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Plant Science 160 (2001) 381– 404
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Review
Calcium: silver bullet in signaling
A.S.N. Reddy *
Department of Biology and Program in Cell and Molecular Biology, Colorado State Uni6ersity, Fort Collins, CO 80523, USA
Received 19 July 2000; received in revised form 5 September 2000; accepted 5 September 2000
Abstract
Accumulating evidence suggests that Ca2 + serves as a messenger in many normal growth and developmental process and in
plant responses to biotic and abiotic stresses. Numerous signals have been shown to induce transient elevation of [Ca2 + ]cyt in
plants. Genetic, biochemical, molecular and cell biological approaches in recent years have resulted in significant progress in
identifying several Ca2 + -sensing proteins in plants and in understanding the function of some of these Ca2 + -regulated proteins
at the cellular and whole plant level. As more and more Ca2 + -sensing proteins are identified it is becoming apparent that plants
have several unique Ca2 + -sensing proteins and that the downstream components of Ca2 + signaling in plants have novel features
and regulatory mechanisms. Although the mechanisms by which Ca2 + regulates diverse biochemical and molecular processes and
eventually physiological processes in response to diverse signals are beginning to be understood, recent studies have raised many
interesting questions. Despite the fact that Ca2 + sensing proteins are being identified at a rapid pace, progress on the function(s)
of many of them is limited. Studies on plant ‘signalome’ — the identification of all signaling components in all messengers
mediated transduction pathways, analysis of their function and regulation, and cross talk among these components — should help
in understanding the inner workings of plant cell responses to diverse signals. New functional genomics approaches such as reverse
genetics, microarray analyses coupled with in vivo protein– protein interaction studies and proteomics should not only permit
functional analysis of various components in Ca2 + signaling but also enable identification of a complex network of interactions.
© 2001 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Calcium; Calcium-binding proteins; Calmodulin; Protein kinase; Cell division; Calmodulin-binding proteins; Cytoplasmic streaming;
Molecular motors; Kinesin; Myosin; Signal transduction; Stress; Plant defense
1. Introduction
Plant growth and development is controlled by
hormonal and environmental signals. Plants, unlike animals, are immobile and therefore have
developed mechanisms to sense and respond to the
Abbre6iations: ABA, abscisic acid; AOS, active oxygen species;
[Ca2 + ]cyt, cytosolic Ca2 + ; CaM, calmodulin; CBP, calmodulin-binding protein; CBD, calmodulin-binding domain; CCaMK, Ca2 + /
CaM-dependent protein kinase; CDPK, Ca2 + -dependent protein
kinase; CRK, CDPK-related protein kinase; CIPK/SIPK, CBL/
SOS3-interacting protein kinase; GAD, glutamate decarboxylase;
KCBP, kinesin-like calmodulin-binding protein; PDE, phosphodiesterase.
* Tel.: + 1-970-4915773; fax: + 1-970-4910649.
E-mail address: [email protected] (A.S.N. Reddy).
biotic and abiotic stresses so that they can better
adapt to their environment. How plants sense
these various signals and produce an appropriate
response has fascinated plant biologists over a
century and has become an area of intense investigation in recent years. Research during the last
two decades has clearly established that Ca2 + acts
as an intracellular messenger in coupling a widerange of extracellular signals to specific responses.
Although Ca2 + is implicated in regulating a number of fundamental cellular processes that are
involved in cytoplasmic streaming, thigmotropism,
gravitropism, cell division, cell elongation, cell differentiation, cell polarity, photomorphogenesis,
plant defense and stress responses, the mechanisms
0168-9452/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved.
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A.S.N. Reddy / Plant Science 160 (2001) 381–404
by which Ca2 + controls these processes are only
beginning to be understood. Because of the space
limitations, my intention here is to summarize
recent progress in understanding Ca2 + -mediated
signal transduction pathways with emphasis on
the current status of research, gaps in our knowledge and future directions.
2. Signals and cytosolic Ca2 +
Improved methods to monitor free [Ca2 + ]cyt
levels, especially using transgenic plants expressing
Ca2 + reporter proteins, have greatly helped in
demonstrating signal-induced changes in free
[Ca2 + ]cyt level [1 –4]. The concentration of Ca2 +
in the cytoplasm of plant cells is maintained low in
the nanomolar range (100 –200 nM) [4,5]. However, Ca2 + concentration in the cell wall and in
organelles is in the millimolar range (Fig. 1) [6,7].
Fig. 1. Schematic diagram illustrating the mechanisms by
which plant cells elevate [Ca2 + ]cyt in response to various
signals and restore Ca2 + concentration to resting level. Ca2 +
channels are shown in red, whereas Ca2 + ATPases and
antiporters are indicated in yellow. Arrows indicate the direction of Ca2 + flow across the plasma membrane, and into and
out of cellular organelles (vacuole, plastids, mitochondria,
endoplasmic reticulum and nucleus). The estimated concentration of resting levels of Ca2 + in different organelles is
indicated [5,6,9,234]. Question marks indicate the lack of
evidence. [Ca2 + ]cyt, cytosolic Ca2 + ; PLC, phospholipase C;
R, receptor, cADPR, cyclic ADP ribose, PIP2, phosphotidyl
inositol-4,5-bisphosphate, DG, diacylglycerol, PKC, protein
kinase C, IP3, inositol-1,4,5-trisphosphate; ER, endoplasmic
reticulum; Mt, mitochondria; Plast, plastids; PM, plasma
membrane.
Despite the existence of a large electrochemical
gradient for Ca2 + entry into the cytoplasm, plant
cells maintain their [Ca2 + ]cyt concentration at low
levels, which requires active pumping of Ca2 + to
the apoplast or organelles.
A wide-range of signals such as light, hormones,
gravity, touch, wind, cold, drought, oxidative
stress and fungal elicitors have been shown to
cause transient elevation of [Ca2 + ]cyt (see Table 1).
A comprehensive discussion of signal-induced
changes in [Ca2 + ]cyt has been published recently
and will not be covered here [4,7 –9]. Current
evidence indicates that 1,4,5-trisphosphate (IP3),
cyclic ADP ribose (cADPR) and Ca2 + channels
play an important role in elevating [Ca2 + ]cyt (Fig.
1). Ca2 + channels have been detected in the
plasma membrane, vacuolar membrane, ER,
chloroplast and nuclear membranes of plant cells
[5]. These channels are classified based on their
voltage dependence. The electrophysiological
properties of all the known Ca2 + channels have
been reviewed recently [5]. However, genes encoding Ca2 + channels in plants have not been
identified.
In plants, as in animals, phospholipase C-mediated hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2) results in the production of
inositol-1,4,5-trisphosphate (IP3) and diacylglycerol. There is some indirect evidence for the involvement of heterotrimeric G proteins in
promoting PIP2 hydrolysis in plants [10,11]. Phosphatidylinositol-specific phospholipase C (PI-PLC)
activity and genes encoding this enzyme have been
characterized from plants [12,13]. Plant PI-PLC
hydrolyzes phosphatidylinositol-4,5-bisphosphate
into IP3 and diacylglycerol with an absolute requirement for Ca2 + (1 mM) [12]. It was shown
that one of the phospholipase C genes (AtPLC1 )
expression is induced by stresses including dehydration, salinity and low temperature [12,14]. Recently, three PI-PLC isoforms (StPLC1 to -3 )
have been isolated from guard cell enriched tissue
of potato [13]. The expression pattern of the
StPLC1 and -2 genes also suggest their involvement in drought stress in potato [13]. The soybean
PLC showed phospholipase activity and complemented the lethal mutant phenotype of yeast lacking PLC activity. Although there is overwhelming
evidence for signal-induced changes in [Ca2 + ]cyt,
the information on the mechanisms by which a
given signal elevates [Ca2 + ]cyt is still limited. Little
A.S.N. Reddy / Plant Science 160 (2001) 381–404
383
Table 1
Effect of signals on cytosolic calcium in plants
Signal
Methods used to measure free
cytosolic Ca2+ a
Effect on
[Ca2]cyt b
Response
References
Abscisic acid
Gibberellic
acid
Auxin
NaCl
1, 2, 3
2
, ¡
Stomatal closure, gene expression
a-Amylase secretion
[3,20–23]
[24,25]
1
1, 2, 3
[36]
[27–31]
Anoxia
1
Touch
Wind
Gravity
Cold
1, 2
1, 2
1
1, 2
Drought
1, 2
Heat Shock
Hypoosmotic
stress
Red light
Ozone stress
Oxidative
stress
(H2O2)
Aluminum
Pathogens
and
elicitors
NOD factors
1, 2
2
Cell elongation and cell division
Gene expression and osmolyte synthesis,
K+ uptake
Gene activation, adaptation to oxygen
deprival
Thigmomorphogenesis
Morphogenesis
Gravitropism
COR gene expression, proline synthesis,
changes in membrane lipid profile and
cold acclimatization
Gene expression, synthesis of
osmoprotectants, and osmotolerance
Thermotolerance
Osmoadaptation
1
2
1, 2
Photomorphogenesis
Production of AOSs
Production of AOSs, HR and cell death
[49]
[50]
[51–55]
1, 2
1, 2
, ¡
Ion imbalance
Phytoalexin biosynthesis and induction of
HR
[56–59]
[1,53,60–62]
1
Nodular formation and root hair curling
[63]
[32,33]
[1,34–36]
[37–39]
[26]
[40–42]
[2,30,43]
[44,45]
[46–48]
a
Cytosolic free calcium levels are measured using injection of fluorescent indicator dyes (1), transgenic plants expressing
aequorin (2) or cameleon (3).
b
Increase ( ) or decrease (¡) in cytosolic free calcium levels. COR, cold-r6 egulated; AOSs, active oxygen species; HR,
hypersensitive response and [Ca2]cyt, cytosolic free calcium.
is known about the mechanisms that are involved
in the activation of phospholipase C and regulation of Ca2 + channels.
IP3 has been shown to stimulate Ca2 + release
from vacuolar store [9,15,16]. In animals, IP3 releases Ca2 + primarily from the endoplasmic
reticulum (ER) whereas in plants Ca2 + is released
from vacuoles. Ca2 + release from the ER elicited
by IP3 has also been reported in plants [9]. IP3 −
and cyclic ADP-ribose (cADPR)-gated channels
that are found on the ER membrane in animals
are found in the vacuolar and ER membranes in
plants [9,17,18]. Recently, nicotinic acid adenine
dinucleotide phosphate has been shown to release
Ca2 + exclusively from plant ER [19]. These results
suggest that multiple Ca2 + mobilization pathways
that are regulated by different agents exist in
plants. In animals, diacylglycerol, the second
product of PIP2 hydrolysis, is an activator for
protein kinase C. Although there are some reports
indicating the presence of protein kinase C-like
activity in plants, the gene encoding it has not
been isolated [64].
Elevation of [Ca2 + ]cyt in response to signals
could be due to influx of Ca2 + from the apoplast
and/or Ca2 + release from intracellular stores (ER,
vacuoles, mitochondria, chloroplasts and nucleus).
Based on the type of signal or cell type internal
and/or external Ca2 + stores could be involved in
raising [Ca2 + ]cyt. The contribution of external
Ca2 + and cellular organelles in elevating cytosolic
Ca2 + in response to various signals is beginning to
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A.S.N. Reddy / Plant Science 160 (2001) 381–404
be identified. Transgenic plants expressing a Ca2 +
reporter targeted to different organelles and the
use of different pharmacological agents that block
or release Ca2 + from internal stores have allowed,
in some cases, analysis of changes in organellar
Ca2 + concentration as well as the contribution of
these organelles in elevating [Ca2 + ]cyt [4,62]. These
studies indicate that different signals use distinct
Ca2 + stores in elevating [Ca2 + ]cyt. Cold-induced
Ca2 + increase is inhibited by plasma membrane
channel blockers, but is not affected by organellar
channel blockers. However, wind-induced Ca2 +
increase is blocked by organellar Ca2 + channel
blockers whereas plasma membrane channel
blockers did not have any effect [37], indicating
that the extracellular Ca2 + contributes to cold-induced elevation of Ca2 + and internal Ca2 + stores
contribute to wind-induced increase in [Ca2 + ]cyt.
By targeting aequorin to cytoplasm and nuclear
organelles, Van der Luit et al. [39] have shown
that distinct cellular Ca2 + pools respond to wind
and cold stimuli. Sodium chloride-induced
[Ca2 + ]cyt is due to Ca2 + release from the vacuole
[1,30]. Transgenic Arabidopsis seedlings in which
aequorin is targeted to the cytoplasmic face of the
tonoplast membrane [2] showed release of vacuolar Ca2 + in response to mannitol treatment [30].
In parsley cells, elicitor-induced sustained increase
in [Ca2 + ]cyt is primarily due to the influx of extracellular Ca2 + [62]. Studies with maize suspensioncultures have shown that mitochondrial Ca2 +
store contributes to anoxia induced [Ca2 + ]cyt [33].
ABA-induced changes in [Ca2 + ]cyt have been attributed to both Ca2 + release from internal stores
and Ca2 + influx from external stores [65,66]. Recently, hydrogen peroxide (H2O2) has been shown
to activate Ca2 + -permeable channels in the
plasma membrane of Arabidopsis guard cells [55].
Furthermore, ABA induced H2O2 production in
guard cells. However, H2O2-induced stomatal closing and activation of Ca2 + channels by H2O2 are
disrupted in an ABA-insensitive mutant, suggesting that ABA-induced H2O2 and the H2O2-activated Ca2 + channels are important in elevating
[Ca2 + ]cyt in response to ABA [55].
It is not yet known to what extent the changes
in the [Ca2 + ]cyt levels are reflected in changes in
free Ca2 + concentration in the nucleus. Recent
studies show that there is a Ca2 + gradient between
the nucleus and cytoplasm indicating the presence
of regulatory mechanisms that control Ca2 +
movement into and out of the nucleus [67]. ATP
stimulates Ca2 + uptake into nuclei and studies
implicate CaM involvement in this uptake process
[67]. Currently, little is known about the participation of nuclear Ca2 + stores in increasing cytosolic
Ca2 + and vice versa. Ca2 + uptake studies with
isolated plant nuclei have shown that the transport
of Ca2 + across nuclear membrane is an ATP-dependent process [68], suggesting that changes in
cytosolic and nuclear Ca2 + may occur independently. Recently, using tobacco plants expressing
Ca2 + reporter either in the cytosol or nucleus, it
has been shown that changes in Ca2 + concentration in the nuclear compartment and cytosol are
controlled independently [69]. These authors have
also shown that the nuclear membrane is not
passively permeable to Ca2 + .
The signal-induced Ca2 + elevations are transient and the level of Ca2 + returns to the resting
level, which requires the removal of Ca2 + from
the cytosol. This requires Ca2 + transport into
organelles against a concentration difference of
103 to 104 fold. Ca2 + homeostasis is achieved by
high affinity Ca2 + -pumps (Ca2 + -ATPases) and
low affinity Ca2 + /H+ antiporters (Fig. 1). These
Ca2 + pumps and antiporters also play a crucial
role in raising and restoring [Ca2 + ]cyt levels in
response to various stimuli. Several Ca2 + -ATPases that belong to E6 R-type C6 a2 + -A6 TPases
(ECA) and a6 utoinihibited C6 a2 + -A6 TPases (ACA)
have been characterized from plants [70]. There
are at least 12 Ca2 + -ATPases in plants of which
eight belong to an ACA-type. In animals, autoinhibited Ca2 + -ATPases, which are regulated by
Ca2 + /CaM, are exclusively in the plasma membrane whereas in plants they are primarily localized in endomembrane systems [70]. Unlike animal
endomembrane Ca2 + -ATPases that lack calmodulin-binding domain, plant counterparts have a
calmodulin-binding domain either at the N- or
C-terminus [70 –73]. Recently, Ca2 + -ATPases that
are regulated by Ca2 + /CaM have been found in
the plant plasma membrane also [74,75]. However,
the structural organization of plasma membranelocalized Ca2 + -ATPases in plants is different from
that of animal counter parts [74,75]. The calmodulin-binding autoinhibitor region in plant plasma
membrane-localized Ca2 + -ATPases is located in
the N-terminus whereas it is in the C-terminal
region in animal plasma membrane-localized
Ca2 + -ATPases, suggesting that plant ATPases
A.S.N. Reddy / Plant Science 160 (2001) 381–404
have unique structural organization. The ACAtype pumps are CaM regulated where, in the
absence of activated CaM, the CaM-binding domain acts as an autoinhibitor. Binding of CaM to
the autoinhibitor region causes activation of the
enzyme, suggesting an important role for Ca2 + in
regulating these pumps. CaM-regulated autoinhibited Ca2 + -ATPases are found in the tonoplast, ER
and most recently in the plasma membrane [70 –
72,74,75]. The large number of Ca2 + -ATPases
indicates complex mechanisms in regulating Ca2 +
homeostasis in plants. Interestingly, the activity of
the CaM-regulated ER ATPase in Arabidopsis is
inhibited by a Ca2 + -dependent protein kinase,
suggesting that Ca2 + both activates (through
CaM) and inhibits (via CDPK) the activity of ER
ATPase [76]. The phosphorylation site on the ATPase was mapped to Ser45 near the CaM-binding
domain. Vacuolar localized Ca2 + /H+ antiporters
(calcium ex6 changer) have been identified from
Arabidopsis (CAX1 and CAX2 ) [77] and mung
bean [78]. The Arabidopsis CAX1 gene functionally complements a yeast mutant defective in its
antiporter activity [77]. CAX1 and CAX2 are high
and low efficiency H+/Ca2 + exchangers, respectively. CAX transcripts are inducible by extracellular Ca2 + levels, Na+, K+, Ni2 + , PEG and Zn2 +
but not by plant hormones. The transgenic plants
expressing the CAX1 gene in sense orientation
showed several abnormalities including stunted
growth with poorly developed root system, necrosis of leaves and apical meristem, hypersensitivity
to K+ and Mg2 + ions, and sensitivity to cold that
could be reversed by Ca2 + supplementation [79].
These results indicate that the increased antiport
activity of the CAX1 gene causes severe depletion
in free cytosolic Ca2 + levels due to pumping of
Ca2 + from cytosol to vacuole. Known Ca2 +
channels, pumps and antiporters that are involved
in Ca2 + homeostasis are shown in the Fig. 1.
3. Ca2 + sensors
Transient Ca2 + increase in the cytoplasm in
response to signals is sensed by an array of Ca2 + sensors (Ca2 + -binding proteins) which decode
Ca2 + signal. Once Ca2 + sensors decode the elevated [Ca2 + ]cyt, Ca2 + efflux into the cell exterior
and/or sequestration into cellular organelles such
as vacuoles, ER and mitochondria restores its
385
levels to resting state. A large number of Ca2 +
sensors have been characterized in plants, which
can be grouped into four major classes [80,81].
These include (A) calmodulin (CaM), (B) CaMlike and other EF-hand containing Ca2 + -binding
proteins, (C) Ca2 + -regulated protein kinases, and
(D) Ca2 + -binding proteins without EF-hand motifs. Members of first three classes of Ca2 + sensors
contain helix-loop-helix motif(s) called EF hands
that bind to Ca2 + with high affinity [82]. However, different Ca2 + -binding proteins differ in the
number of EF hand motifs and their affinity to
Ca2 + with dissociating constants (Kds) ranging
from 10 − 5 to 10 − 9 M. Binding of Ca2 + to the
Ca2 + sensor results in a conformational change in
the sensor resulting in modulation of its activity or
its ability to interact with other proteins and modulate their function/activity.
3.1. Calmodulin
CaM is a highly conserved, well-characterized
and ubiquitous Ca2 + receptor in eukaryotes
[82,83]. It is a small molecular weight acidic
protein of 148 amino acids with four EF-hand
motifs that bind to four Ca2 + ions (Fig. 2A). The
crystal structure of CaM indicates that it has two
globular domains, each with a pair of EF hands,
connected by a central helix [84]. The binding of
Ca2 + to CaM results in a conformational change
in such a way that the hydrophobic pockets of
CaM are exposed in each globular end which can
then interact with target proteins [85,86]. In plants,
there are multiple CaM genes that code for either
identical proteins or contain a few conservative
changes [81,83,87,88]. These small changes in
amino acid composition of CaM isoforms may
contribute to differential interaction of each CaM
isoform with target proteins. There is considerable
evidence to indicate that CaM genes are differentially expressed in response to different stimuli
[81,83]. Such differential regulation is likely to be
one of the mechanisms for cells to fine-tune Ca2 +
signaling.
Although it is preliminary, recent studies on
CaM genes expression in response to different
stimuli indicate that different CaM isoforms are
involved in mediating a specific signal [81]. Three
of the six Arabidopsis Cam genes (Cam1, -2 and
-3 ) are inducible by touch stimulation [81] indicating the presence of different cis-regulatory ele-
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A.S.N. Reddy / Plant Science 160 (2001) 381–404
ments in their promoters. The presence of multiple
CaM isoforms in plants adds further complexity to
the Ca2 + mediated network and points to their
differential sensitivity to elevated [Ca2 + ]cyt levels
in response to different stress stimuli. In potato,
only one of the eight CaM isoforms (PCaM1) is
inducible by touch [87]. The striking example for
differential regulation of CaMs comes from the
studies with soybean CaM isoforms. In soybean
there are five CaM isoforms (SCaM1 to −5).
SCaM1, -2 and -3 are highly conserved compared
to other plant CaM isoforms including Arabidopsis CaM isoforms whereas SCaM4 and -5 are
divergent and showed differences in 32 amino
Fig. 2.
A.S.N. Reddy / Plant Science 160 (2001) 381–404
acids with the conserved group [89]. Surprisingly,
these divergent CaM isoforms are specifically induced by fungal elicitors or pathogen [90]. These
results provided evidence for the differential regulation of CaM isoforms in plants. Soybean isoforms show differences in their relative abundance
in vivo. The conserved isoforms are relatively
abundant in their expression compared to divergent forms. All CaM isoforms activate phosphodiesterase (PDE) but differ in their activation of
NAD kinase, calcineurin and nitricoxide synthase
indicating Ca2 + /CaM specificity between CaM
isoforms and target proteins [91]. Differential regulation of other enzymes by soybean divergent
and conserved CaM isoforms has also been reported [92]. Although SCaM isoforms show similar patterns in blot overlay assays, they differ in
their relative affinity in interacting with CaMbinding proteins [93]. Two divergent CaM isoforms that are found in Arabidopsis do not
interact with proteins that bind to conserved CaM
isoforms [94]. These studies suggest that conserved
and divergent CaM isoforms may interact with
different target proteins.
Using agonists and antagonists of the Ca2 +
signaling pathway, Ca2 + and CaM have been
implicated in plant defense against pathogens
[7,95]. Because the pharmacological agents have
non-specific effects and because the targets of
these agents are not clearly defined some of these
387
studies are not conclusive. More recent studies
with transgenic plants overexpressing CaM and
CaM isoforms have provided strong evidence for
the involvement of CaM in plant defense responses. Transgenic plants expressing a mutated
CaM with a single amino acid change (K115 to R)
in the CaM sequence, which abolishes trimethylation of lysine at 115, showed increased levels of
resistance to a variety of stimuli such as bacterial
derived elicitor harpin, mechanical stress, and osmotic stress [96]. Furthermore, transgenic cell lines
treated with cellulase or mechanical stress produced increased levels of AOSs compared to control cell lines. Production of AOSs has been shown
to initiate a battery of defense responses against
invading pathogens [53,60,61,97]. The enhanced
resistance in transgenic plants is primarily attributed to their ability to hyperactivate NAD
kinase [96].
Constitutive expression of divergent CaM isoforms SCaM4 and -5 in tobacco has also indicated
a role of some CaM isoforms in plant defense
against fungi and TMV. Conserved CaMs
(SCaM1, -2 and -3) are not inducible by stress
where as divergent CaM isoforms are normally
expressed at a low level and are highly inducible in
response to pathogen attack or elicitors. Transgenic plants expressing high levels of SCaM4 and
-5 showed constitutive expression of salicylic acid
related gene expression independent of SA
Fig. 2. (A) CaM and other EF-hand motif-containing proteins from plants. AtCaM2 [98]; one of the six isoforms of CaM from
Arabidopsis; At Centrin, one of the three centrins from Arabidopsis [186]; CCD-1, C-terminal centrin-like domain [235];
AtCBP22, Arabidopsis C6 alcium-B6 inding P6 rotein 22 [236]; AtTCH2 Arabidopsis T6 ouch 26 [237]; AtTCH3, Arabidopsis T6 ouch 36
[169]; PhCaM53, Petunia hybrida Calm6 odulin 53 [165]; ABI1, Arabidopsis ABA i6 nsensitive 16 [171,172], AtCBL1 to 3, Arabidopsis
C6 alcineurin B6 -L6 ike-16 , -26 and -36 proteins [157]; AtCBL4, Arabidopsis CBL4 or S6 alt-O6 verly-S6 ensitive3 (SOS3) protein [31]; AtCP1,
salt-induced Arabidopsis C6 alcium-Binding P6 rotein [173]; OsEFA27, rice EF hand protein responsive to A6 bscisic acid [170];
PvHRA32, bean H6 ypersensitive R6 eaction A6 ssociated protein [178], AtRBOHA, Arabidopsis R6 espiratory B6 urst O6 xidase
H6 omologue A6 and BetV-4, birch pollen allergen [238]. Asterisks in CBLs indicate the myristoylation motif. Interruptions in the
AtRBOHA protein are denoted by ‘//’. (B) Schematic representation of Ca2 + -regulated protein kinases and CBPs from plants.
CDPK, C6 alcium-d6 ependent and calmodulin-independent p6 rotein k6 inase [194,196]; C6 RK, C6 DPK-r6 elated protein kinase [239];
CaMK (MCK1), m6 aize C6 aM-dependent protein k6 inase [104], CaMK (CB1), apple CaM-dependent p6 rotein kinase [203]; CCaMK,
c6 alcium and calcium/calm6 odulin-dependent protein k6 inase [107]; SIP, one of the eight S6 OS3-i6 nteracting p6 roteins [162]; PhGAD,
P6 etunia h6 ybrida g6 lutamic a6 cid d6 ecarboxylase [102]; Ca2+ ATPase (BCA1), B6 rassica C6 a2 + -A6 TPase 1 [72,112]; Ca2+ ATPase
(ACA2), A6 rabidopsis C6 a2 + -A6 TPase 2 [71]; AtKCBP, Arabidopsis k6 inesin-like c6 almodulin-b6 inding p6 rotein [109]; At cNGC1, one
of the six c6 yclic n6 ucleotide g6 ated c6 hannels from Arabidopsis [94,115]; HvCBT1, H6 ordeum 66 ulgare C6 aM b6 inding t6 ransporter;
NtCBP4 N6 icotiana t6 abacum c6 almodulin-b6 inding p6 rotein 46 [116,117]; Zm SAUR1, Z6 ea m6 ays s6 mall a6 uxin u6 p R6 NA; PMDR1, p6 otato
m6 ultidrug r6 esistance protein [119]; BjGLY I, B6 rassica j6 uncea glyoxalase I [118]; NtCB48, N6 icotiana t6 abacum c6 almodulin-b6 inding
p6 rotein 48 [106]; MPCBP, m6 aize p6 ollen-specific c6 almodulin-b6 inding p6 rotein [121]. Calmodulin-binding sites that are predicted
based on secondary structure but not verified experimentally are denoted with a question mark. Three other calmodulin-binding
C6 a2 + -A6 TPases from plants that have been reported recently [73– 75] are not shown in this figure. (C) Putative CaM-binding
myosins from Arabidopsis and their structural organization. Myosins in the Arabidopsis genome were identified by searching the
database with the conserved motor domain of a myosin. The sequences that contained the motor domain of myosin were analyzed
by the Simple Modular Architecture Research Tool (SMART) program.
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A.S.N. Reddy / Plant Science 160 (2001) 381–404
throughout their life cycle and showed enhanced
disease resistance to a wide spectrum of fungal
pathogens and tobacco mosaic virus [90]. These
studies indicate CaM isoform specificity in their
target activation in Ca2 + mediated signal transduction processes in plants. Furthermore, recent
studies revealed that the CaM isoforms differ in
their affinity to the same target protein [91,98 –
100] highlighting the significance of the existence
of multiple CaM isoforms in mediating specific
Ca2 + -mediated signal transduction pathways.
CaM regulates various cellular activities by
modulating the activity or function of a number of
proteins. In most cases, only the activated form of
CaM (Ca2 + bound CaM) interacts with its target
proteins. However, in some cases CaM can interact with its target proteins in the absence of Ca2 +
[86].
3.1.1. Ca 2 + -dependent CaM-binding proteins
CaM is multifunctional because of its ability to
interact with and control the activity of a variety
of target proteins. The role of CaM in a given cell
or a tissue is determined by the presence of its
target proteins. In recent years efforts to dissect
Ca2 + /CaM mediated signaling pathways in plants
have focused on identification and characterization of CaM-binding proteins (CBPs) in plants.
The amino acid sequences of the CBD in different
CaM target proteins is not conserved [86]. However, CaM-binding motifs from different CaMbinding proteins form characteristic basic
amphipathic a-helices with several positive
residues on one side and hydrophobic residues on
the other side [86].
Using biochemical assays and protein-protein
interaction-based screening of expression libraries
with labeled CaM : 24 proteins that interact with
CaM in a Ca2 + -dependent manner have been
identified from plants (Fig. 2B,C). These include
NAD kinase [101], glutamate decarboxylase [102],
Ca2 + /CaM kinases [103,104], elongation factor-1a
[105], heat shock inducible proteins [106], CCaMK
[107], transcription factor [108], kinesin-like
protein [109 – 111], Ca2 + -ATPase [71,112], nuclear
NTPases [113], ion transporters [94,114 –117], glyoxalase I [118], multidrug resistant protein [119],
SAUR, an auxin-induced gene [120], pollen specific CBP [121] and some other proteins of unknown function [122].
The identity of the known CBPs indicate that
diverse proteins implicated in a wide variety of
processes including ion transport, gene regulation,
cytoskeletal organization, cell division, disease resistance and stress tolerance interact with CaM
(see Fig. 2B,C and Fig. 3). Interestingly, many of
these CBPs have no homologs in animals. Some of
these (e.g. glutamate decarboxylase, GAD) have
homologues in animals but lack CaM binding
motif, suggesting that they are regulated by CaM
only in plants. In some cases plants and animals
have similar proteins but the location of the CaM
binding domain is different between plant and
animal proteins (e.g. Ca2 + ATPase).
Although the catalytic core in GAD, which
catalyzes the conversion of glutamate into g-amino
butyric acid (GABA) and CO2 is conserved across
bacteria, plants and animals, the Ca2 + /CaM regulation of GAD is unique to plants [123]. GAD has
been cloned and characterized from a number of
plant species. Although the CBD is not conserved,
all plant GADs isolated so far posses a CBD and
are regulated by Ca2 + /CaM [123]. In Arabidopsis
there are two GAD isoforms (GAD1 and GAD2)
and the CBD of these two isoforms are different,
raising the possibility for functional diversity and
regulation by Ca2 + /CaM [124]. Plants produce
increased levels of GABA in response to mechanical and cold stresses [125]. These environmental
stresses also elevate the [Ca2 + ]cyt levels raising the
possibility that elevated Ca2 + activates GAD,
which in turn increases GABA levels. Transgenic
tobacco plants expressing full-length GAD showed
normal wild type phenotype with moderately increased GABA and reduced Glu. However, the
transgenic plants expressing GAD minus CBD
showed severe anatomical abnormalities in cell
elongation in stem cortex and parenchyma tissues.
This phenotype is shown to correlate with accumulation of extremely high levels of GABA and
low levels of Glu [126].
The NAD kinase, which catalyses the conversion of NAD to NADP [101,127], is vital to living
organisms especially when energy is in demand
under stress conditions. Recently, it has been reported that NAD kinase plays a role in oxidative
burst and in the formation of active oxygen species (AOSs) [96], which are known for their involvement in plant defense against pathogens.
Several CBPs that show similarities to ion channels have been isolated from barley [114], Ara-
A.S.N. Reddy / Plant Science 160 (2001) 381–404
bidopsis [94,115] and tobacco [116,117]. These
proteins showed sequence similarity to cyclic
nucleotide gated channels from animals and
inward rectifying K+ channels from plants.
These proteins have been shown to reside in the
plasma membrane. Constitutive over expression of one of these proteins, NtCBP4, in sense
389
and antisense orientation in tobacco exhibited a
normal phenotype under normal growth conditions. However, the NtCBP4 sense transgenic
plants showed improved tolerance to Ni2 + and
hypersensitivity to Pb2 + [116], suggesting a role
for this CaM-binding channels in metal tolerance.
Fig. 3. Ca2 + sensing proteins and their functions in plants. Four major groups of Ca2 + sensors (indicated in four boxes) have
been described in plants. These include (A) Ca2 + -dependent protein kinases, (B) CaM, (C) other EF-hand motif-containing
Ca2 + -binding proteins, and (D) Ca2 + -binding proteins without EF-hand motifs. Inhibition of enzyme activity is shown by ‘Þ’.
CDPK, Ca2 + -d6 ependent and calmodulin-independent p6 rotein k6 inase [194,196]; Centrin from Arabidopsis [186]; AtTCH2
Arabidopsis T6 ouch 26 [237]; AtTCH3, Arabidopsis T6 ouch 36 [169]; AtCBP22, Arabidopsis C6 alcium-B6 inding P6 rotein 22 [236]; ABI1,
ABA i6 nsensitive 16 [171,172]; PhCaM53, P6 etunia h6 ybrida Calm6 odulin 53 [165]; AtCBL1 to 3, Arabidopsis C6 alcineurin B6 -L6 ike-16 , -26
and -36 proteins [157]; AtCBL4, Arabidopsis CBL4 or S6 alt-O6 verly-S6 ensitive3 (SOS3) protein [31]; AtCP1, salt-induced Arabidopsis
C6 alcium-Binding P6 rotein [173]; OsEFA27, rice EF hand protein responsive to A6 bscisic acid [170]; PvHRA32, bean H6 ypersensitive
R6 eaction A6 ssociated [178]; CCD-1, C6 -terminal c6 entrin-like d6 omain [235]; AtRBOHA, Arabidopsis R6 espiratory B6 urst O6 xidase
H6 omologue A6 [183]; BetV-4, birch pollen allergen [238]; Myosins [151,152]; s6 mall a6 uxin u6 p R6 NA [120]; EF-1 alpha, elongation
factor-1 a [143]; CCaMK, c6 alcium and calcium/calm6 odulin-dependent protein k6 inase [107]; CaMK calcium/calmodulin-dependent
protein kinase [104,203]; CBP1, c6 almodulin-binding p6 rotein 1 [122]; CBP5, c6 almodulin-b6 inding protein 5 [122]; MPCBP, m6 aize
p6 ollen-specific c6 almodulin-b6 inding p6 rotein [121]; StCBP S6 olanum t6 uberosum c6 almodulin-b6 inding p6 rotein (Reddy, unpublished
results); BjGLY I, glyoxalase I [118,138]; NAD kinase [127], NtCB48, N6 icotiana t6 abacum c6 almodulin-binding p6 rotein 48 [106];
MDR1, m6 ultidrug r6 esistance protein [119]; GAD, g6 lutamic a6 cid d6 ecarboxylase; PR proteins, p6 athogenesis r6 elated proteins [90];
cNGCs, c6 yclic n6 ucleotide g6 ated c6 hannels [94,114–117]; Ca2+ ATPase [70,71,73– 75]]; TGA, DNA binding protein [108]; NTPase,
nucleoside triphosphatase [113]; KCBP, k6 inesin-like c6 almodulin-b6 inding p6 rotein [109]; PCP, p6 istil-expressed c6 alcium-binding
p6 rotein.
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A.S.N. Reddy / Plant Science 160 (2001) 381–404
A novel CaM-binding microtubule motor
protein (KCBP, kinesin-like calmodulin-binding
protein) was isolated from Arabidopsis and other
plants [109 – 111]. This motor is unique among
kinesins and kinesin-like proteins in having a
CaM-binding domain adjacent to the motor domain at the C-terminus and a myosin tail homology region in the N-terminal tail [128]. KCBP
binds CaM in a Ca2 + dependent manner at physiological Ca2 + concentration. KCBP binds bovine
CaM and three CaM isoforms (CaM-2, -4 and -6)
of Arabidopsis. However, CaM-2 showed 2-fold
higher affinity to KCBP as compared with other
isoforms [99]. Although, a homologue of KCBP
has not been found in S. cere6isiae, C. elegans and
Drosophila whose genomes have been sequenced,
recently a CaM-binding C-terminal kinesin (kinesin C) was cloned from sea urchin [129]. The
CBD of kinesin C shared 35% sequence identity
with the CaM binding domain in KCBP. However, kinesin C differs considerably from KCBP
and does not show any sequence similarity in the
stalk and tail regions. The amino-terminal tail and
stalk regions of KCBPs from different plant systems are highly conserved and contain myosin tail
homology (MyTH4) and talin-like regions that are
not present in kinesin C [130]. Further analysis of
CaM-binding kinesin-like proteins from a number
of phylogenetically divergent plants and animals
including the primitive eukaryotes is needed to
understand their origin and evolution.
Motility studies with the KCBP have shown
that it is a minus-end directed microtubule motor
[131]. Binding of activated CaM to KCBP inhibits
its interaction with microtubules [131 –133]. The
CaM effect on KCBP suggests that activated CaM
can act as a molecular switch to down-regulate the
activity of KCBP. Among motor proteins, heavy
chains of unconventional myosins also bind CaM
where the inhibition of the enzymatic activity and
motility by Ca2 + is similar to the effect of Ca2 +
on a CaM binding KCBP, although the mechanism of inhibition is very different between these
proteins [134]. Based on these studies with KCBP
it is reasonable to speculate that spatial and temporal changes in free [Ca2 + ]cyt levels in response to
signals are likely to regulate KCBP activity in the
cell. Current evidence indicates the involvement of
KCBP in trichome morphogenesis and cell division [135 – 137,240].
Glyoxalase I, which catalyses the conversion of
toxic methylglyoxal to a nontoxic metabolite, expression is induced by NaCl, mannitol or abscisic
acid [138]. Glyoxylase I from Brassica juncea
(BjGly I) binds CaM-Sepharose and its activity is
stimulated by Ca2 + /CaM [139]. Leaf discs from
transgenic plants expressing BjGly I showed tolerance to methylglyoxal and salt. In contrast, the
control antisense and wild type leaf discs showed
loss of chlorophyll and did not survive these
stresses, suggesting that BjGly I plays a role in
conferring tolerance to salt stress in plants.
Two nuclear proteins, a chromatin associated
nucleotide triphosphatase (NTPase) [140] and a
DNA binding protein (TGA3) that binds to CaM3 promoter [108], are also regulated by CaM. The
binding of CaM to TGA3 enhances its binding
with the promoter. In plants, Ca2 + /CaM is involved in stabilizing cortical microtubules at low
Ca2 + concentrations and destabilizing the same at
higher Ca2 + concentrations [141,142]. Two CBPs
that show different sensitivities to Ca2 + are implicated in evoking these two opposing effects of
CaM on cortical microtubules. Elongation factor1a, a CaM-binding microtubule associated
protein, stabilizes microtubules and Ca2 + /CaM
has been shown to inhibit elongation factor-1apromoted microtubule stabilization [143]. An
auxin-induced gene product has been shown to
bind CaM, suggesting the involvement of CaM in
auxin action [120]. CBPs that are induced by heat
shock have been isolated from tobacco [106]. One
of these CBPs (NtCBP48) contains a centrally
located putative transmembrane domain and a
nuclear localization sequence motif [106]. Two
maize proteins (CBP-1 and CBP-5) and a multidrug resistant protein also bind CaM in a Ca2 + dependent manner [119,122]. However, the
functions of these are not known. A pollen specific
CaM-binding protein from maize, MPCBP, [121]
and CBP from developing tubers (StCBP) (Reddy,
unpublished results) are also novel CBPs and have
no homologs in non-plant systems. Binding of
superoxide dismutase to CaM-Sepharose column
suggests that it might be regulated by CaM [144].
3.1.2. Ca 2 + -independent CaM-binding proteins
Although CaM, in most cases, binds to its
target proteins in the presence of Ca2 + , some
CaM-binding proteins interact with CaM in the
absence of Ca2 + [86]. In animals, myosins, a
A.S.N. Reddy / Plant Science 160 (2001) 381–404
superfamily of actin-based motors that perform a
broad array of cellular functions, bind CaM in the
absence of Ca2 + [145]. The CaM-binding domain
(also called ‘IQ motif’) in these myosins has a
consensus sequence ‘IQXXXRGXXXR’ (I,
isoleucine, Q, glutamine, R, arginine, G, glycine)
which forms an a-helix [145]. Although there is
compelling indirect evidence for the role of actin
and actin-based motors in various transport processes in plants, little is known about plant
myosins and their regulation [134]. Myosins containing IQ domains, which are typically CaM-sensitive, have been identified in plants but their
interaction with, or regulation by CaM has not yet
been demonstrated in these proteins [134]. The
deduced amino acid sequence of plant myosins,
like other known myosins, contain ‘IQ’ motifs in
the neck region [146,147]. Phylogenetic analyses
using the conserved motor domain grouped
known myosins into 15 distinct classes [134]. Of
these, published plant myosins fall into two separate groups — myosin VIII (e.g. Arabidopsis
myosins ATM1 and ATM2) and myosin XI (e.g.
Arabidopsis myosins MYA1 and MYA2). These
two subgroups contain only plant myosins, suggesting that plants may have a unique set of
myosins. By comparing the sequences in the Arabidopsis genome database with the motor domain
of myosins, I have identified 17 myosin-like
proteins in 60% of the sequenced genome. All
Arabidopsis myosin-like proteins that are identified so far possess two to six putative CaM-binding ‘IQ’ motifs (Fig. 2C). Although the binding of
CaM to any of these proteins has not been demonstrated experimentally, the presence of these motifs suggests that myosins in plants are likely to be
regulated by CaM in response to changes in intracellular Ca2 + .
A number of reports have shown that the cytoplasmic streaming in plant cells is regulated by the
[Ca2 + ]cyt concentration [148]. Elevated levels of
Ca2 + inhibits cytoplasmic streaming and the
movement of organelles [149,150]. However, specific motors involved in these processes remain to
be identified. The mechanisms by which Ca2 +
regulates the activity of these motors is just beginning to emerge. Both CaM and CDPKs are likely
to mediate Ca2 + effects on cytoplasmic streaming
and organelle transport. Recently, Yokota et al.
[151,152] purified two myosins from plants and
demonstrated that CaM associates with the
391
purified myosin and regulates its motor activity.
Ca2 + inhibits the myosin motor activity as well as
myosin-activated ATPase activity in plants. The
inhibition of myosin activity in the presence of
Ca2 + appears to be due to dissociation of CaM
from the heavy chain since Ca2 + inhibition of
motor activity can be restored by exogenous addition of CaM. Some studies suggest that, in some
cases, Ca2 + can effect myosins indirectly through
another mechanism [153]. There is some indirect
evidence in support of a role of phosphorylation
catalyzed by CDPK in regulating myosin activity
[154,155]. A CDPK, which has been shown to
bind actin filaments, phosphorylates a putative
myosin light chain in Chara [155,156]. However,
the effect of such a phosphorylation is not known.
3.2. CaM-like and other EF-hand containing
proteins
In addition to CaM, recent studies indicate the
presence of numerous CaM-like proteins in plants
(Fig. 2A). However, the function of these proteins
in Ca2 + signaling pathway(s) is not fully characterized as compared to that of CaM. These CaMlike proteins differ from the CaM in containing
more than 148 amino acids, and one to six EFhand motifs with limited homology to CaM [83].
Hence, it is likely that these proteins are functionally distinct from CaM and are involved in controlling
different
Ca2 + -mediated
cellular
functions.
Recently, a new family of Ca2 + -binding
proteins, called calcineurin B-like (CBL) proteins,
has been identified [31,157,158]. Studies with saltoverly-sensitive (SOS) mutants of Arabidopsis indicate a key role for Ca2 + in salt stress signaling
[159 –161]. These mutants are hypersensitive to
Na+ and Li+ and are unable to grow on low-K+
culture medium. These abnormal growth patterns
in the presence of NaCl could be mitigated by the
addition of increased levels of Ca2 + in the same
medium. The SOS3 gene encodes a Ca2 + sensor
protein similar to calcineurin B, a regulatory subunit of Ca2 + -dependent protein phosphatase and
neural Ca2 + sensors (CNS) of animals and raises
the possibility that SOS3 gene product might control the K+/Na+ transport system via a Ca2 + -regulated pathway [31]. In Arabidopsis there are at
least six CBL genes encoding highly similar but
functionally distinct Ca2 + -binding proteins [157].
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A.S.N. Reddy / Plant Science 160 (2001) 381–404
Drought, cold and wound stress signals induce
AtCBL1 gene transcripts, whereas AtCBL2 and
AtCBL3 are constitutively expressed [157]. Further, C6 BL-i6 nteracting p6 rotein k6 inases (CIPK1 to
4) belong to the serine/threonine class of kinases
and show high homology to protein kinases SNF1
and AMPK from yeast and mammalian systems,
respectively. SOS3 interacts with SOS2, which is
also a member of CIPKs (also called SIPKs,
S6 OS3-i6 nteracting p6 rotein k6 inases), and seven other
CIPKs [162]. CIPKs interact with CBLs, but not
with CaMs, in a Ca2 + -dependent manner [158].
The activity of CIPK/SIPK is activated by SOS2
(CBL4) and other CBLs in a Ca2 + -dependent
manner, suggesting that CBL/SIPK complex is
likely to regulate Na+ and K+ homeostasis
through phosphorylation. CIPKs/SIPKs represent
a novel family of Ca2 + -regulated protein kinases
in plants. The interaction of calcineurin B-like
(CBL) proteins with protein kinases is very surprising since the CBL proteins are known to activate a protein phosphatase in animals. In addition
to Ca2 + -regulated protein kinase, there is some
evidence for the role of a Ca2 + -regulated phosphatase also in salt tolerance. Ectopic expression
of constitutively active yeast calcineurin has been
shown to confer salt tolerance in tobacco
[163,164].
Certain CaM-like proteins contain a 34 amino
acid Caax-box motif (prenylation domain) at their
C-termini (CTIL in PhCaM53) [165] or (CVIL in
OsCaM63) [166]. Transient expression of GFP
fused with the full-length CaM53 or mutated
CaM53 (without CaaX-box motif) in tobacco and
petunia protoplasts showed that the full-length
protein localizes to plasma membrane whereas
mutant CaM53 localizes to the nucleus [165,167],
suggesting an important role for the prenylation
domain in CaM53 localization. Ectopically expressed CaM53 with a prenylation domain in tobacco showed stunted growth and a necrotic
phenotype. However, ectopic expression of neither
non-prenylated form nor Caax-box motif alone
showed such altered morphogenic alterations indicating the importance of the prenylation domain
[165].
Arabidopsis, in addition to six CaMs, has other
CaM-like genes including the TCH genes that are
induced in response to various mechanical, chemical and environmental stimuli (Fig. 2A) [168].
TCH3 is 324 amino acids long and contains six EF
hand motifs [169]. A cDNA sequence encoding a
27 kDa CaM-like protein (EFA27) has been isolated from ABA-treated rice seedlings [170]. It
contains a single EF hand motif and the expression of EFA27 is induced in response to salt and
dehydration stress, and to ABA signal. In Arabidopsis there are several EFA27 gene homologues
[170], suggesting the existence of similar proteins
in phylogenetically distant species. EF hand motif
containing protein phosphatases were also identified in an ABA-signaling pathway (Fig. 2A)
[171,172]. Another Ca2 + -binding protein, AtCP1,
from Arabidopsis contains three EF hand motifs
(Fig. 2A) and binds Ca2 + [173]. The AtCP1 gene
transcripts are also highly inducible by NaCl treatment but not by ABA treatment indicating the
specificity of this unique Ca2 + -binding protein in
responding to stress factors.
Several studies implicate the involvement of
Ca2 + signal in plant defense responses such as
phytoalexin biosynthesis, induction of defense-related genes and hypersensitive cell death
[7,174,175]. Ca2 + is implicated in mediating systemin, a peptide involved wound-induced activation of defense genes in tomato [176]. Further,
Flego et al. [177] showed a correlation between
increased Ca2 + concentration in plants and increased resistance to bacterial pathogen Erwinia
caraoto6ora. P6Hra32, a gene that is highly expressed during hypersensitive reaction in bean tissue challenged with Pseudomonas syringae, has
been shown to encode a small Ca2 + -binding
protein (161 aa) with four EF motifs [178]. The
AOSs stimulate rapid influx of Ca2 + into the site
of infection and initiate the hypersensitive response in order to develop resistance against invading pathogens [53,60]. The plasma membrane
located Ca2 + channel (LEAC, l6 arge conductance
e6 licitor-a6 ctivated ion c6 hannel) is likely to be involved in elevating [Ca2 + ]cyt in response to AOSs
[61]. During the interaction of Arabidopsis and P.
syringae, the resistance gene product, RPM1, functions immediately and elevates the Ca2 + levels
[179,180]. Similar results were obtained from the
C. ful6um/tomato interaction [181]. Furthermore,
it was shown that the resistance gene activates a
CDPK [182]. Identification and characterization of
human gp91 phox (phox for phagocyte oxidase) homologues from Arabidopsis and rice (RbohA,
r6 espiratory b6 urst o6 xidase h6 omologue A6 ), provided
evidence for the downstream target for the ele-
A.S.N. Reddy / Plant Science 160 (2001) 381–404
vated levels of Ca2 + in oxidative burst [183]. The
RbohA shows high similarity to human gp91 phox, a
plasma membrane bound neutrophil phagocyte
oxidase that is involved in the generation of superoxide radicles via its NADPH oxidase activity. In
addition to the gp91 phox region, the plant gp91 phox
(RbohA) also contains two EF hand motifs which
are not present in human gp91 phox suggesting that
Ca2 + modulates the formation of superoxide radicles through RbohA during oxidative burst in
plants [183].
Centrin (also known as caltractin) is a small
(: 20 kDa) acidic protein with four Ca2 + -binding
EF-hand motifs [184]. Genes encoding centrins
were isolated from the salt marsh plant, Atriplex
nummularia [185], Arabidopsis [186] and tobacco
BY-2 cells [187]. In plants, centrins are 167 –177
amino acids long. The Arabidopsis centrin was
isolated as an early-induced gene in response to
bacterial inoculation. The expression of this centrin is induced when the plants are inoculated with
avirulent strains of bacteria but not with virulent
strains, suggesting a role for this centrin in plant
defense [186]. Based on the localization of plant
centrins it was suggested that centrins are involved
in cell plate formation [187] and Ca2 + -regualted
transport through plasmodesmata [188].
3.3. Ca 2 + -regulated protein kinases
By manipulating cellular Ca2 + levels with pharmacological agents, Ca2 + -regulated protein phosphorylation has been implicated in a variety of
responses including host –pathogen interactions
[182,189], cold stress [190], nuclear migration during fungal infection [97], gravitropism [80], lightregulated gene expression [191], thigmotropism
[192] and hypoosmatic shock [48,193]. Studies during the last decade indicate that there are at least
five types of Ca2 + -regulated protein kinases in
plants: (i) a C6 a2 + -d6 ependent and CaM-independent p6 rotein k6 inases (CDPKs); (ii) C6 DPK-r6 elated
k6 inases (CRKs); (iii) CaM-dependent protein
k6 inases (CaMKs); (iv) C6 a2 + /CaM-dependent
protein k6 inases (CCaMK); and (v) S6 OS3/C6 BLi6 nteracting p6 rotein k6 inases (SIPKs/CIPKs) (Fig.
2B).
CDPKs, a new family of protein kinases, are
most abundant and ubiquitous in plants [194].
Apart from plants, CDPKs are found in protozoans and algae. Ca2 + directly binds to the CDPK
393
and stimulates the kinase activity whereas CaM
does not have any significant effect on the kinase
activity [195]. CDPKs have a unique structural
organization in which a protein kinase catalytic
domain is followed by CaM-like region with four
Ca2 + binding motifs (Fig. 2B). The kinase domain
of CDPK shows significant homology with the
mammalian Ca2 + /CaM-dependent protein kinase
II (CaM K II) catalytic domain. The region that
joins the kinase domain to the CaM-like region
(junction region) corresponds to the autoinhibitory/CaM-binding region of CaM K II and
prevents kinase activity in the absence of Ca2 +
[196]. The autoinhibitory domain located in the
junction region of CDPKs inhibits the kinase activity in the absence of Ca2 + . Ca2 + binding to
CDPK effects confirmation of the kinase and relieves the inhibition caused by the autoinhibitory
region [194]. CDPKs are found in different cellular
locations. Several CDPKs have putative myristoylation sites indicating that myristoylation of
CDPKs may regulate the association of CDPKs
with membranes [194].
The transcripts of two Arabidopsis CDPK genes
(AtCDPK1 and AtCDPK2 ) are highly inducible by
drought and high salt but not by low temperature
or heat stress suggesting the specificity of CDPK’s
induction in response to different stress factors
[197]. A CDPK from Vigna radiata is highly inducible by wounding, CaCl2, IAA and NaCl treatments [198]. There are over 40 CDPKs in the
Arabidopsis genome [194] (http://plantsp.sdsc.edu/
cdpk/). CDPKs are classified into several groups
based on their sequence domain organization
(myristolyation, PEST and the number of EF
hand motifs). Furthermore, CDPKs differ in their
affinity for Ca2 + . For example, AtCDPK1 differs
from AtCDPK2 in its Ca2 + stimulated activity
although both of them possess four EF hand
motifs. Recent studies indicate that, besides Ca2 + ,
lipids are involved in the regulation of CDPK
activity [199,200]. A carrot CaM-like domain
protein kinase, DcCPK1, resembles animal PKC
in its activation by Ca2 + and certain phospholipids suggesting that lipids regulate the activity of
some CDPKs and perform specific biological functions in plants [201]. Ion channels, enzymes involved in metabolism, cytoskeletal proteins, and
DNA binding proteins have been identified as
CDPKs substrates, suggesting their role in diverse
cellular processes ranging from ion transport to
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A.S.N. Reddy / Plant Science 160 (2001) 381–404
gene expression [194]. Sheen [202] has shown that
Arabidopsis AtCDPK1 and AtCDPK1a are involved in regulating the expression of stress-inducible
genes.
Furthermore,
phosphatases
counteract these responses suggesting that involvement of Ca2 + -regulated phosphorylation is necessary for stress-induced gene expression (see
Section 3.1.1 on Ca2 + and gene expression).
CRKs are similar to CDPKs except that the
CaM-like region is poorly conserved with degenerate EF-hands. There are at least seven CRKs in
Arabidopsis. However, regulation and function of
these kinases are not known [194]. A cDNA with
significant sequence similarity to mammalian CaM
K II has been isolated from apple [203] and maize
[104] (Fig. 2B). However, the biochemical properties and regulation of these kinases are not known.
A Ca2 + /CaM-dependent protein kinase (CCaMK)
was characterized from lily and other plants
[104,107]. Sequence analysis revealed the presence
of an N-terminal catalytic domain, a centrally
located CaM-binding domain and a C-terminal
visinin-like domain containing only three EF
hands (Fig. 2B). Biochemical studies of CCaMK
established that Ca2 + /CaM stimulates CCaMK
activity. In the absence of CaM, Ca2 + promotes
autophosphorylation of CCaMK. The phosphorylated form of CCaMK possesses more kinase activity than the non-phosphorylated form [204].
CCaMK phosphorylates elongation factor-1a
[205]. However, the physiological significance of
this phosphorylation is not yet known. The
affinity of CCaMK to CaM is regulated by Ca2 + stimulated autophosphorylation [206].
3.4. Ca 2 + -binding proteins without EF-hand
motifs
There are several proteins that bind Ca2 + but
do not contain EF-hand motifs (Fig. 3). These
include annexins, calreticulin and p6 istil-expressed
C6 a2 + -binding p6 rotein (PCP). Annexins are a family of proteins in plants and animals that bind
phospholipid in a Ca2 + -dependent manner and
contain four to eight repeats of : 70 amino acids
[207]. Although the exact function of annexin is
not known, plant annexins are implicated in secretory processes and some have ATPase, peroxidase
or F-actin binding activities [208]. Calreticulin is a
Ca2 + sequestering protein in the ER and functions
as a chaperone [209]. In addition, it is implicated
in Ca2 + homeostasis and other functions [210]. A
19 kDa novel Ca2 + -binding protein (PCP) that is
expressed in anthers and pistil was isolated recently [211]. PCP is a high capacity (binds 20 mol
of Ca2 + per mol of PCP), low affinity Ca2 + -binding protein. The pattern of PCP expression suggests a role for it in pollen-pistil interactions
and/or pollen development. A cysteine class of
Ca2 + -dependent protease (CDP) has been purified
from Arabidopsis root cultures [212]. Its activity is
specifically dependent on Ca2 + but not on other
divalent cations. Structural organization of CDP is
not known since the gene encoding the CDP has
not been isolated.
4. Ca2 + and gene expression
Although there is a great deal of information on
the involvement of Ca2 + in regulating various
physiological processes [213,214], the role of Ca2 +
in regulating gene expression in plants is forthcoming only recently. Manipulation of [Ca2 + ]cyt
by various means is shown to affect the expression
of specific genes in plants. Mannitol-induced expression of RAB and AtP5CS1 genes is blocked in
the presence Ca2 + channel blockers like verapamil
or lanthanum or the Ca2 + chelator EGTA
[30,215]. The expression of these genes in treated
cultures and plants is less than that of untreated
counterparts, indicating a role of Ca2 + in drought
tolerance. Increased external Ca2 + or heat shock
rapidly induce the expression of touch-induced
CaM-related genes (TCH2, TCH3 and TCH4 )
whereas TCH1 gene, which codes for CaM, is not
significantly induced [216]. Heat shock, in the
presence of EGTA, a Ca2 + chelator, did not show
induction of TCH genes. This EGTA effect is
reversed by Ca2 + replenishment. Based on these
results it was suggested that heat shock elevates
[Ca2 + ]cyt levels which in turn regulates the expression of TCH2, -3 and -4 genes. Ca2 + effect on
touch-induced CaM-related genes is specific since
magnesium, another divalent ion, did not have any
effect. Furthermore, increased Ca2 + did not effect
the expression of a heat shock induced gene. Depletion of Ca2 + by Ca2 + chelator blocked ethylene-induced chitinase synthesis whereas artificial
elevation of cytosolic Ca2 + with Ca2 + ionophore
(ionomycin) or an inhibitor of microsomal Ca2 + ATPase induced chitinase synthesis in the absence
A.S.N. Reddy / Plant Science 160 (2001) 381–404
of ethylene [217]. Accumulation of cold-induced
mRNAs in alfalfa was also partially blocked by
lanthanum, a Ca2 + channel blocker and a CaM
inhibitor (W7) completely blocked the expression
of cold-regulated genes [218]. Lam et al., [219]
have shown that light induced chlorophyll a/b
binding (cab) transcripts could be induced in the
dark by increasing the intracellular level of Ca2 +
using ionomycin. Compelling evidence for the role
Ca2 + and CaM in the regulation of gene expression came from microinjection studies with phytochrome mutant (aurea) [220]. In this mutant,
light-regulated genes are not expressed. Using a
reporter gene fused to light-regulated promoter, it
was shown that microinjection of reporter gene
construct with Ca2 + or Ca2 + -activated CaM resulted in the expression of reporter gene. Partial
development of chloroplasts in the aurea mutant,
which requires the expression of several genes,
could also be obtained by microinjection of Ca2 +
and CaM into these cells [10]. In some cases, the
increased levels of [Ca2 + ]cyt induced the expression
of some genes and repressed others [221,222]. In
tobacco, expression of one specific CaM isoform is
inducible by cold and wind. By targeting aequorin
to cytoplasm and nucleus, Van der Luit et al. [39]
showed distinct cellular Ca2 + pools respond to
wind and cold stimuli in the expression of NpCaM1 gene. Wind and cold stimuli induce [Ca2 + ]n
and [Ca2 + ]cyt, respectively, suggesting that different Ca2 + transients employ distinct signal pathways in NpCaM1 gene expression [39].
Sheen [202] has elegantly demonstrated the involvement of CDPKs in stress-induced gene expression. A reporter gene (GFP/LUC) fused to a
stress-inducible promoter was used in transient
assays to monitor its expression under different
stress conditions and in the presence of the constitutively expressed kinase domain of several CDPK
isoforms. Cold, salt, dark and ABA induced the
expression of the reporter gene driven by the
stress-inducible promoter. The reporter gene was
also expressed in the absence of stress treatments
but in the presence of both Ca2 + and Ca2 +
ionophore, suggesting that stress-induced gene expression is mediated by Ca2 + . Protoplasts transformed with a control construct (a reporter gene
fused to stress nonresponsive promoter) did not
show reporter gene expression in response to
stresses or Ca2 + [202]. Since Ca2 + is able to
induce the expression of a stress inducible gene,
395
Sheen tested the effect of CDPKs on the expression of stress promoter-reporter construct. The
protoplasts were cotransformed with the reporter
construct along with the kinase domain of eight
CDPK isoforms individually. Of the eight Arabidopsis CDPKs tested, only ATCDPK1 and
ATCDPK1a activated the expression of the reporter gene, indicating that specific CDPK isoforms mediate the effects of stresses on gene
expression. Cotransformation of the reporter gene
construct along with the ATCDPK1a and PP2C, a
protein phosphatase, decreased the reporter gene
expression significantly whereas cotransformation
of ATCDPK1a with PP2C null mutant had no
effect on the CDPK-induced reporter gene expression. These results establish an important role for
specific CDPKs in stress-induced gene expression.
5. Specificity in decoding Ca2 + signal
The fact that Ca2 + is a messenger in transducing a wide range of signals into diverse responses
raises an important question — How does Ca2 +
achieve specificity in eliciting a response to a given
signal? A number of factors are likely to be involved in controlling the specificity. First, competence of an organ, a tissue or a cell type within the
tissue to respond to a given stimulus. In vivo
imaging of cold-induced changes in [Ca2 + ]cyt indicate that cotyledons and roots of a seedling are
highly responsive whereas hypocotyls are relatively
insensitive to cold shock [223]. It has also been
shown that a given signal may induce a different
Ca2 + signature in different cell types [224]. For
example, the Ca2 + response in the endodermis
and pericycle in the presence of salt and osmotic
stress is different from other cell types [224]. Second, temporal and spatial changes of Ca2 + together with the extent of increase (amplitude) are
likely to contribute significantly in achieving the
specificity in eliciting appropriate physiological responses [225,226]. It is becoming clear that different signals cause distinct spatial and temporal
changes in Ca2 + and this Ca2 + signature is likely
to be important in achieving the specificity
[3,6,225,227,228]. In pollen tubes, changes in cytosolic Ca2 + are limited to the tip with characteristic
oscillations [226]. Some signals have been shown
to cause wave-like Ca2 + increases in the cells of
cotyledons. Using luminescence-imaging technol-
396
A.S.N. Reddy / Plant Science 160 (2001) 381–404
ogy, the spatial and temporal pattern of elevated
[Ca2 + ]cyt in response to low temperature has been
demonstrated in transgenic plants expressing aequorin [40]. These authors showed that cold-induced signal was transmitted from root to aerial
tissues with a lag period of 3 min. Ozone has been
shown to cause biphasic [Ca2 + ]cyt transients [50].
Using lanthanum chloride and EGTA, the activity
of AOS-induced glutathione synthase was found
to require a second [Ca2 + ]cyt transient peak [50].
In parsley cells, a fungal elicitor induces a biphasic
Ca2 + signature and it was shown that the sustained concentrations of Ca2 + but not the rapidly
induced transient peak are necessary to activate
defense-associated responses [62]. The magnitude
and kinetics of Ca2 + transients induced by touch,
cold and fungal elicitors are also found to be
different [1,37,223]. The strength of artificial wind
application on transgenic aequorin seedlings correlated with the amount of [Ca2 + ]cyt levels [229]. The
spatio-temporal pattern of [Ca2 + ]cyt changes could
be unique to each signal [3,6,225]. Localized
changes may occur in the cytoplasm or in one
compartment of the cell (e.g. signal-induced
changes in the cytoplasm but not in the nucleus or
vice versa). Heterogeneous nature of signal-induced changes has been well documented in guard
cells and in other plants cells [225]. Oscillations
and waves of Ca2 + have also been reported in
plants. Third, the type of Ca2 + sensors expressed
in a cell and their cellular location should also
contribute to the specificity. The plethora of Ca2 +
sensors and their tissue specific expression and
differential response to signals indicate that Ca2 +
specificity is in part accomplished by the type of
Ca2 + sensors and their target proteins.
6. Future directions
During the last decade, significant progress has
been made in demonstrating that signals not only
elevate [Ca2 + ]cyt but the Ca2 + signature generated
by each signal is likely to be different. Based on
what is already known, it is clear that plants
contain many unique Ca2 + sensing proteins with
novel regulatory mechanisms that have evolved to
perform plant-specific functions. It is likely that
many more novel Ca2 + sensing proteins will be
identified, especially as the Arabidopsis genome
sequence is completed. So far : 150 proteins that
are involved in Ca2 + mediated signal transduction
pathways have been identified in Arabidopsis. This
number is likely to reach 300 –400 in the next few
years, suggesting that up to 2% of the expected
number of genes in Arabidopsis encode proteins
involved in Ca2 + signaling. Considering the involvement of Ca2 + in so many diverse processes it
is not surprising that plants contain such a large
number of proteins involved in Ca2 + signaling.
Although the specific functions of most of these
remain to be elucidated, a large body of indirect
evidence indicates the involvement of Ca2 + sensing proteins in many cellular processes such as
cytoplasmic streaming, organelle and vesicle transport, microtubule dynamics, cell division, chromosome segregation, cell elongation, tip growth and
morphogenesis including some plant-specific processes. Recent studies have produced many surprises
and
significantly
advanced
our
understanding of Ca2 + signaling in plants and
raised many important questions. What are all the
proteins involved in maintaining Ca2 + homeostasis and in elevating [Ca2 + ]cyt in response to signals? How the proteins involved in Ca2 + influx
and efflux are regulated? How elevation of Ca2 +
evokes different responses to different signals?
What is the nature of Ca2 + signatures that are
produced in response to a given signal? Are these
changes confined to a subcellular compartment?
What are the targets of numerous CaM-like
proteins that have been identified? The next
decade should bring answers to these interesting
questions. The challenge now is to elucidate the
role(s) of numerous Ca2 + sensing proteins and
cross-talk among various components of Ca2 +
signaling and other messenger mediated signaling
pathways. Molecular, cell biological and genetic
analysis of various proteins involved in Ca2 + signaling should permit elucidation of functional
analysis of these proteins. In planta protein –
protein interaction studies using new strategies
such as fluorescence resonance energy transfer are
necessary to verify and extend the results obtained
with in vitro studies [230]. Loss-of-function experiments with individual components of Ca2 + signaling will help in understanding the function(s) of
each protein involved in Ca2 + signaling. Because
of the large gene families of some Ca2 + sensing
proteins and likely functional redundancy or overlap with other members of the family, it will be
necessary to create double or triple mutants. New
A.S.N. Reddy / Plant Science 160 (2001) 381–404
technologies such as reverse genetics [231] to create knock out mutants coupled with the analysis
of cell- and tissue-specific expression of individual
members Ca2 + signaling pathway using microarrays [232], and non-destructive visualization of
proteins in live cells using fluorescent reporters
should help in gaining new insights into the function(s) of proteins involved in Ca2 + signaling and
in deciphering signaling networks. Because of the
involvement of Ca2 + signaling in many stress
responses it is possible to generate plants that are
more tolerant to biotic and abiotic stresses by
manipulating one or more components of Ca2 +
signaling pathway. Although some progress has
already been made in this area [90,96,116,233],
there is a great potential in producing crop plants
with desirable traits by engineering Ca2 + signaling
pathway.
Acknowledgements
I would like to thank Dr Irene Day and Dr
Vaka Reddy for critically reading the manuscript;
Bryan Criswell for his help in preparing the
figures. My apologies to those colleagues in the
field whose work was not mentioned due to space
limitations. Research on Ca2 + signaling in my
laboratory is supported by grants from NSF,
Agricultural Experiment Station and NASA.
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