REVIEW ARTICLE
Calcium as environmental sensor in plants
Harsh Nayyar
Department of Botany, Panjab University, Chandigarh 160 014, India
Plants are constantly exposed to a changing and often
unfavourable environment which has led to the evolution of adaptive strategies that permit plant cells to
sense environmental stimuli and to activate responses
that allow avoidance or survival of environmental
stresses. A central theme in the current research in
stress biology has been the elucidation of signalling
pathways and their components controlling the stressresponse regulons. Efforts have been directed by several laboratories across the globe to understand how
environmental cues are sensed and transduced to the
level of gene expression. A number of interacting signal pathways appear to control the activation of stressresponsive genes. Sensing environment by plants must
involve a highly precise, specific and finely-regulated
mechanism in the form of ‘language’. Over the years,
search for such a ‘language’ has narrowed down to a
divalent cation, calcium (Ca2+), that acts as a ubiquitous messenger in the responses of a cell to the environment. With the advent of some novel techniques,
the measurement of intracellular Ca2+ concentration
has become easy. This has led to unequivocal evidences
for involvement of Ca2+ in several plant responses to
the environment. Since development of crops tolerant
to abiotic stresses is an important aspect in agricultural research, understanding the involvement of Ca2+
in facing these challenges becomes imperative and
worthwhile. Hence, the present review focuses on
some recent aspects of Ca2+ functioning in relation to
some selective abitoic factors.
CALCIUM (Ca2+) has many important structural and physiological roles in plants. It is important in maintaining the
stability of the cell walls, membranes and membranebound proteins, due to its ability to bridge chemical residues among these structures1. It is absorbed from the soil
solution and transported to various sites in the plants
through the xylem, like other minerals. Its mobility is low
and its concentration in the cell is kept to the minimum,
generally due to active sequestration into cell organelles
and rapid chelation2. In phloem, Ca2+ concentrations are
minimized by phosphate and other chelators3. Recently, it
has been found to regulate the phloem P-proteins in legumes4. Ca2+ mediates several plant processes like cytoplasmic streaming, thigmotropism, gravitropism, cell division, cell elongation, cell differentiation, cell polarity,
e-mail: [email protected]
CURRENT SCIENCE, VOL. 84, NO. 7, 10 APRIL 2003
photomorphogenesis1, plant defence, stress-responses5
and stress protection6–11, and the list is growing rapidly.
Among all its roles, being a second messenger in several
plant processes has attracted major attention and recent
research has revolutionized our thinking about the physiology of plant development, as we realize the involvement of Ca2+ in mediating diverse processes2,12,13. Now, it
has been firmly established as an important component of
a diverse array of plant signal transduction pathways14–18.
Due to advancement in tecniques to precisely measure
and monitor expression of intracellular Ca2+, like use of
fluorescent dyes and luminescent protein aequorin, the
progress in the search of a role for Ca2+ in cell signalling
has been exciting, and almost every process involving
cell–cell communication has been found14,15 to depend
upon Ca2+. Among the various plant processes involving
Ca2+, responses of plants to their ever-changing environment draws particular attention16,19. Intracellular Ca2+
concentration is kept low (100 to 200 nM) and is precisely regulated in order to save the cell from toxicity15.
Various environmental stimuli affect Ca2+ channels located at the plasma membrane and organellar membranes
to elevate its levels in the cytosol, which may serve to
transduce messages and amplify signals by triggering an
intracellular cascade of biochemical processes16,20,21.
ATP-dependent Ca2+-pumps (Figure 1) or H+ gradientdriven Ca2+/H+ antiports located variously at the plasma
and intracellular membranes maintain low cytosolic Ca2+
by translocating Ca2+ into the external space and internal
pools. Opening of Ca2+ permeable channels in the plasma
or intracellular membranes therefore allows Ca2+ to move
down its concentration gradient into the cytosol, and
hence to generate a Ca2+ signal. Ca2+ is mobilized from
its stores by two major types of Ca2+-releasing ligands,
inositol 1,4,5-triphosphate (IP3)22 and cyclic ADP-ribose
(cADPR)23. Since different Ca2+ pools may be accessed
in response to external stimuli to generate various types
of intracellular responses varying in magnitude and duration (Figure 1), the spatial localization of Ca2+ influx into
the cytosol may contain the key information within the
general Ca2+ signalling response15. In fact, Ca2+ alterations have been found to be highly specific for different
environmental variations and have even been observed to
be cell24- and organalle25-specific. For example, the response in the endodermis and pericycle in the presence of
salt and osmotic stress is different from other cell types24.
The stimulus-specific elevations in cytosolic Ca2+, called
Ca2+ signatures24 are exhibited in the form of waves25 or
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oscillations26. A temporal and spatial change of Ca2+ and
its amplitude seems to determine the Ca2+ signatures precisely and is probably responsible for specificity27. The
change in cytosolic Ca2+ concentration as a result of
stimulus-sensing can initiate a cascade of downstream
events involving binding of Ca2+ with some specific effector proteins, which activate a sequence of reactions leading
to gene activation in the nucleus to respond to external
stimuli. These proteins are also referred to as Ca2+ sensors and include calmodulin (CaM)28, Ca2+-dependent
protein kinases (CDPKs)29, Ca2+-regulated phosphatases,
annexins and integrins30. The Ca2+ sensors (CaM, CDPK
and other Ca2+-binding proteins) do exhibit tissuespecific expression and are likely to be important in decoding the Ca2+ signals. Detailed description about these
proteins is provided elsewhere17,29,30.
Understanding the components of Ca2+ signalling pathways that mediate environmental stress responses is essential to enhance future genetic-engineering strategies for
developing stress-tolerant crops. In this context, the recent status of Ca2+ in relation to the following abiotic
factors has been reviewed here.
Gravisensing
Graviperception is believed to involve the positioning of
statoliths, measuring their actual positions by cytoskeltonmediated changes, which must be transformed into an
endocellular signalling pathway31. The actin cytoskelton
is linked with the signalling system of the plasma membrane through its G-proteins32. The actin-binding protein,
profilin, was reported to be closely associated with the
phosphoinositide signal-transduction system, directly controlling the availability of polyphosphoinositides for second-messenger production33. Inositol 1,4,5-triphosphate
(IP3) is known to be involved in the mobilization of Ca2+
from intracellular stores34, while IP3 receptors have also
been identified as Ca2+ channels at the plasma membrane.
These IP3 receptors are reported to be connected through
F-actin network to intracellular Ca2+ channels35. It was
proposed that gravity-induced structural changes in actinbased cytoskelton activate Ca2+ channels, resulting in
quick release of Ca2+ from internal stores. Moreover, Factin-dependent plasma-membrane adhesion sites (responsible for perception of gravisensing) are closely asso-
Figure 1. Hypothetical model showing the effect of various stimuli on Ca2+ release channels located on
plasma membrane (PM) and organelles. The specificity of different signals in releasing Ca2+ from intracellular stores is also indicated. (See text for details and abbreviations.) The question mark on some arrows indicates unknown mechanisms causing cytosolic Ca2+ elevation.
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REVIEW ARTICLE
ciated with transmembrane integrins (I) that co-distribute
with stretch-activated Ca2+ channels (SAC; Figure 1) in
tip-growing lower plant cells36. The components of phosphoinositol signalling pathway have been recently investigated in maize nodes after their gravistimulation37. IP3
increases transiently in the faster-growing lower half
within 10 s after gravistimulation, and may act by releasing Ca2+ from internal stores. The root tips sense gravity
through specialized columella cells having amyloplasts38
and transduce the response as a signal to the elongation
zone of the root. Ca2+ and phosphoinositides are suggested to be involved directly in the transmission of signal
between the root tip and elongation zone. A marked asymmetric distribution of Ca2+ within 10 min across gravistimulated oat coleoptiles was demonstrated by Slocum
and Roux39. A rapid depolarization of gravity-stimulated
roots has been observed within 2 s, which may open voltage-gated Ca2+ channels (VGC) causing influx of Ca2+
into the cytosol, which subsequently may stimulate Ca2+
release from intracellular stores5. Changes in cytosolic
free Ca2+ have been documented for Arabidoposis roots
after stimulation by touch40 and gravity41, and similar
changes were observed for CaM too42. Cyclopiazonic
acid, a specific inhibitor of ER- Ca2+-ATPases inhibited
the graviresponse of cress roots, but not their growth43.
The involvement of CaM in graviresponse had also been
demonstrated in Arabidopsis, where wild-type plants
showed a three-fold increase in CaM expression in early
stages of graviresponse, while a decrease in CaM expression was observed in gravity-insensitive mutants44. In
order to observe auxin and Ca2+ interaction, Young and
Evans45 applied Ca2+ chelators to roots and found inhibition of auxin redistribution, showing involvement of Ca2+
in gravitropic response. They suggested that Ca2+ distribution was essential for development of auxin gradient.
Friedman et al.46 have provided evidence for involvement
of Ca2+ in controlling gravitropic bending of cut snapdragon (Antirrhinum majus L.) spikes by using Ca2+ inhibitor, lanthanum chloride. Gravistimulus may be perceived by mechanosensitive ion channels or voltagedependent Ca2+ channels that affect Ca2+ fluxes and alter
protein kinase activities, which in turn may regulate the
activities of enzymes affecting cell-wall extensibility.
This aspect needs further attention to investigate the
ivolvement of Ca2+. Although Ca2+ has been found to be
the key component in graviresponse, several questions
still remain regarding the downstream components of
Ca2+ signalling.
Photosensing
Light-stimulated responses in plants are diverse and involve photoreceptors like phytochrome (PhyA, PhyB,
PhyC, PhyD and PhyE forms in Arabidopsis)47, cryptochrome (CRY1, CRY2)48 and phototropin (NPH1)49. Phytochrome is one of the key photoreceptors for perceiving
CURRENT SCIENCE, VOL. 84, NO. 7, 10 APRIL 2003
red and far-red wavelengths and significant progress has
been made in dissecting its signal transduction pathways50. Red light triggers many developmental events
requiring Ca2+. Some of these have been studied in the
past, including chloroplast rotation in Mougeotia, spore
germination and cell expansion in Onoclea, leaflet closure
in Mimosa, membrane depolarization in Nitella, activation of NAD kinase and inhibition of mitochondrial
ATPase1. Light-mediated responses have been shown to
involve changes in free cytosolic Ca2+ levels51. A direct
link between phytochrome and transient rise in cytosolic
free Ca2+ in leaf chloroplasts of wheat52 as well as in protoplasts of dark-grown wheat seedlings53, was demonstrated.
Bowler et al.54, while examining phytochrome-deficient
mutants of tomato studied the greening of etioplasts to
form fully functional chloroplasts and concluded that
there are three separate but interactive signalling pathways operating via G-proteins, cGMP, Ca2+ and calmodulin. The development of chloroplasts involved the activation of G-protein and combination of cGMP and Ca2+,
and the process was inhibited in the presence of Ca2+ and
calmodulin alone. Earlier, Neuhaus et al.55 obtained similar results using a GUS reporter system which demonstrated that phytochrome phototransduction first involved
the activation of two or more G-proteins which activated
separate signal transduction pathways, one requiring Ca2+
and activated calmodulin, the other being Ca2+-independent. Sopory and Chandok56 suggested that phytochrome alters the levels of cGMP or Ca2+ by interacting
with a G-protein, which could then activate an enzyme
such as guanyl cyclase or phospholipase-C which in turn
produces IP3 and DAG, resulting in increase in cytosolic
Ca2+ and activation of protein kinases (Figure 1). Subsequently, Sanan and Sopory57 reported a role of G-proteins
and Ca2+ in phytochrome signal transduction during primary leaf development in Sorghum bicolor. The leaf
development was observed to be arrested at the stage of
leaf emergence or leaf expansion by the addition of
inhibitors of G-proteins or by Ca2+ channel blockers.
Reports on modulation of Ca2+/CaM protein kinases by
light have been rare. Recently, a novel Ca2+/calmodulindependent protein kinase (ZmCCaMK), characterized
from Zea mays58, was found to be autophosphorylated
and regulated its kinase activity towards substrate phosphorylation in response to red light59. In Arabidopsis
thaliana, AtSR1, a protein kinase which belongs to the
SNF1-related protein kinase subfamily-3 showed accumulation of its transcripts in response to light. It was
also observed to interact with six calcineurin-B-like proteins of Arabidopsis and especially with AtCBL2. The
physiological meaning of the interaction of AtSR1 and
AtCBL2 is not clear, but they presumably function in
signal transduction of light60. Using epidermal strips of
‘Argenteum’ mutant of Pisum sativum, Elzenga et al.61
provided evidence for Ca2+ and CaM involvement in red
and blue light signal transduction pathways. Ca2+-channel
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blockers inhibited the response to both blue and red light,
while Ca2+-ionophores, ionomycin and A23187 reduced
the light response. Furthermore, the light-induced acidification was inhibited by the calmodulin antagonists W-7
and trifluoperazine, but not by W-5. These calmodulin
inhibitors completely inhibited the red light-induced acidi
fication, but inhibited the response to blue light by only
60–70%. This differential effect on red and blue lightinduced responses indicates a role for Ca2+–CaM signalling
in both the red and blue light response, while a second
process, independent of Ca2+ is activated by blue light.
Blue-light regulates plant growth and development and
three photoreceptors, CRY1, CRY2, and NPH1, have been
identified48,49,61,62. The transduction pathways of these
receptors are poorly understood. Baum et al.63 used
transgenic plants containing aequorin to dissect the involvement of these three receptors in the regulation of
intracellular Ca2+. Pulses of blue light-induced cytosolic
Ca2+ transients lasting about 80 s in Arabidopsis and
tobacco seedlings. Use of organelle-targetted aequorin
showed that Ca2+ increases are limited to the cytoplasm.
Blue-light treatment of cry1, cry2 and nph1 mutants
showed that NPH1, which regulates phototropism, is
largely responsible for the Ca2+ transients. The special
response of the Ca2+ transient is similar to that of phototropism, further supporting NPH1 involvement. Christie
and Jenkins64 examined the induction of chalcone synthase (CHS) gene expression by UV-B and UV-A/blue
light in Arabidopsis cell-suspension culture. Both the UV-B
and UV-A/blue phototransduction processes involved
Ca2+, although the elevation of cytosolic Ca2+ was observed to be insufficient on its own to stimulate CHS expression. The UV-A/blue-light induction of CHS expression
did not appear to involve calmodulin, whereas the UV-B
response did involve calmodulin indicating that the signal
transduction pathways were at least in part, distinct. The
UV-B and UV-A/blue-light signalling pathways are
therefore different from the phytochrome signal transduction pathway regulating CHS expression. Long and Jenkins65 investigated the effect of UV and blue light in
signal transduction processes involved in the induction of
CHS and phenylalanine ammonia lyase (PAL) gene expression in Arabidopsis cell-suspension culture. Experiments with electron transport inhibitors indicated that
plasma membrane redox activity is involved in both signal transduction pathways and is coupled to the regulation of Ca2+ release from an intracellular store, generating
a Ca2+ signal that is required to induce CHS expression.
Although participation of Ca2+ and CaM has been established in light-mediated signalling, direct involvement of
protein kinases associated with Ca2+/CaM is awaited.
tant factor by which a living plant adapts itself to these
stresses66. Mechanical signals are important both as environmental and endogenous developmental cues in plants.
During the past few years, Knight and coworkers20, and
Knight and Knight67 have been able to measure shortduration transient rise in cytosolic Ca2+ in plant cells by
developing recombinant tobacco plants that express the
coelenterate Ca2+-indicator, aequorin. Stimulation by wind
resulted in an immediate increase in aequorin luminescence68, probably reflecting increased cytosolic Ca2+.
Some other mechanical stimuli that result in transient
increase in Ca2+, like wounding, also induce a rise in Ca2+
in small, isolated groups of cells some distance from the
wound, as well as at the wound site itself68. Little is known
about the signalling events and components that link perception of mechanical signals to gene expression in
plants. Ca2+ has been identified previously as being
potentially involved, and a role for ethylene has also been
suggested. In Arabidopsis, the expression of some specific genes, like TCH3 was found to be upregulated by
mechanical stimlus21. A role of ethylene, besides Ca2+,
has been suggested from studies using Arabidopsis
mutants ein6, which indicated the requirement of EIN6
protein; but its role in mechanically induced TCH3 expression appears to be independent of ethylene. However,
EIN6 and protein kinase activity operated downstream of
Ca2+ to mediate mechanically stimulated TCH3 expression. The mechanism(s) whereby a plant perceives mechanical stimuli remains almost unresolved. These stimuli,
working on membrane systems at cell level, play an important role not only in thigmomorphogenesis but also in
various responses to environmental factors, including
water, temperature and gravity. This has led to the exploration of a mechano-sensor or a mechano-receptor at
membrane level that perceives these stimuli. The Ca2+
elevation appears to result from movement of the tissue
probably due to mechanosensory Ca2+ channels that detect
flexing and compression of cells. Ca2+ channels in plasma
membranes may be implicated as Ca2+ sensors and evidence favours the organellar channels in this context69.
The stretch-activated Ca2+ channel (Figure 1) was observed
to cluster in response to mechanical stimulus and make
a close association with plant integrin-like proteins70.
Integrins, a type of transmembrane glcoproteins, have
been established to act as mechanosensors in animals71
and their possible action in plants is being explored70. It
appears that a plant has mechanosensors in both the
plasma membrane and the intracellular membrane. In
future studies, it will be necessary to prove this hypothesis as well as to elucidate the mechanism whereby such
mechanosensors can induce multiple responses of a plant to
the environment.
Mechanosensing
Drought sensing
The response to various mechanical stimuli like touch,
wind and injuries due to biotic interference is an impor-
Among the various abiotic stresses, signal transduction of
water stress has invited more attention. Molecules func-
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tioning as osmosensors and abscisic acid (ABA) receptors have not been identified in higher plants as yet and
based upon knowledge of osmosensors in yeast and bacteria, cloning of homologues of the two component histidine kinase as an osmosensor is in progress in higher
plants. ABA occupies a central place in almost all the
stresses causing dehydration of cells and its signal transduction has been the subject of recent interest72,73. ABAsignalling74 has been found to be dependent or independent of Ca2+. Ca2+ involvement in water-stress response
has been shown by identification of stress- and ABAinducible mRNAs that code for a Ca2+-binding membrane
protein in rice75 and for a phosphatidylinositol-specific
phospholipase-C in Arabidopsis76. ABA raised the levels
of cytosolic Ca2+ in hypocotyls and roots of parsley and
coleoptiles of corn77. The link between ABA and Ca2+
was further proved in a study on maize leaf protoplasts,
where a barley HVA promoter was fused to reporter genes
that could be activated by applied ABA or by various
stress conditions78. The expression of the reporter gene
could also be induced by treating the protoplasts with
1 mM Ca2+ and Ca2+ ionophores, or by overexpressing
constitutively active forms of the normally Ca2+dependent protein kinases from Arabidopsis ATCDPK1
and ATCDPK1a, even in the absence of these stimuli.
Stomatal response to water stress has been extensively
studied to investigate the signal transduction pathways in
guard cell79. Stress-induced ABA is perceived at receptors located in the plasma membrane of guard cells or in
the cytosol. ABA elevates the cytosolic Ca2+, (ref. 74),
but the origin of Ca2+ required to increase its cellular
level is unclear80. Evidence has been presented for influx
of Ca2+ across plasma membrane81 or its release from
intracellular stores mediated by IP3 and/or cADPR73.
ABA activates the S-type anion channels through increase in Ca2+ levels and inhibits the inward-rectifying
K+ channels at the same time82. Evidence for Ca2+independent signalling pathways directing stomatal control also exists, and involves rise in pH by ABA83. The
involvement of kinases and phosphatases has been
proved by inhibitor studies in stomatal regulation in Vicia
faba and Commelina84. Both Ca2+-dependent protein
kinases85 and phosphatases86 have been inferred to be
potential candidates in modulating the activity of various
ion channels in guard cells, but their precise roles in
ABA-signalling have not been clearly established at present. In a recent study on a facultative CAM plant, Mesembryanthemum crystallinum, Cushman87 observed that
water stress can induce the CAM pathway and Ca2+ plays
a central role in the signalling events following stress
perception88. The functional role of stress-induced
CDPKs (McCPK1) from M. crystallinum by defining
proteins with which they interact, such as two-component
pseudo-response regulators89, is currently under investigation and will lead to a better understanding of this
adaptation.
CURRENT SCIENCE, VOL. 84, NO. 7, 10 APRIL 2003
Salt sensing
Salinization is a major environmental stress limiting crop
production90. Salt stress disrupts ion homeostasis in plants,
resulting in excess toxic Na+ in the cytoplasm and a deficiency of essential ions such as K+ (ref. 90). Various ion
transporters function to limit Na+ entry into and exit out
of plant cells, to regulate Na+ compartmentation in the
vacuole, and to selectively import K+ over Na+ into plant
cells91. When the plants are subjected to salt stress, some
of the ion transporters need to be activated or to have
their activities enhanced, whereas others (e.g. Na+ influx
transporters) may need to have their activities suppressed. In addition, the transcript levels of many of the
transporters are increased or decreased in response to salt
stress90. A signalling pathway for regulation of ion
homeostasis and salt tolerance has emerged following the
recent cloning and biochemical characterization of several SOS (salt overly sensitive) pathway genes and gene
products from Arabidopsis (Figure 1). The initial receptor for Na+ sensing remains to be identified. SOS1 encodes a plasma membrane-localized Na+/H+ antiporter92.
Thus, the biochemical and physiological function of
SOS1 is to remove Na+ from the cytoplasm and export it
to the extracellular space or the root medium. SOS1 is
indicated to be Na+ sensor and has been suggested to
have a long cytosolic tail, besides having transmembrnae
domains92. The role of SOS1 in K+ acquisition may be
indirect and could possibly arise through H+ coupling
with H+–K+ co-transporters92. The SOS2 gene encodes a
serine/threonine protein kinase with an amino-terminal
catalytic domain and a carboxy-terminal regulatory
domain93. SOS2 has been found to negatively regulate the
expression of AtPLC1 (Arabidopsis phospholipase-C)94.
The SOS3 gene encodes a myristoylated Ca2+-binding
protein95 with a sequence similarity to the regulatory Bsubunit of calcineurin (a Ca2+-dependent protein phosphatase-2B) and neuronal Ca2+ sensors96. SOS3 does not
appear to function through a protein phosphatase. SOS3
physically interacts with and activates a protein kinase
encoded by SOS2 (ref. 97).The sequence of signal events
begins with elevation of Ca2+ in response to salt stress
and its binding with SOS3, which leads to interaction
with SOS2 and activation of the kinases. The components
of the SOS pathway, either SOS3 or upstream elements,
might be associated with an osmotically responsive
channel through which Ca2+ influx could initiate the signalling. The SOS3–SOS2 kinase complex regulates the
transcript levels of SOS1 and activates the Na+/H+ antiporter activity, as observed in isolated plasma membrane
vesicles under salt stress98. It is yet to be ascertained
whether or not SOS3–SOS2 directly regulates the activities of SOS1 and other transporters through phosphorylation. The SOS3/SOS2/SOS1 interaction seems to be
operative only in plants and differs from yeast that has
calcineurin as a downstream target of Ca2+ signalling,
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though a similarity exists between this calcineurin and
SOS3. Among the SOS signal-pathway outputs are transport systems that facilitate ion homeostasis. Ca2+ appears
to have at least two roles in salt tolerance, a pivotal signalling function in the salt-stress response leading to adaptation and a direct inhibitory effect on a Na+ entry
system.
Thermosensing
The understanding of temperature sensing and signalling
is also rudimentary99. Identity of a low temperature (LT)
sensor or receptor linked with elevating cytosolic Ca2+
concentration is not known. Ding and Pickard100 proposed that sensor-receptor for LT may be a Ca2+ channel
in the membrane. Based upon these studies, it was hypothesized that proteins such as annexins, which are Ca2+
and phospholipid-binding proteins, capable of forming
Ca2+ channels in vitro, may be involved in the LT signaltransduction pathway101. Recently, two new isoforms of
wheat annexin protein with molecular mass of 39 and
22.5 kDa have been identified by Breton et al.102, and the
level of both proteins increased rapidly in response to
LT. It has been suggested that a change in membrane
fluidity in response to temperature be used as a biological
thermometer by Synechocystis. Suzuki et al.103 identified
Hik33 and Hik19 as two histidine kinases, necessary for
low temperature induction of desB (one of the three fatty
acid desaturase genes in Synechocystis). The inactivation
of these kinases results in a reduction of transcription of
several low temperature-inducible genes103. Subsequent
experiments by these researchers led to the identification
of a gene, Rer1 (response regulator) that encodes for a
putative protein having domains typical of a response
element in a two-component regulator. The sequence of
events begins with change in membrane fluidity which
activates Hik33 that gets autophosphorylated and transfers the phosphate group to Hik19 (Figure 1) and finally
to Rer1 and induces the expression of desB. Low temperature alters membrane fluidity104 that leads to cold
acclimation through changes in cytoskeleton organization
and the induction of Ca2+ fluxes into the cells105. Whether
these types of sensors are also operational in plants
remains to be determined. Investigations on the chillingsensitive species, Nicotiana plumbaginifolia, demonstrated that cold shock can induce a large transient rise in
cytosolic Ca2+ (ref. 106) and pharmacological studies
suggest the possible involvement of membrane fluidity,
cytoskeleton arrangement and protein phosphorylation in
plant cold responses107. Murata and Los108 suggested that
a primary signal might be the change in membrane fluidity, which is one of the most rapid effects of temperature
on the plasma membrane. The mechanisms by which
reduction in membrane fluidity would lead to gene activation are not understood. A relationship between the
898
membrane fluidity and Ca2+-channel activity at low temperature was proposed108; but so far, no such mediator
has been detected. However, a clear correlation between
temperature and Ca2+ influx into the cells has been demonstrated in Arabidopsis, suggesting that a temperaturemodulated Ca2+ channel could indeed be involved in
temperature sensing. Mechanosensitive Ca2+ channels
exhibiting temperature-dependent modulation have been
identified in plants and they might be involved in lowtemperature sensing70. Further evidence for the role of
plasma-membrane fluidity in cold sensing has come from
experiments in alfalfa and Brassica107, where chemical
agents were used to modulate membrane fluidity. Membrane fluidization during cold treatment inhibited the
induction of cold-inducible genes and the development of
freezing tolerance. The effects of gene expression were
apparently mediated by reorganization of cytoskeleton
and a following Ca2+ influx channel activity at low temperature. The presence of low temperature-induced Ca2+dependent protein kinases (CDPKs) in Arabidopsis109 and
alfalfa110, and the fact that their inhibition prevents cold
acclimation111 supports the connection between changes
in cytosolic Ca2+ levels and protein phosphorylation.
ABA has been implicated in several stress responses and
is involved in low-temperature response too that leads to
its transient increase112, which in turn triggers downstream events leading to target gene expression. Ca2+ is
suggested to act as a signal transducer, which is released
through cADPR-controlled release from intracellular
stores113. Both protein kinases and phosphatases have
been involved in these events, as observed in phosphatase
mutants114. Calmodulin acts as a signalling component
during cold induction of COR (cold on regulated) genes
(LTI78 and KIN1/2) in Arabidopsis109 and COR genes
require Ca2+ for expression106. A recent study by Townley
and Knight115 has shown that overexpression of calmodulin in plants causes inhibition of COR gene expression,
indicating that CaM may negatively regulate the COR
gene expression. Thus, the possibility is raised that
calmodulin might also act as a negative agent with respect to COR gene expression. In this case, in addition to
positive Ca2+-dependent pathways leading to increased
COR gene expression at low temperature106,109, there
would also be negative Ca2+-dependent pathways involving calmodulin. The mechanism of action of CaM under
such circumstances may involve activation of Ca2+ATPases, causing Ca2+ efflux and reducing its intracellular concentration116. This observation is of immense importance in signal engineering for developing LT-tolerant
plants.
Time sensing
Plants show many rhythmic phenomena like leaflet and
petal movement, stomatal conductance, photosynthetic
CURRENT SCIENCE, VOL. 84, NO. 7, 10 APRIL 2003
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rate, ion flux and gene expression117. The biological
clock has been suggested to be comprised of three components. The first component (the entrainment pathway)
couples the second component (the autonomous regulator) to the environmental periodicity and determines the
phase of the free-running rhythm. The third component
(the output pathway directed by the oscillator) gives rise
to the overt biological rhythm118. Johnson et al.119, while
working on transgenic tobacco and Arabidopsis plants
(having aequorin), observed circadian oscillations in free
cytosolic Ca2+ that can be phase-shifted by light–dark
signals. When apoaequorin was targetted to the chloroplast, Ca2+ rhythms were likewise observed after transfer
of seedlings of these plants to constant darkness. Circadian oscillations in free Ca2+ concentrations can be
expected to control many Ca2+-dependent enzymes and
processes accounting for circadian outputs. In an attempt
to find out whether the plants possess a master clock directing a multitude of diverse rhythmic outputs or whether
multiple circadian oscillators exist, either within single
cells or distinct morphological structures, Wood et al.118
used transgenic plants of tobacco (Nicotiana plumbaginifolia) containing aequorin to investigate the involvement
of cytosolic or nuclear Ca2+ in circadian rhythms. These
plants showed luminescence, thereby directly indicating
Ca2+ oscillations when challenged with environmental
stimuli. Measurement of cytosolic and nuclear Ca2+
showed that circadian variations in the cytoplasm are not
expressed in the nucleus. The Ca2+ rhythmicities of cells
and tissues oscillated with distinct differences in phase
and this might represent different underlying cellular control mechanisms. Circadian changes in Ca2+ are likely to
emanate from a number of different cell or tissue locations within the plant and different tissues and cells generate different rhythmic pattern in Ca2+ (ref. 118). Further, experiments have indicated that circadian variation
in Ca2+-regulated genes is not transcriptionally controlled
by nuclear Ca2+, although a cytoplasmic transduction
mechanism operating through Ca2+ was not precluded.
There may be multiple copies of independent oscillators
in different plant tissues and expressing circadian controls through regulation of Ca2+ rhythmicities.
CAM pathway is controlled by a circadian rhythm and
Taybi et al.120 working on this metabolism have recently
cloned and characterized a CDPK-related protein kinase
responsible for phosphorylating phosphoenolpyruvate
carboxylase (PEPC) kinase, a key component of the CO2
pump. In CAM plants, nocturnal phosphorylation renders
PEPC considerably less sensitive to inhibition by negative effectors, but both more active and more sensitive to
activation by positive effectors. Expression of PEPC
kinase is controlled by a circadian oscillator that largely
restricts its own mRNA and protein expression to the
night. Circadian control of PEPC kinase provides one of
the key regulatory steps in controlling the competing
actions of PEPC and ribulose 1,5-bisphosphate carboxyCURRENT SCIENCE, VOL. 84, NO. 7, 10 APRIL 2003
lase/oxygenase. The role of calcium in entrainment and
resetting the clock requires to be examined.
Engineering Ca2+ expression
Efforts are being made to manipulate signalling components to develop stress-tolerant plants121. It may be possible by regulating signal pathways that control tolerance
effectors or modulate effector activity or efficacy. Ca2+
expression is also being modulated by targetting Ca2+
transporters and protein kinases to alter their stress sensitivity. An enzyme glyoxalase I, which catalyses the conversion of toxic methylglyoxal to a nontoxic metabolite,
is expressed in response to NaCl, mannitol or abscisic
acid122. Glyoxylase I from Braasica juncea (BjGly I)
binds CaM-sepharose and its activity is stimulated by
Ca/CaM123. Leaf discs from transgenic plants expressing
BjGly I showed tolerance to methylglyoxal and salt compared to control antisense and wild-type leaf discs, indicating that BjGly I plays a role in conferring tolerance to
salt stress in plants123. Another example is of inducing
salt tolerance by affecting SOS signalling pathway,
which, as described above, is Ca2+-activated and maintains Na+ and K+ homeostasis. SOS2 kinase requires Ca2+
for activation and its kinase activity is essential for salttolerance determinant function124. The SOS2 C-terminal
regulatory domain interacts with the kinase domain to
cause auto-inhibition. Deletion of its auto-inhibitory
domain or site-specific modifications (Thr to Asp mutation) to the catalytic domain resulted in constitutive expression of SOS2 kinase94, opening the possibility of
regulation of stress signalling that controls ion homeostasis. Likewise, modulating the expression or activation
of SOS1 (Na+/H+ antiporter on plasma membrane) would
increase salt tolerance. The SOS1 gene has been shown to
encode a putative plasma membrane Na+–H+ antiporter in
Arabidopsis92. Mutations in SOS1 result in increasing the
sensitivity to Na+ stress, while its over-expression lowers
the Na+ content in shoots and improves salt tolerance.
Calcineurin, a Ca2+- and CaM-dependent protein phosphatase, as studied in yeast, is required for regulating
homeostasis of Na+, K+ and Ca2+. Loss of function mutations in CnB (a regulatory subunit of calcineurin) makes
the yeast cells more sensitive to Na+ inhibition125. Calcineurin regulates the K+ transport system under salt
stress in yeast by directly or indirectly regulating the
phosphorylation of TRK1, a high-affinity K+ transporter
in yeast cells125. In higher plants, calcineurin-like activity
has been reported and appears to regulate the inward K+
channel activity86 in guard cells, which may lead to
modulation of stomatal control by manipulation of this
putative phosphatase. Constitutive activation of yeast
calcineurin in the host or in plants increased salt tolerance by predisposing the plants to survive stress125,126. In
a recent study, a cDNA encoding a novel Ca2+-binding
899
REVIEW ARTICLE
protein from Entamoeba histolytica (EhCaBP) was transferred into tobacco, resulting in enhanced tolerance to
salt127. Cold-tolerance response has been increased by
antisense inhibition of the gene for protein phosphatase
2CA(PP2CA)128. Transgenic plants altered for components of Ca2+ signalling have been observed to show
enhanced production of activated oxygen species129,
tolerance to heavy metals130, cold, salt and drought in
rice126. Engineering IP3 functioning has been a target to
control Ca2+ expression131. The genes encoding two of
the phosphatases have been cloned and transgenic plants
constructed having altered levels of inositol phosphatases
and presumably of IP3 levels as well. This work is exciting in that IP3, as a common component of signalling, is
an excellent target for manipulating signals from a variety of pathways, and it may be possible to modulate
responses of plants to a variety of environmental stresses
and several other responses like disease resistance,
fertility and hormone perception. In an interesting and
novel study being undertaken in the North Carolina
State University (NCSU) laboratory of Robertson, the
utility of a high-capacity Ca2+-binding domain of calreticulin has been demonstrated, which might increase
Ca2+ stores in plants. The Arabidopsis plants expressing a
fusion protein of the C-domain of maize calreticulin and
the green fluorescent protein (GFP) showed a delayed
loss of chlorophyll after transfer to Ca2+-depleted medium, 9–35% increase in total Ca2+ for induced C-domain
plants compared to controls and, unexpectedly, a significantly increased salt and heavy-metal (aluminum) tolerance132.
Manipulation of mechanisms regulating heavy-metal
transport was conducted by transferring an Arabidopsis
gene, CAX1 (for Ca2+ exchanger 1) into tobacco, which
resulted in increasing stress sensitivity133. Subsequently,
CAX2 (having low affinity for Ca2+ in yeast) was introduced into tobacco, which resulted in increased tolerance
to high manganese levels, and plants were also able to
accumulate more Ca2+, cadmium and manganese in the
media in which they were grown; but they remained
healthy. The CAX2-modified plants also had much higher
cadmium and manganese transport in isolated root cell
membrane vesicles. The present study has an important
implication for developing tolerant plants effective in
phytoremediation.
Perspectives
Calcium research has entered a ‘golden age’ and tremendous advances are being made in our understanding of
the involvement of Ca2+ in cellular responses to environmental factors. The next few years will witness the identification of many novel stimulus-specific Ca2+/CaMdependent protein kinases and their genes, which will
immensely increase our knowledge about functioning of
900
Ca2+ in plants. The genes for various Ca2+ transporters
are being identified and will hold the key to regulate
cytosolic Ca2+ expression. The connections between
membrane receptors and intracellular Ca2+ regulation still
remain enigmatic and will be an important subject of
research in future. ‘Listening’ to ‘cross-talk’ involving
Ca2+ within cells, between cells, tissues and organs will
be exciting, to know the coordination among them in response to various stimuli. The complex web of calcium
signalling in plants has been equated with neural network
of animals, and it may be a part of ‘intelligence’ and
‘learning’ behaviour of the plants to decide their response
to at least 17 different environmental factors and their
combinations134. As the specificity of Ca2+ signalling for
specific environmental signals gets unveiled, it will be
possible to precisely engineer the Ca2+ expression in
plants to make them more tolerant to a stressful environment.
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