations,
which can have nonspecific effects on cell metabolism
and the Ca2+-regulatory system (Gilroy, Hughes &
Trewavas, 1987). Taken as a whole, although admittedly incomplete, the evidence points to Ca 2+ regulation of a wide range of physiological processes.
What plant physiological processes are
regulated by calcium?
It should be obvious even from this brief account that
much in the area of plant calcium is still based on
inspired imagination and the haziest of actual knowledge rather than solid well-established fact. And yet
the promise is still there; enough information has
emerged to confirm what, 5-8 years ago, was then
only an intuitive assessment of the importance of
cytoplasmic calcium in plant growth and development.
Plant calcium channels are a major area of uncertainty. Recent evidence showing that phytochrome (a
plasma-membrane-located protein in certain organisms) may have protein kinase activity, could be
significant here (Wong, Cheng, Walsh & Lagarias,
1986). Phosphorylation of calcium channels certainly
modifies animal channel activity and could explain
the tie up between red light and enhanced calcium
entry. The relationship of calcium to cell growth
needs detailed examination. High concentrations of
calcium directly inhibit cell extension and these high
concentrations probably elevate cytoplasmic calcium
levels. A further gross area of ignorance concerns the
substrates for calcium-regulated protein kinases in
plants. We have little or no information as to their
putative identity.
In assessing the evidence for Ca2+ regulation of a
physiological process we would do well to remember
the three criteria put forward by Jaffe for the rigorous
identification of such regulation: (1) the response
should be accompanied by a change in intracellular
[Ca 2+ ]; (2) artificial induction of such a change should
stimulate the process and (3) blockage of the change
should block the process. In the case of Ca2+dependent inhibition of cytoplasmic streaming (Williamson & Ashley, 1982; Hepler & Wayne, 1985) and
a requirement for sustained intracellular Ca 2+ gradients for polarized growth (Brownlee & Wood, 1986;
Reiss & Nobiling, 1986; Hepler & Wayne, 1985) the
fragmentary data have met all of Jaffe's rules. Whilst
in the case of mitosis in plant cells we lack only a
direct quantification of the changes in Ca 2+ level that
have been tentatively observed during mitotic progression (Keith, Raten, Maxfield, Bajer & Selanski,
1985; Hepler & Wayne, 1985).
In many other processes the evidence for Ca2+
regulation is less complete but nonetheless compelling. Intracellular calcium levels have been artificially
Future prospects
5. Gilroy, D. P. Blowers and A. J. Trewavas
Probably the major impact of plant calcium work
will still come in studies on polarity particularly
polarized cell division, morphogenesis and cell wall
secretion and patterning. Plants are organisms in
which growth and development are pronounced polar
phenomena, much more obviously so than in many
animals. These processes in one way or another
depend on cytoskeletal rearrangements and restructuring in numerous organisms, e.g. Fucus or Acetabularia. Clearly the relationship of calcium-regulated
processes to cytoskeletal structure should prove exceptionally fertile ground. Since these phenomena
require differential calcium levels in various parts of
the same cell, the new techniques of fluorescence
ratio imaging coupled with digital image processing
seem ideal investigative tools (Tsien & Poenie, 1986).
However, biochemical approaches to these problems
are equally necessary and are likely to prove equally
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