Download Calcium Homeostasis in Plants: Role of Calcium Binding Proteins in

Indian Journal of Biotechnology
Vol I, April 2002, pp. 135-157
Calcium Homeostasis in Plants: Role of Calcium Binding Proteins in Abiotic
Stress Tolerance
Giridhar Pande/ M K Reddyl , Sudhir K Sopor/ and Sneh lata Singla-Pareek l*
IPlant Molecu lar Biology Laboratory, International Centre for Genetic Engineering and Biotechnology,
Aruna Asaf Ali Marg, New Delhi 110067, India
2Department of Plant and Microbial Biology, University of California, Berkeley, CA, USA
A majority of environmental signals of varied types as well as varied intensities are perceived at the membrane
level in a cell. In case of multicellular organisms like plants or animals, this 'perception' not only needs to be transferred to the actual centre of controlling and responding unit i.e. the nucleus but sometimes from one cell to another
cell which may just be lying close enough or even at an appreciable distance e.g. 'root-shoot communications'. In
contrast to processes of cell-to-cell communication in a plant system, which is just beginning to be elucidated, the
mechanism of 'transfer' of this information from outer surface to the core controlling units has been an active area
of research since past few decades. Abundant reports do exit in literature, which support a kind of 'cascading
mechanism' for this purpose. It is now a well-established fact that a divalent cation i.e. Ca 2+ plays an extremely important role in this process. The fluctuations in the level of Ca 2+ at a given time in a given cell organelle is the crucial
factor determining the activation stage of some special proteins, which have been proposed to have a high affinity for
Ca 2+. Such proteins are known as Ca 2+ -binding proteins (CaBPs). Under environmental abuses, the level and activation of CaBPs play an important role in bringing about the 'ignition' of 'protective' as well as 'defensive' mechanisms, which ultimately are reflected in the form of the physiological adaptations in the plant as a whole system. The
present review is an attempt to highlight the importance of CaBPs in plants under abiotic stress conditions.
Keywords: abiotic stresses, adaptations, Ca 2+, CaBPs, signal transduction, stress tolerance, transgenic plants
Introduction
Calcium plays a key role in plant growth and
development. Changes in cellular Ca2+ are perceived
by specific Ca2+ binding proteins (CaBPs), which by
modulating their targets regulate an astonishing
variety of cellular processes. The Ca2+ cation is now
firmly established as an intracellular second
messenger that couples a wide range of extra cellular
stimuli to characteristic responses in plants . Ever
since, a stimulus induced increase in the concentration
of cytosolic Ca 2+ (represented as [Ca2+] Cyt ) in higher
plants has been reported, there has been a massive
increase in the number of signalling systems known to
use [Ca2+] CYt as an intracellular second messenger
(Trewavas & Malho, 1998; Sander et ai. 1999 ;
Trewavas, 1999; Pandey et ai, 2000; Reddy, 2001;
Rudd & Franklin-Tong, 2001) . The implication is th at
calcium flow constitutes a large network which
actually connects the environment, cytoplasm,
vesicles, organelles, nucleus and in hi gher speciesthe organs.
*Author for correspondence:
Tel: +91-1 1-61 8 1242; Fax: +91-11-6162316
E- mail: snehpareek@ hotmail.com
The changes in the [Ca 2+JCYt in response to any specific signal vary in the frequency, amplitude and spatial domains - leading to the concept of 'calcium signatures'. This term is used to describe the specific
pattern or 'finger print' of Ca 2+ release and propagation. 'Calcium signatures' have been proposed to allow signal specificity by precise control of alterations
2
in spatial, temporal and concentration of [Ca +] Cyt.
They encode specific information, which is relayed to
the down stream elements (or effectors) for translation
into an appropriate cellular response.
The levels of calcium are delicately balanced by
the presence of "calcium stores" like vacuoles, which
release calcium whenever required. Several specific
pumps/channel (through which Ca2+ can move in and
out), and a plethora of CaBPs (also called as Ca2+ sensors), which are involved in either performing some
complex functions or involved in buffering' of calcium
(i .e. calcium homeostasis) have been identified. The
role of calcium as an important signal molecule and
the various components involved in maintaining calcium homeostasis has been reviewed by several
workers (Sander et ai, 1999; Trewavas, 1999; Kni ght,
2000; Pandey et aI, 2000; Reddy, 2001; Rudd &
Franklin-Tong, 2001).
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INDIAN J BIOTEC HNOL, APRIL 2002
At a given tim e, even under favourable condition s,
the physiological reactions taki ng place in a cell (be it
uni- or multi-cellular) are very co mpl ex, which in turn
indicate the in volvemen t of physio logical cation in a
host of cellular proceedings. Nonetheless, the cellul ar
ph ys iology becomes even more complex under the
indu ced conditi ons.
Environmental stresses are known to affec t crop
yield in a maj or way. Such stresses co mmonly enco untered by plants lead to a rapid transient elevation
in concentrati on of [Ca 2+] cy! (Kni ght et ai, 1991 ; Bush,
1995), which then transduces the signal further by
actin g as a seco nd messenger. The alteration in cellular calcium signals lead ultimately to the in creased
expression of stress res ponsive genes th at encode
proteins of protective function and hence confer stress
tolerance (Knight et al, 1996, 1997). In thi s context,
understanding of ci+ homeos tas is under unfavo urab le enviro nmen tal co nditi ons presents a maj or challenge. A number of studi es have been attempted to
address to the fo ll o wing questio ns:
(a)
(b)
(c)
(d)
How Ca 2 + concentrations are affected in a microenv ironment under stress conditions?
How thi s change in Ca2+ concentration, in
turn, bri ngs about changes in concentration of
downstream molecules i.e. Ca BPs?
How perturbations in the level of Ca BPs help
the system to counterac t the damaging effects
of envi ronmental stresses?
Ultimately, how can this response be manipulated to address to the problem of increas in g the crop yield under stress conditions?
The authors have attempted to review the work
done to elucidate the nature and function of various
CaBPs with special reference to their role in abiotic
stress tolerance.
Calcium Signalling in Plants: An Overview
In thi s part, the authors have briefly prefaced o n the
aspect as to how the Ca 2+ homeostas is is brought
about under favourable environmental conditions. The
later part of this section describes how this machinery
is affected under stress conditions.
Mechanism of Ca 2+ Regulation
An ensemble of Ca2 + transporter proteins maintains
cellul ar Ca 2+ homeostas is. Functionally, these transporters fall into two classes-those that mediate efflux from the cytoplasm (the Ca 2+ ATPase and
Ca 2+/H+ anti porters) and -those that mediate influx
into the cytoplasm (the Ca 2+ channels) as follows:
(a) Efflux Transporters
Bas ically , two types of efflu x tran sporters ope rate
in a cell: th e Ca 2+ ATPase and the Ca 2+/H+ anti porter.
A brief di sc uss ion o n eac h has been prov ided below.
2
(i) Ca + A TPases. Plant cells co ntain a diverse
group of pri mary ion-pumps, the Ca 2+ ATPases,
which belong to the super fami ly of P-type ATPases
that directl y use ATP to drive ion translocation. Two
distinct Ca 2+ pump families have been proposed based
on protein sequence identities (Axelsen & Palmgren,
1998). Members of the type II A and II B families ,
include the ER-ty pe and plasma membrane-type Ca 2+
pumps, respectively. Previously, ER and plasma
membrane-type pumps were distinguished by three
cri teri a: (a) locali zation to either th e ER or the plasma
membrane; (b) differential sensitivity to inhibitors
(e.g. ER-type is inhibited by cyclopiazonic ac id and
thapsigargin); and (c) direct activation of plas ma
membrane type pumps by CaM (Evans & Willi ams,
1998).
Several genes encodi ng type II A (ER type) pumps
have been cloned from pl ants, inclu di ng LCA 1 (Lycopersicllm Ca 2+ ATPase) fro m tomato (Wimmers et
aI, 1992), OCA I fro m rice (Chen et ai, 1997) and
2
ECAI/ACA3 from A rabidopsis (ER -type Ca + ATP2
ase/Arabidopsis Ca + ATPase isoform 3; Liang et al,
1997) . There is evidence th at ECAlp/ACA3p is located in the ER. A unique sub-family of CaMdependent Ca 2+ ATPases has been recently identified.
One of the isoforms of thi s family, ACA2p from
Arabidopsis, has been also found to resi de in the ER.
This was confirmed by taggi ng the gene with GFP
and transforming the Arabidopsis plants. Confocal
and computational anal ys is co nfirmed that it was located in the ER which makes it distinct from all other
CaM -regulated pumps identified in plants and animals (Hong et ai, 1999)
Further, three pl ant genes encod ing type II B
(plasma membrane-type) pumps have been reported:
ACA 1 and ACA2 fro m Arabidopsis (Huang et al,
1993a; Harper et al, 1998) and BCA 1 from B. oleracea (Malmstrom et al, 1997). In general, the plant
proteins are distingui shed from ani mal plas ma membrane-type pumps by (l) localization at membranes
other th an the plas ma membrane, and (2) a unique
structural arrangement with putative auto-inhibitory
domains at the N-terminus instead of the C-terminus.
PANDEY et al: ABIOTIC STRESS TOLERANCE IN PLANTS
Based on membrane fractionation and immunodetection with an anti-ACA 1 polyclonal antibody (Huang
et al, 1993a), it has been shown that ACAlp is localized in the plastid inner envelope membrane. BCA 1P
appears to be present in the vacuolar membrane, since
it showed correspondence with a peptide sequence
obtained from a purified vacuolar-ATPase (Malmstrom et al, 1997). ACA2p was shown to fractionate
with the ER, as indicated by immunodetection of
ACA2p in membrane fractions and this was confirmed by cytological visualization of an ACA2p
tagged with a C-terminal green fluorescent protein
(Harper et ai, 1998; Hong et ai, 1999).
(ii) Ca 2 +/H+ antiporters. They are different from
Ca 2+-ATPases as they do not require ATP and are not
sensitive to vanadate. The first plant Ca 2+/H+ antiporter to be cloned and functionally expressed was
CAX1p (Calcium exchanger 1; Hirschi et al, 1996).
The gene was identified by its ability to restore
2
growth on a high Ca +medium to a yeast mutant defective in vacuolar Ca 2+ transport. Antiporters are
shown to exist mainly on the tonoplast (vacuolar
membrane) and are involved in the maintenance of
vacuolar Ca 2+ stores (Blackfords et al, 1990). There
are so me evidences for the presence of Ca 2+/H+ antiporters on other membrane, such as the plasma
membrane (Kasai & Muto, 1990).
(b) Influx Transporters
The steep electrochemical gradient for Ca 2+ influx
into the cytosol is known to be tightly regulated. Although Ca2+ influx can occur through a pump or an
anti porter operating in reverse, transporters appear to
operate far away from thermodynamic equilibrium
and are likely to function only in the export of Ca2+. A
wheat cDNA clone, LCn (low-affinity cation transporter 1), complements yeast mutants defective in
2
Ca + influx (Schachtman et ai, 1997; Clemens et ai,
1998). Although, the sequence of LCn provides no
clues to its likely relationship to previously identified
ion-channels, an exciting possibility is that LCn
provides a physiologically significant pathway for
Ca2+ uptake in plants. Based on the state of the channel, influx transporters are classified into five classes
of ci+ channels in the animal system (Tsien & Tsien,
1990). Of these, voltage-operated, second messengeroperated, and mechanically operated, have been identified in plants and are as follows:
(i) Voltage gated channels. At least two major
classes of Ca2+ channels reside on the plasma mem-
137
brane (White, 1998): (l) Maxi-cation channels- are
relatively nonselective with respect to cation and possess a high single channel conductance (White, 1993,
1994) ; and (2) Voltage-dependent cation channel 2- is
relatively more selective for cations and exhibits as
walker single-channel conductance (White, 1994;
Pineros & Tester, 1995, 1997; Sander et al, 1999) .
Both the channels have been characterized most thoroughly in cereal crops. They exist in a variety of other
cell types and tissues like carrot, parsley suspension
cultures (Thuleau et al, 1994) and Arabidopsis mesophyll and root cells (Ping et al, 1992; Thion et al,
1998). Different pharmacological compounds like
verapamil, bepridil, nifedipine, etc. either block or
promote specifically the activities of these channels in
various calcium mediated responses.
(ii) Ligand gated channels. These channels are predominantly localized on the vacuolar endomembranes. At least four different Ca2+ permeable channel
types have been localized in the vacuolar membrane
(Allen & Sanders, 1997). Two of these channels are
ligand gated; one by inositol 1,4,5-triphosphate (lP 3 )
(Schumaker & Sze, 1987; Alexandre et ai, 1990) and
the other by cADP-ribose (cADPR) (Allen et al, 1995).
The pharmacological properties of plant cADPR
gated channels resemble those of ryanodine receptors
(Muir & Sanders, 1996). Microinjection of IP 3 and
cADPR into guard cells revealed that both compounds
have the capacity to elevate cytosolic calcium,
thereby demonstrating that IP3 and cADPR channels
are functional in plants (Gilroy et ai, 1991; Leckie et
al, 1998). A property of IP3 receptors and ryanodine
receptors in animal cells is their capacity to get acti2
vated by [Ca +]CYt. This response is thought to underlie
2
Ca + induced Ca2+ release (CICR), which can be fun2
damental to the amplification of Ca + signals (Taylor
& Traynor, 1995). Neither IP 3 nor cADPR gated currents across the vacuolar membrane of plants are activated by [Ca2+]CYt (Allen & Sanders, 1994; Leckie et
al, 1998), despite the presence of waves, oscillation
and spikes of [Ca 2+]CYb which are suggestive of CICR .
(iii) Stretch activated channels. These are Ca2+
permeable channels activated by tension and found on
the plasma membrane of plants (Cosgrove & Hedrich,
1991; Ding & Pickard, 1993; Pickard & Ding, 1993).
They have been proposed to be involved in turgor
regulation (Cosgrove & Hedrich, 1991), thigmotropic
responses (Braam, 1992; Braam & Davis, 1990) and
responses to temperature (Ding & Pickard, 1993) and
hormones (Pickard & Ding, 1993; Bush, 1995).
138
INDIAN J BIOTECHNOL, APRIL 2002
Ca 2+ Homeostasis under Stress Conditions
The authors have discussed in detail the various
types of cellular machineries and their functioning,
which control the entry and exit of Ca 2+ in the cytoplasm. Basically, for signal transduction, small bursts
2
of Ca + are flushed into the cytoplasm from two large
2
Ca + sto res; one is extracellular (cell wall, which is a
huge reserve of Ca 2+) and other is intracellular (a minor ER component and a major vacuolar store). However, under stress conditions, a major challenge for
the cell will be to maintain these machineries equally
functional. The survival of a cell will ultimately depends on to what an extent or a degree, a cell is able
to maintain these processes functionally close to the
unstressed conditions .
The changes in the cytosolic calcium concentration
have been reported in various stress conditions. Involvement of calcium has also been shown to modulate the expression of virulence genes of the pathogen
(Flego et ai, 1997) and in cell-to-cell communication
(Trucker & Boss, 1996). Cold shock response has
been studied in case of maize suspension culture cells
(Campbell et ai, 1996) and Arabidopsis seedlings
(Knight et aI, 1996) where a biphasic i.e. fast and
slow prolonged increase in calcium concentration was
observed. In cold sensitive tobacco and cold resistant
Arabidopsis seedlings, the plant seems to have a cold
calcium memory and depicted a plant specific calcium
signature in response to cold acclimation . Heat shock
induced changes in Ca2+ level have also been recorded
and correlated with thermotolerance in tobacco seedlings (Gong et ai, 1998).
Response of plants to hypoxi a, studied in Arabidopsis seedlings, was a biphasic pattern of calcium
change (Sedbrook et ai, 1996). Similarly, a change in
cytoplasmic calcium level was seen during water
stress in ageotropic pea mutants (Takano et ai, 1997)
and senescence in detached parsley leaves (Huang et
ai, 1997). Hypoosmotic shock to tobacco cells induces a biphasic cytosolic response of calcium change
(Cessna et ai, 1998).
In Arabidopsis seedlings, response to drought and
stress was mediated via changes in calcium level. A
transient increase in calcium could be blocked with
calcium chelator - EGT A or channel blocker - lanthanum. Elicitor induced signalling was also mediated
via calcium ions (Gelli & Blumwald, 1997) into tomato protoplasts . Also, oligonucleotide elicitor mediated signalling is mediated via calcium ion (Ebel,
1998). Even ozone mediated signalling inv olved a
transient increase in cytosolic calcium level (Clayton
et ai, 1999).
Several mech anical stimuli like wind, touch and
gravity are known to mediate a change in cytoplasmic
calcium concentration (Bjorkman & Cleland, 1991;
Halley et ai, 1995). A regress test to show the in2
volvement of cytosolic Ca + in gravitropic responses
has been performed in Arabidopsis roots (Legue et ai,
1997; Sinclair et ai, 1996).
Not only increase in calcium, but also its relationship to gene expression has been shown in a number
of instances. Salt or mannitol induces expression of
three genes, p5cs (which encodes ~ l -pyrroline-5carboxylate synthetase-the first enzyme of the proline biosynthesis pathway), rab 18 and Hi 78 (both of
these encode protein of unknown function). Mannitol
stimulated expression could be inhibited by pretreatment with lanthanum, but in case of salt stimulated expression, lanthanum could inhibit expression
of p5cs only. Calcium chelator - EGTA and calcium
channel blocker - gadolinium and verapamil blocked
mannitol induction of p5cs thus further confirming the
role of calcium in this response. Also, the involvement of calcium via IP 3 mediated release from vacuoles has been shown in thi s case (Knight et ai, 1997).
A transgenic approach has utilized the sea urchin
photoprotein, aequorin, as a calcium indicator in plant
tissues (Knight et ai, 1991). This protein consists of
an apoprotein and a luminophore and is, therefore, a
proteinaceous luminiscent reporter of Ca2+. Apoaequorin has been introduced into tobacco using the
cDNA and constitutively expressed under the CaMV
35S promoter. Using these plants, it has been possible
to determine changes in [Ca 2+]j (intracellular free calcium) in response to a number of external stimuli .
Using various Ca2+ channel blockers, the signals, such
as cold shock, fungal elicitor, touch and wind, leads to
a transient increase in [Ca 2+]; but the source of Ca2+,
either extracellular or intracellular, is dependent on
stimulus (Knight et ai, 1992).
Cold calcium signalling in Arabidopsis has been
involved in two cellular pools and a change in calcium signature after acclimation to give rise to "cold
memory" (Knight et ai, 1996, 1997). Intracellular
Ca2 + increase following cold shock requires extracellular calcium and may be derived from a Ca2+ influx
2
mediated by plasma membrane Ca + channels (Polinsensky & Braam, 1996). The cold up-regUlation of at
least a subset of TCH genes, which encode CaM related proteins, req uires an intracellular Ca 2+increase.
PANDEY el af: ABIOTIC STRESS TOLERANCE IN PLANTS
In apoaequorin transformed tobacco cells, influx of
2
Ca + was required to communicate oxidative burst
signal but not maintain the defense response, which
2
suggest that Ca + pulses serve frequently, but not invariably, to transduce an oxidative burst signal
(Chandra et aI, 1997 ).
Ca2+mediated signal transduction in stomata guard
cells has also been addressed. In Nicotiana plumbaginijolia, aequorin expression was specifically targeted to the guard cells. Changes in the Ca 2+ levels in
response to ABA, mechanical and low temperature
promoted rapid stomatal closure were monitored. Ele2
vations of guard cell [Ca +] CYI play a key role in the
transduction of all the stimuli (Wood et al. 2000).
However, there was a striking difference in the magnitude and kinetic of all the responses. Studies using
Ca2+ channel blockers and calcium chelator further
suggested that mechanical and ABA signals primarily
mobilize ci+ from the intracellular store(s) whereas
the influx of extracellular Ca2+ is a key component in
the transduction of low temperature signals.
Although the roots respond to cold, drought and
salt stress with increase in cytoplasmic free calcium.
the role(s) of various functionally diverse cell types
that comprise the root is not known. Transgenic
Arabidopsis with enhancer trapped GAL4 expression
in specific cell types was used to target the aequorin,
fused to a modified yellow fluorescent protein (YFP)
- to enable in vivo measurement of changes in cyto2
solic free Ca + concentrations in specific cell types
during acute cold, osmotic and salt stresses. In response to acute cold stress, all cell types displayed
2
rapid [Ca +]CYI peaks while the endodermis and pericy2
cle displayed prolonged oscill ations in [Ca +]CYI in
response to osmotic and salt stress (Kiegle et al.
2000).
Thus, different abiotic stresses can elicit very dis2
tinct Ca + signatures that are affected by the cell type,
kinetics, amplitude and duration of the increased Ca 2+
and the source of the mobilizable Ca2+ store. These
studies highlight the importance of Ca 2+signalling in a
wide range of plant responses to perturbations in the
environmental conditions. Still much remains to be
learned in signal perception mechanisms, particularly
the nature of receptors for these types of signals, it is
clear that they can be sensed at the cell surface or intracellularly. In both cases, the information can somehow be transduced into a Ca2 + signature that is in
some way used to direct the specific responses of the
plant to the stimulus.
139
Calcium Binding Proteins (CaBPs) in Plants
2
The conversion of the Ca + signal into gene expression or a physiological response is dependent on the
nature of the Ca 2+ signal and on downstream response
components. Thi s involves various CaBPs and kinases. Thus, a Ca2+ dependent 'conformation switch'
performs these functions: activation of enzymatic or
other biochemical activities of the proteins and association or dissociation with their target molecules .
The diverse functions mediated by Ca2+ are can-ied
out by a large number of CaBPs, which serve as Ca 2+
sensors (Ohta et al. 1995; Reddy, 2001). CaBPs sense
an increase in the [Ca2+]Cyl levels which decode Ca2+
signal. Once the Ca2+ sensors decode the elevated
[Ca 2+]CYI, the Ca 2+ levels are restored back to its resting
state by either efflux of Ca2+ to the cell exterior and/or
sequestration into cellular organelles such as vacuoles, ER and mitochondria. Thus, CaBPs playa key
role in decoding Ca 2+ signatures and transducing signals by activating specific targets and pathways.
Several CaBPs have been identified and characterised in both the animal (Celio, 1996) and the plant
(Poovaiah & Reddy, 1993; Zielinski, 1998; Reddy,
2
2001) systems. In plants, Ca + sensors can be grouped
into the following five major classes as follows: (a)
CaM; (b) CaM-like and other EF-hand containing
CaBPs; (c) Ca2+-regulated protein kinases; (d) Ca 2+_
modulated protein phosphatases; and, (e) CaBPs
without EF-hand motifs. The members of first three
2
classes of Ca + sensors contain a common structural
motif(s), 'EF hand', which is a helix-loop-helix
structure that binds a single Ca2+ ion with high affinity
(Roberts & Harmon, 1992). These motifs typically
occur in closely linked pairs, interacting through antiparallel ~-sheets. This arrangement is actually the basis for cooperativity in Ca 2+ binding. Different CaBPs
differ in the number of EF hand motifs and their affinity to bind Ca2+; some of the CaBPs have Ca 2+_
binding affinities in the nanomolar to micro molar
range. Binding of Ca2+ to the Ca2+ sensor results in a
conformational change in the sensor that alters its interaction with the other proteins, which modulate their
function/activity or modulates its own activity .
(a) Calmodulin (CaM)
CaM is a highly conserved, most well characterized, ubiquitously expressed CaBP in eukaryotes
(Snedden & Fromm, 1998 ; 2001). It is a small molecular weight (16.7 kDa), acidic, heat-stable protein
of 148 amino acids with four EF-hand motifs that
140
INDIAN J BIOTECHNOL, APRIL 2002
bind . to four Ca2+ ions. The binding of Ca2+ to CaM
results in 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 (0' Neil & DeGrado, 1990; Rhoads & Friedberg, 1997).
CaM has been shown to be a multi-functional protein. The comparison of various cDNA sequences and
alignment of amino acid sequences from various plant
and animal CaMs shows a very high degree of homology, indicating existence of a parallel Ca2+/CaM
signalling pathway in plants and animals. The involvement of CaM has been shown during processes
of cell cycle (Vantard et al, 1985), cell growth and
embryogenesis (Oh et al, 1992), germination of seed
embryo (Cocucci & Negrini, 1988), differentiation of
treachery elements (Kobayashi & Fukuda, 1994), cell
proliferation (Perera & Zielinski, 1992) and phytochrome mediated signalling pathways (Lam et ai,
1989; Neuhaus et al, 1993). Besides, responsiveness
of CaM gene(s) to various stimuli and their spatially
and temporally regulated expression confirms their
importance in Ca2+ signalling pathways (Galaud et ai,
1993),
Plants have a large number of CaM isoforms
whereas no isoform could be detected in animals despite the presence of a multigene family. Presence of
multigene family of CaM in plants i.e. two each in
rice, Petunia and Vigna; three in maize; five in soybean; six in Arabidopsis, eight in potato and existence
of a number of isoforms that contain a few conservative changes i.e. four in Arabidopsis; three in wheat
and four in soybean (Ling et aL, 1991; Liu et aL, 1991;
Gawienoiwski et aL, 1993; Lee et al, 1995; Takezawa
et aL, 1995; Yuang et al, 1996), possibly contributes
to the diversity and specificity of Ca 2+/CaM mediated
signalling. These small changes in the CaM isoforms
may contribute to differential interaction of each isoform with the target protein. CaM genes are expressed
differentially in response to different stimuli (Snedden
& Fromm, 1998 ; Zielinski, 1998). Such differential
regulation is possibly one of the mechanisms, for the
cells to fine-tune Ca2+ signalling.
In plants, the expression pattern of CaM isoforms
is spatially regulated. In Arabidopsis, ACAM 1 was
most abundant transcript in leaves and developing
siliques (Ling et ai, 1991) while ACAM 3 was expressed preferentiaIIy in aerial tissues except floral
buds (Perera & Zielinski, 1992; Antosiewicz et ai,
1995). The levels of ACAM 4, 5 and 6 were consid-
erably lower than the other isoforms. In roots, only
ACAM 1 could be detected (Perera & Zielinski, 1992;
Gawienoiwski et aL, 1993). In Brassica also, the level
of CaM transcript was higher in leaves and shoot api cal meristem than in root tips or root elongation zone
(Chye et ai, 1995). Maize CaM isoforms, ZMCAM 1
and ZMCAM2, were differentially expressed in different tissues (Breton et al, J 995) . In potato, PCM 1
isoform was highly expressed in stolon tip, moderately in roots and very low in leaves, while PCM6
showed a steady state expression level in all the tissues except roots (Takezawa et aL, 1995).
The expression of CaM mRNA was also found to
be different in different tissues of soybean (Lee et al,
1995). In Arabidopsis, in a study of transcriptional
activation of all the six CaM genes in response to
touch show that two of the isoforms are not regulated
by touch whereas the other four show differences in
their response (Verma & Upadhy aya, 1998), This
shows that the level of expression of CaM varies in
different parts of a plant, and the isoforms also behave
differently thus providing the much needed diversity
and specificity to Ca2+ signal.
Various other external stimuli as light (lena et al,
1989; Braam & Davis, 1990; Botella & Arteca, 1994),
auxin (lena et al, 1989; Botella & Arteca, 1994;
Okamoto et al, 1995) and ethylene (Braam & Davis,
1990), also bring about differenti al expression of
CaM and related gene(s). Similarly, in barley aleurone
protoplasts, where Ca 2+ mediates GA and ABA effects, a modulation of CaM gene expression and protein level could be detected (Gilroy, 1996). Specific
soybean CaM isoforms, SCaM-4 and SCaM-5, are
activated by infection or pathogen derived elicitors
and participate in Ca2+ mediated induction of plant
disease resistance response, whereas other SCaM
genes encoding highly conserved CaM isoforms
showed no effect indicating strikingly the differential
regulation of CaMs in plants (Heo et ai, 1999).
A strategy for elucidating specific molecular targets of Ca2+ and CaM in plant defense responses has
been developed . A dominant-acting CaM mutant,
VU-3, was used to investigate the oxidative burst and
nicotinamide coenzyme fluxes after various stimuli
(cellulase, harpin, incompatible bacteria, osmotic and
mechanical) that elicit plant defense responses in
transgenic tobacco cell cultures. VU-3 CaM differs
from endogenous plant CaM in that it cannot be
methylated post-translationally, and as a result, it hyperactivates CaM-dependent NAD kinase. CeIIs ex-
PANDEY et al: ABIOTIC STRESS TOLERANCE IN PLANTS
pressing VU-3 CaM exhibited a stro nger active oxygen burst that occuned more rapidly than in normal
control cells challenged with the same stimuli. Increase in NADPH levels were also greater in VU-3
cells and coincided both in timing and magnitude with
development of the active oxygen species (AOS)
burst. These data show that CaM is target of calcium
fluxes in response to elicitor or environmental stress,
and provided the first evidence that plant NAD kinase
may be a downstream target which potentiates AOS
production by altering NAD(H)/NADP(H) homeostasis (Harding et al, 1997).
CaM binding proteins (CBPs). CaM is multifunctional because of its ability to interact with and regu'late the activity of various target proteins, which is
mediated by CBPs in plants. The CaM-binding motifs
from different CBPs form characteristic basic amphipathic a-helices with several positive residues on
one side and hydrophobic residues on the other side.
However, the amino acid seq uences of the CBP in
different CaM target proteins are not conserved per se
(Rhoads & Friedberg, 1997).
CaM regulates the activation of large number of
diverse proteins implicated in a wide variety of cellular processes such as kinases, nitric oxide synthase,
myosin light chain kinase, phosphorylase kinase, calcineurin phosphatase, plasma membrane Ca2+
ATPases and many other enzymes (Billingsley et al,
1990). Role of these proteins has been extensively
reviewed (Zeilinski, 1998; Reddy, 2001). Interestingly, many CBPs have no homologs in animals.
CaM stimulates small nuclear NTPases. As nuclear
NTPases also get stimulated during phytochrome signalling, it was postulated that CaM may be playing a
role along with Ca2+ in phytochrome mediated signalling through nuclear NTPases (Hsieh et al, 1996).
Another important protein with which CaM binds
to and stimulates its activity is glutamate decarboxylase (GAD) . This enzyme has homologues in animals
but lack CaM binding motif, suggesting that they are
regulated by CaM only in plants. GAD has been purified from different plants (Ling et ai, 1994) and exists
as many isoforms . It catalyses decarboxylation of
glutamate, thereby producing CO 2 and gammaaminobutyrate, a very important component of many
metabolic pathways. Transgenic Petunia plants, harbouring a mutan t GAD lacking the CaM binding site,
showed several abnormalities confirming CaM regulation of GAD activity (Baum et ai, 1996). As GAD
141
activity gets stimulated by hypoxia and some other
stress signals, it indicates that besides its role in controlling various metabolic processes, it might also be
involved in CaM regulated stress signalling (Baum et
al, 1993, 1996; Gallego et ai, 1995; Snedden et al,
1996).
NAD-kinase, which catalyzes the conversion of
NAD to NADP, was shown to be regulated by CaM in
plants, whereas in animal systems no such regulation
was observed. is vital to living organisms especially
when energy is in demand under stress conditions . It
plays a role in oxidative burst and in the formation of
active oxygen species, which are involved in plant
defen se against pathogens (Harding et al. 1997). NAD
kinases are also regulated by CaM isoforms (Lee et
al, 1995). In certain cases, light and some other stimuli have been shown to stimulate the activity of NAD
kinases. Using transgenic plants which over express
CaM and its mutants and by giving different stress or
elicitor signals such as cellulase, heparin and osmotic
or mechanical stress to such transformed plants, it was
found that CaM is the target of Ca2+ fluxes, and NAD
kinase could be the downstream target for this
Ca 2+ICaM mediated signalling pathways (Harding et
al, 1997; Lee et al, 1997) .
The other important CBPs are the endoplasmic reticulum and tonoplast located Ca2+ ATPases and slow
vacuolar ion channels which have been involved in a
number of calcium signalling processes (Askerlund,
1997; Harper et al. 1998). Several CBPs that show
similarities to ion channels have been isolated from
barley (Schuurink et al. 1998), tobacco (Arazi et al,
1999, 2000) and Arabiodopsis (Kohler et al. 1999;
Kohler & Neuhaus, 2000). Such proteins reside in the
plasma membrane and show sequence similarity to
cyclic nucleotide gated channels from animals and
inward rectifying K+ channels from plants . Constitutive overexpression of one of these proteins, NtCBP4,
in sense and antisense orientation in tobacco, exhibited a normal phenotype under normal growth conditions. However, NtCBP4 sense transgenic plants
showed improved tolerance to Ni 2+ and hypersensitivity to Pb 2+ (Arazi et ai, 1999, 2000) suggesting a role
for this CaM-binding channel in metal tolerance.
By using 3sS-labeled CaM, it was found that it can
bind to various microtubular motors in specific ways.
A novel CaM-binding microtubule motor protein was
isolated from A rabidopsis (Reddy et ai, 1996a;
1996b) and tobacco (Wang et al. 1996a). This kinesin-like CaM-binding protein (KCBP) is distinct from
It
142
INDIAN J BIOTECHNOL, APRIL 2002
all other KLPs in having a CaM binding domain adjacent to its motor domain and appears to be ubiquitous
in plants. KCBP interacts with tubulin subunits (Song
et ai, 1997). The binding of KCBP with tubulin'is
regulated by Ca 2+/CaM i.e. in presence of Ca2+/CaM,
the motor domain with the CaM binding domain does
not bind to tubulin and this modulation is abolished in
the presence of antibodies specific to CaM binding
domain of KCBP. This CaM-dependent modulation of
KCBP interaction with tubulin suggests regulation of
KCBP function by calcium (Narasimhulu et ai, 1997)
as well as in cell division. During cell division, these
proteins have been found to be associated with preprophase band, mitotic spindle and phragmoplast. Association with microtubular motor arrays in dividing
cells suggests that this negative end directed motor
protein is likely to be involved in the formation of
microtubular arrays and/or functions associated with
these structures (Bowser & Reddy, 1997) The myosin
heavy chain binding cDNA also contains a putative
CaM binding site (Kinkema & Schiefelbein, 1994).
This shows that CaM is involved in intracellular
transport processes in cells .
Heat shock proteins also contain CaM binding domains (Li et ai, 1994; Lu et ai, 1995). One of such
CBPs, NtCBP48, contains a centrally located putative
transmembrane domain and a nuclear localization sequence motif (Lu et ai, 1995). Besides, few transcription factors of basic helix-loop-helix family (Corneliussen et ai, 1994), basic amphipathic a-helix (Dash et
ai, 1997) also contain CaM binding domains . In
Arabidopsis, ACAM-3 promoter binds to the leucine
zipper family of transcription factor TGA3, while
CaM itself acts as an enhancer of TGA3 binding with
its own cis-elements (Szymanski et ai, 1996). Different CaM isoforms differentially enhance binding of
TGA3 promoter to ACAM 3 and also to cauliflower
2
nuclear proteins . All this suggests that Ca + mediated
signalling coupled to gene expression could be mediated via CaM and CaM binding transcription factors
and could lead to specificity of the response.
AtCNGCl and AtCNGC2 have CaM and a · putative cyclic nucleotide-binding domain (Kohler et at,
1999), and interact with yeast CaM. In plants,
Ca 2+/CaM is involved in stabilizing cortical microtubuies at low Ca 2+ concentration and destabilizing the
same at higher Ca 2+ concentration (Cyr 1991, Fisher
et at, 1996). Two CBPs that show different sensitivities to Ca 2+ are implicated in evoking these two opposing effects of CaM on cortical microtubules. Elon-
gation factor-l a (EF-la), a CaM-binding microtubule associated protein, stabilizes microtubules .
2
Ca +/CaM has been shown to inhibit
EFla-promoted microtubule stabilization (Moore et ai,
1998). An auxin-induced gene product binds CaM ,
suggesting the involvement of CaM in auxin action
(Yang & Poovaiah, 2000) . Two maize proteins, CBP1 and CBP-5, and a multidrug resistant protein also
2
bind CaM in a Ca +-dependent manner (Reddy et ai,
1993; Wang et ai, 1996b). However, their function is
not known. A pollen specific CBP from maize,
MPCBP (Safadi et ai, 2000), is a novel CBP and has
no homolog in non-plant systems. Binding of superoxide dismutase to CaM-Sepharose column is indicative of its regulation by CaM (Gong & Li, 1995).
(b) CaM like Ca 2+ Regulated Proteins
In addition to CaM, plants contain numerous CaMlike proteins whose function in Ca2+ signalling pathway(s) is not fully characterized as compared to that
of CaM. CaM-like proteins differ from CaM in containing more than 148 amino acids and one to six EF~
hand motifs with limited homology to CaM (Snedden
& Fromm, 1998). Hence, it is likely that such proteins
are functionally distinct from CaM and are involved
in controlling different Ca2+ mediated cellular functions .
Calcineurin in calcineurin B-like (CBL) protein, a
new family of CaBPs, is a Ca2+/CaM dependent protein phosphatase involved in Ca2+ signalling in ani mals and yeast. However, the biochemical identity of
calcineurin remains elusive. In Arabidopsis, AtCBL
(Kudla et ai, 1999), which shares maximum homology to the regulatory subunit of mammalian calcineurin B, shows significant similarity with another CaBP,
the neuronal calcium sensor in animals . AtCBL contains typical EF-hands motif. Interaction of AtCBLl
and calcineurin A complemented the salt sensitive
phenotype in a yeast calcineurin B mutant. Cloning of
cDNA revealed a family of at least six genes in
Arabidopsis encoding highly similar but functionally
distinct CaBPs (AtCBL proteins) .
(c) Ca 2+ Modulated Protein Kinases in Plants
The role of protein kinases and phosphatases in
plant signal transduction has been extensively reviewed (Stone & Walker, 1995; Sopory & Munshi,
1998). Kinases are the main trigger proteins, which
transduce signal by phosphorylating other proteins/kinase(s) . A large number of protein kinases
PANDEY et al: ABIOTIC STRESS TOLERANCE IN PLANTS
ex ist in plants. Some of these kinases are similar to
the kinases present in animal systems while others are
specific to plants. Calcium regulates following three
different families of protein kinases in plants: (i) calcium dependent protein kinases (CDPKs) which require only Ca2+ for their activity; (ii) Ca2+/CaM dependent protein kinases (CCaMKs) which along with
Ca2+ also require CaM for their activity; and, (iii)
Ca2+/lipid dependent protein kinases (PKCs) which
require lipids along with Ca2+ for activity .
(i) CDPKs. These are widely distributed most
abundant and well-characterized kinases and their
existence is ubiquitous in plants (Sopory & Munshi,
1998 ; Harmon et at, 2000). CDPKs are plant specific
protein kinases as no CDPK homologue has been reported from the animal systems as yet. These kinases
require micromolar concentration Ca 2+ for their activity and have no requirement of CaM or lipids.
The CDPKs have a unique structure as the Nterminal protein kinase domain is fused with Cterminal auto-regulatory domain and a CaM like domain(CaMLD), which has four Ca2+ binding EF-hand
or helix-loop-helix motif (BaIty & Venis, 1988;
Harper et at, 1991; Suen & Choi, 1991). The region
that joins the kinase domain to the CaM-like region
Uunction region) corresponds to the autoinhibitory/CaM-binding region of CaM K II (Ca"2+/CaMdependent protein kinase II) and prevents kinase activity in the absence of Ca 2+ (Harper et ai, 1991).
Binding of Ca2+ to CDPK affects conformation of the
kinase and relieves the inhibition caused by the autoinhibitory region (Harmon et at, 2000).
The N-terminal domain of CDPKs is variable and
provides specificity to different CDPK isoforms. Ca2+
directly binds to the CDPK and stimulates the kinase
activity . These enzymes show autophosphorylation
and many fold stimulation with Ca2+. The CDPK from
groundnut shows that autophosphorylation is a prerequisite for the activation of GnCDPK (Dasgupta,
1994; Chaudhari et at, 1999). They are both soluble as
well as membrane bound and have been reported from
organelles also. Several CDPKs have putative myristorylation sites indicating that myristoylation of
CDPKs may regulate the association of CDPKs with
membranes (Harmon et ai, 2000). Although CaM
does not stimulate these kinases, different CaM inhibitors affect the activity of these CDPKs, possibly
due to the existence of CaMLD. The autoregulatory
domain keeps the activity of the enzyme at basal
level.
143
Under in vitro conditions, the kinases get activated
by binding to Ca 2+ and use various proteins as substrates like histone, casein, phosvitin, BSA and few
synthetic peptides but in vivo substrates are not
known in many cases. Genes for various CDPKs have
been cloned and some of them belong to mUltigene
family (Biermann et ai, 1990; Ali et at, 1994; Breviario et ai, 1995; Thummler et ai, 1995; Hrabak et ai,
1996; Redhead & Pal me, 1996). There are over 40
CDPKs in the Arabidopsis genome . Furthermore,
CDPKs differ in their affinity for Ca2+. In Arabidopsis, the AtCDPKl differs from AtCDPK2 in its Ca2+
stimulated activity although both of them possess four
EF hand m9tifs.
Besides Ca 2+, lipids are involved in the regulation
of CDPK activity (Harper et at, 1993; Binder et at,
1994). 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 (Farmer &
Choi, 1999). CDPKs have been reported to playa role
in diverse cellular processes ranging from ion transport to gene expression. Ion channels, enzymes involved in metabolism, cytoskeletal proteins and DNA
binding proteins have been identified as CDPK substrates (Harmon et ai, 2000).
Further, there are certain CDPK-related kinases
(CRKs) that are similar to CDPKs except that the
CaM -like region is poorly conserved with degenerate
EF-hands. Atleast seven CRKs have been found in
Arabidopsis, however, regulation and function of
these kinases are not yet known (Harmon et at, 2000).
(ii) CCaMKs. It is a group of calcium-dependent
kinases, which in addition to calcium also require
CaM for their activity. Thus CaM, besides acting directly, could also exert its effect by binding to protein
kinases and modulating their activities. In animal
systems, CaM activates Ca2+/CaM dependent kinase I,
II and III, which regulate a wide variety of physiological processes involving Ca2+ mediated signalling
(Colbran et at, 1989).
Since, importance of Ca 2+/CaM kinases in animal
systems is very well established, attempts were made
to look for a parallel signalling pathway in plant systems as well. Some indirect studies (Salimath &
Marme, 1983; Blowers & Trewavas, 1987) predicted
existence of kinases family in plants which was
eventually confirmed by cloning of various cDNA
homologues from different plant systems e.g. carrot
144
INDIAN J BlOTECHNOL, APRIL 2002
(Suen & Choi, 1991), apple (Watillon et ai, 1992,
1993, 1995), lily (Patil et al, 1995; Takezawa et ai,
1996) and maize (Lu et al, 1996). All homologues
show a considerable similarity with their animal system counterparts at the cDNA level. 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. Biochemically, Ca 2+/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 (Takezawa et
al, 1996).
The kinase cDNA homologue from apple encodes
a single polypeptide (mol wt, 46.5 kDa) with serine/threonine catalytic domain and an adjacent
2
Ca +/CaM binding regulatory domain. It shows considerable homology to corresponding regions of
mammalian multi-functional Ca2+/CaM protein kinase
II (Watillion et ai, 1995). Ca 2+/CaM dependent kinase
gene from lily anthers is a chimeric gene containing a
2
neural visinin like Ca + binding domain fused with a
CaM binding domain . The amino-terminal region of
the encoded protein contains all the eleven conserved
sub-domains characteristics of serine/threonine protein kinase. The CaM binding region has high homology (79 %) to the subunit of mammalian
Ca 2+/CaM dependent protein kinase (Patil et al, 1995).
Biochemical properties of lily Ca2+/CaM dependent
kinase, studied by over-expressing its cDNA in E.coli,
show that it is a non-conventional, novel Ca2+/CaM
2
kinase, which shows a dual regulation with Ca + and
CaM . Autophosporylation of the protein is only Ca2+
dependent while both Ca2+ and CaM regulate substrate phosphorylation, though neither of them modulates the kinase activity individually (Takezawa et al,
1996). Using the yeast two-hybrid system, to obtain
genes coding for the proteins interacting with this kinase, a cDNA clone, which shows very high similarity to EF-l ex, has been obtained. The kinase phosphorylates EF-1 ex in a calcium/CaM dependent manner
suggesting its direct role in the regulation of gene expression (Wang & Poovaiah, 1999). Similar CCaMK
genes (TCCaMK-l and TCCaMK-2) cloned from tobacco show differential regulation by CaM isoforms
(Liu et al, 1998 ).
MCKI, a Ca 2+/CaM kinase homologue from maize
roots (Lu et al, 1996; Lu & Feldman, 1997), contains
all the eleven conserved subdomains, characteristic of
protein kinase catalytic domain and all the conserved
amino acid residues . It shares sequence homology
with yeast CMK1 (42%), rat CaMK II (37 %) and apple CBI (34%). It is expressed in root caps, which is
the site of perception of both light and gravity signals.
Since MCKI is expressed both in light and dark
grown tissue, it appears not to be directly regulated by
light but has been shown to be involved in gravitropic
responses. However, biochemical properties of this
kinase have not been studied till now. A kinase (mol
wt, 72 kDa), characterized biochemically from Zea
mays, belongs to the seri ne/threonine family of protein kinases and shows a dual regulation by calcium
and CaM. The substrate phosphorylation is calcium
dependent and addition of exogenous CaM stimulates
it further, whereas autophosphorylation is only calcium dependent (Pandey & Sopory, 1998).
Thus, homologues of both conventional and novel
Ca2+/CaM dependent protein kinases exist in plants.
As these kinases are involved in a wide range of signalling processes in animals also, hence their impor2
tant role is envisaged in Ca + signalling pathways in
plants.
(iii) PKCs. These kinases belong to the serine/threonine family of protein kinases, and in addition to Ca2+ require phospholipids for their activity . In
mammalian system, they are basically involved in
various regulatory processes (Nishizuka, 1992),
playing a pivotal role in signal transduction involving
receptor-mediated hydrolysis of PIP2 , which produces
IP) and DAG. DAG is an activator of PKC type kinases, while IP) releases calcium from intracellular
stores. Such kinases are present in both membrane
and soluble fraction, and show some selectivity towards the lipids, which are required for their stimulation.
In plants, some of these types of purified kinases
have similar activities as their animal system counterparts (Baron-Marting & Scherer, 1989; Komatsu &
Hirano, 1993; Honda et ai, 1994; Nanmori et ai,
1994; Karibe et al, 1995; Chandok & Sopory, 1998).
Although these kinases have been reported from various plant sys tems, their exact role in calcium mediated signalling is not known and further work is
needed in this direction. A cPKC activity in maize has
been characterized in great detail and its role in light
mediated nitrate reductase (NR) gene induction has
been studied (Chandok & Sopory, 1998).
(d) Ca 2+ Modulated Protein Phosphatases in Plants
A Ca 2+ modulated protein phosphatase has been
PANDEY er al: ABIOTIC STRESS TOLERANCE IN PLANTS
discovered through the analysis of a calcium signalling pathway mutant. A mutation at the ABIl (absci sic acid in sensitive) locus in Arabic/apsis thaiiana
caused a reduction in the sensitivity to the plant
hormone, absci sic acid. The sequence of ABI I predicts a protein composed of an N-terminal domain
that contains motif for an EF-hand Ca 2+ binding site
and a C-terminal domain with similarities to protein
serine/threonine phosphatase 2C. The C-terminal of
ABU can partially complement temperature sensitive growth defect of a Saccharomyces cerevisiae
protein phosphatase 2C mutant and this also shows
phosphatase activity in vitro (Leung et ai, 1994;
Meyer et ai, 1994; Bertauche et ai, 1996). These
suggest that the ABIl protein is a Ca2+_ modulated
phosphatase and functions to integrate ABA and
ci+ -signals with phosphorylation dependent response pathway .
Protein phosphatase 2C (PP2C) is a class of ubiquitous and evolutionarily conserved serine/threonine
PP involved in stress responses in yeasts, mammals,
and plants (Shenolikar, 1994; Hunter, 1995). Mutational analysis of two Arabic/apsis thaliana PP2Cs,
encoded by ABH and AtPP2C, involved in the plant
stress hormone abscisic acid (ABA) signalling in
maize mesophyll protoplasts . Consistent with the
crystal structure of the human PP2C, the mutation of
two conserved motifs in ABIl (predicted to be involved in metal binding and catalysis) abolished
PP2C activity. Surprisingly, although the OGHI77179KLN mutant lost the ability to be a negative
regulator in ABA signalling, the MEOI41-143IGH
mutant still inhibited ABA-inducible transcription,
perhaps through a dominant interfering effect.
Moreover, two G to 0 mutations near the OGH motif
eliminated PP2C activity but displayed opposite effects on ABA signalling . The G 1740 mutant had no
effect but the G 1800 mutant showed strong inhibitory
effect on ABA-inducible transcription. Based on the
results that a constitutive PP2C blocks but constitutive
2
Ca + dependent protein kinases (COPKs) activate
ABA responses, the MEOI41-143IGH and G1800
dominant mutants are unlikely to impede the wild
type PP2C and cause hyper phosphorylation of substrates . In contrast, these dominant mutants could trap
cellular targets and prevent phosphorylation by PKs
required for ABA signalling. The equivalent mutations in AtPP2C showed similar effects on ABA responses. This study suggests a mechanism for the action of dominant PP2C mutants that could serve as
145
valuable tools to understand protein-protein interactions mediating ABA signal transduction in higher
plants (Sheen, 1998). Stress response in plants involves changes in the tran scription of specific genes.
2
The constitutively active mutants of two related Ca +
dependent protein kinases, COPK 1 and COPK 1a, activate a stress-inducible promoter, by passing stress
signals. Six other plant protein kinases, including two
distinct COPKs, fail to mimic this stress signalling
(Sheen, 1996). Furthermore, the activation is abolished by a COPKI mutation in the kinase domain and
diminished by a constitutively active protein phosphatase 2C that is capable of blocking response to the
stress hormone, abscisic acid.
(e) CaBPs without EF-Hand Motifs
There are several proteins that bind Ca2+ but do not
contain EF-hand motifs, like calreticulin, centrin, annexin, etc.
2
(i) Caireticulin(CRT). It is a Ca + sequestering
protein in the ER and functions as a chaperone
(Michalak et at, 1998; Baluska et at, 1999). It is a
major calcium storage protein (mol wt, 48 - 55 kOa)
located mainly in lumen of the endoplasmic reticulum
and also in the nucleus and/or cytoplasm of some
cells. The cONA for several CRT have been cloned
from maize, barley, tobacco and Ricinus communis
(Chen et at, 1994; Kwiatkowski et at, 1995; Oresselhaus et at, 1996; Coughlan et ai, 1997; Borisjuk et ai,
1998). Several other homologues have been detected
in other plants e.g. CRT-like protein in tobacco cells
(Oenecke et at, 1995; Oroillard et at, 1997), spinach
(Navazio et at, 1996) and pea (Hassan et ai, 1995).
The functional role of CRT in post-translational processing and translocation processes has been suggested
apart from its postulated function in cellular Ca 2+ homeostasis (Borisjuk et ai, 1998).
The Ca2+ status of the endoplasmic reticulum can
be altered by the overexpression of a CaBP(CRT) in
transgenic plants. A 2.5-fold increase in CRT led to a
2-fold increase in ATP-dependent 45Ca2+ accumulation in the ER-enriched fraction of the transgenic
plant indicating that altering the production of CRT
affects the ER Ca2+ pool (Persson et ai, 2001). Such
plants were able to retain chlorophyll when transfen'ed from Ca2+-containing medium to Ca2+-depleted
medium thereby enhancing the survival of plants
grown in low Ca 2+ medium.
(ii) Arabic/apsis GF14 protein. It shows more than
60% identity with mammalian brain 14-3-3 proteins at
146
INDIAN J BIOTECHNOL. APRIL 2002
the amino acid sequence level and is apparently associated with G-box DNA binding protein complex (Lu
et ai. 1994). Arabidopsis GFI4w binds calcium and
its C-terminal domain contains a potential EF-hand
motif. GF14w is phosphorylated by Arabidopsis protein kinase activity at a serine residue(s) in vitro .
Therefore, GF14w protein has biochemical property
consistent with potential signalling roles in plants.
(iii) Centrill. It is an acidic CaBP of 20 kDa and
was first identified in the green alga, Tetraseimis striata, as a component of striated flagellar roots. Basal
body complexes isolated from Chiamydomonas reil1hardtii contain protein whose cDNA shows high homology to centrin, which is strongly related to CDC
31 gene product of S. cerevisiae (Schiebel & Bornens,
1995). Centrin has been reported from higher plants,
Atripiex l1ummularia (Zhu et ai, 1992) and animals,
mouse (Ogawa & Shimizu. 1993) and humans (Lee &
Huang, 1993). In some organisms, centrins are also
known as caltractins. Although centrin from different
species seems to have different functions, it could
well be that they act by a common underlying molecular mechanism . Centrins probably drastically
2
change their conformation upon binding to Ca + and
either performs contraction (in Chiamydomonas) or
transduction of signal for duplication of the SPB (in S.
cerevisiae) (Schiebel & Bornens, 1995).
(f) Other CaBPs
CaBPs have also been identified as caldesmon like
protein from pollen tubes of Ornithogalum virel1s
(Krauze et aI, 1998) and from vacuoles of celery
(Apium graveoiens L.) (Randall, 1992). The genes
(cDNA) for other CaBPs from several plants have
also been cloned. Some of these cDNA are novel e.g.
NaCI induced CaBP from Arabidopsis, AtCP, (lang et
al. 1998), a CaBP from Brassica, PCP, which is predominantly expressed in pistil and anthers (Furuyama
& Dzelzkalns, 1999), ABA and osmotic stress induced CaBP from germinating rice seedlings (Frandsen et ai, 1996), a 22 kDa CaBP (CaBP-22) from
Arabidopsis, which is related to CaM (Ling & Zielinski, 1993).
In Atripiex Ilummularia, multiple transcripts of
CaBP showed differential regulation by environmental stimuli and development (Zhu et al. 1996). In
Arabidopsis, a homologue of neutrophil NADPH oxi dase gp91phox subunit encoding a plasma membrane
CaBP containing EF-hand motif (respiratory burst
oxidase homologue A-rbohA) (Keller et ai, 1998)
and in slime mold, Dictyostelium discoideum, a novel
CaBP, Calfumirin-l (CAF-l), which shows specific
expression during transition of cells from growth to
differentiation, have been reported.
In bean, Hra 32 (hypersensitive reaction associated), a CaBP (mol wt, 17 kDa) that specifically accumulates during HR (hypersensitive response), has
been identified. Hra 32 has four putative EF-hand calcium -binding domains (lakobek el ai, 1999). Therefore, several CaBPs have been identified from plants
but their in vivo function has not been shown .
(i) Annexin. Higher plants contain annexins
(Boustead et aI, 1989), which have been purified and
characterized from a variety of plant sources . Analyses of the deduced proteins encoded by annexin
cDNA indicate that the majority of these annexins
possess the characteristic four repeats of 70 to 75
amino acids and motif proposed to be involved in
Ca 2+ binding. Like animal annexins, plant annexins
bind Ca2+ and phospholipids and are abundant proteins, but the number of distinct plant annexin genes
may be considerably fewer than that found in animals.
Various members of the annexin family in plants may
play roles in secretion and/or fruit ripening, as they
show interaction with the enzyme callose (1, 3-betaglucan) synthase, possess intrinsic nucleotide phosphodiesterase activity, bind to F-actin and/or have
peroxidase activity (Clark & Roux, 1995).
(ii) Calnexin. Of the few calnexin genes identified
in plants, the first calnexin(CNX 1P), reported in
Arabidopsis, encodes for a protein of 65.5 kDa, and is
48 % identical to dog calnexin (Huang et ai, 1993b).
Calnexin is 64 kDa protein, which co-purified with
oat vacuolar ATPase B subunit and interacts with
both subunit A and B of vacuolar ATPase (Li et ai.
1998). A pea calnexin, PsCNX, cloned and characterized recently (Ehtesham et aI, 1999), binds to ATP
and contain ATPase activity. In addition, casein kinase II phosphorylates it in vitro. The function of
calnexin in plants is not yet known .
(iii) Phospholipases (PLA, PLC, PLD). Phospholipid catabolism is essential for cell function and encompasses a variety of processes including metabolic
channelling of unusual fatty acids, membrane reorganization and degradation, and the production of
secondary messenger. Phospholipases are grouped
into classes depending upon their general site of
cleavage. Multiple isoforms of two phospholipases
have been identified. Their different activities were
observed during different physiological consequences
PANDEY
el al:
ABIOTIC STRESS TOLERANCE IN PLANTS
in the context of the plant development and in response to environmental stress (Chapman, 1998). Of
the several phospholipases reported from plants, almost all of them have C2-domain and require Ca 2+ for
their activities (Hirayama et aI, 1995 , 1997; Shi et aI,
1995; Singer et aI, 1997; Wang, 1997; Munnik et aI,
1998).
(iv) Copines. Paramaecium tetraurelia copine (mol
wt, 55 kDa) is calcium dependent phospholipid binding protein, which contains C2 domain . In plants,
copine gene is reported in Arabidopsis thaliana . In
ciliates, the role of copine is suggested in the membrane trafficking. Current sequence databases indicate
the presence of multiple copine homologues in green
plants, Arabidopsis thaliana, nematodes, and human s
(Creutz et aI, 1998).
Role of CaBPs in Stress Tolerance
Various stimuli, which act by modulation of Ca 2+
concentration, affect the expression of different CaM
related gene(s). The presence of their multiple isoforms in plants add further complexity to the Ca 2+
mediated network and points to their differential sensitivity to elevated cytosolic Ca 2+ levels in response to
different stress stimuli. For example, in case of mechanical signals such as rain, wind and touch , differential expression of various CaM related genes was
observed and the pattern of expression of such genes
was reported to be spatially and temporally regulated
(Braam & Davis, 1990; Ito et aI, 1995; Polisensky &
Braam, 1996). Other CaBPs have also been shown to
be responsive to various environmental stimuli. The
possible involvement of CaBPs in stress response is
summarized below.
• Studies with salt overly sensitive (SOS) mutants
of Arabidopsis indicate a key role for Ca 2+ in salt
stress signalling (Wu et ai, 1996, Liu & Zhu 1997,
Liu et ai, 2000). SOS mutants are hypersensitive
to Na+ and Li+ and are unable to grow on low K+
culture medium . The abnormal growth patterns in
the presence of NaCI could be ameliorated by the
addition of increased levels of Ca 2+ in the same
medium. The deduced amino acid sequence of
SOS3 gene product shows its close affinity to
CaBPs, specifically calcineurin B like Ca 2+ sensor
and neuronal calcium sensors (NCS) of animals,
which can stimulate protein phosphatases or inhibit protein kinases (Liu & Zhu, 1998). Also,
SOS3 gene is predicted to contain three potential
2
Ca + binding sites. These versatile proteins par-
147
tlclpate in ion transport phenomena in other organisms, which concluded that SOS3 mediates
the interact ion of potassium, sod ium and calcium
i.e. control the K+/Na+ transport system via a Ca2+
-regulated pathway (Bressan et aI, 1998; Epstein ,
1998; Liu & Zhu, 1998). The SOS3 has been
found to physically interact with and to activate a
protein kinase SOS2 (Shi et aI, 1999; Halfter et
aI, 2000). Without SOS3, SOS2 has virtually no
activity as tested on several peptide substrates,
while it is able to phosphorylate pep tides in the
presence of SOS3. The binding of SOS3 and
SOS2 appears to be independent of free calcium,
however, the SOS2 phosphorylation of peptide
substrates requires calcium. Mutations in the
Arabidopsis salt genes, SOS3 and SOS2, cause
Na+ and K+ imbalance and render plants more
sensitive towards growth inhibition by high Na+
and low K+ environments. Also in these mutants,
the operation of high affinity potassium transporter is suppressed (Liu & Zhu, 1998). Both abnormal functions are mitigated or abolished by
high external calcium concentration, suggesting
that in mutants there is impairment in the signalling pathway essential for the normal function of
calcium in mitigating salt stress. The SOS3 gene
is predicted to encode a CaBP with an Nmyristoylation signature sequence. Both Nmyristoylation and calcium binding are crucial for
SOS3 function in plant salt tolerance (Ishitani et
ai, 2000). The SOS2 gene has been isolated and
identified as a serine/threonine type protein kin ase
(Liu et ai, 2000), which forms another potential
candidate for engineering abiotic stress tolerance.
The functional domains in protein kinase SOS2,
that interacts with SOS3 and is required for plant
salt tolerance, have also been identified (Guo et
ai, 2001). SOS3 interaction with and activation of
SOS2 kinase is very consistent with genetic evidence that the two genes are both the positive
regulators of salt tolerance and function in the
same pathway (Halfter et aI, 2000). These two
proteins together define a previously unknown
regulatory pathway, the SOS pathway, for plant
Na+ tolerance. It has been suggested that high Na+
stress is sensed either externally or internally and
somehow leads to an increase in cytosolic free
Ca 2+ concentration. SOS3 binds to Ca 2+ and activates the protein kinase SOS2. Activated SOS3SOS2 kinase complex is necessary for increased
148
INDIAN 1 BIOTECHNOL, APRIL 2002
expression of SOS I-a putative Na+/H+ antiporter
at plasma membrane (Shi et ai, 2000), and perhaps the other transporter genes under salt stress.
The SOS3/S0S2 pathway may also regulate the
activities of SOS 1 and other transporters at the
post-translational level. This gene expression and
transporter activity regulation brings about homeostasis of ions such as Na+ and K+ and consequently plant tolerance to Na+ stress (Zhu, 2000).
•
•
In Arabidopsis (apart from 6 CaMs), there are
other CaM-like genes including the TCH genes
that are induced in response to various mechanical, chemical and environmental stimuli (Braam
et ai, 1997). Further, a protein, TCH3 , has been
found to be 324 amino acids long and contains six
EF hand motifs (Sistrunk et ai, 1994).
A cDNA sequence encoding a 27 kDa CaM-like
protein, EFA27, has been isolated from ABAtreated rice seedlings (Frandsen et ai, 1996). It
contains a single EF hand motif and the expressio n of EFA 27 is induced in response to salt and
dehydration stress, and to ABA signal. In Arabidopsis, there are several EFA27 gene homologous
(Frandsen et ai, 1996), suggesting the existence of
similar proteins in phylogenetically distant species.
•
Another CaBP, AtCP 1, from Arabidopsis contains three EF hand motifs and binds Ca2 + (Jang et
ai, 1998). The AtCPI gene transcripts are also
highly inducible by NaCl treatment but not by
ABA treatment indicating the specificity of this
unique CaBP in responding to stress factors.
•
The transcripts of Arabidopsis CDPK genes,
AtCDPKI and AtCDPK2, are highly inducible by
drought and salinity but not by low temperature or
heat stress, suggesting the specificity of CDPK's
induction in response to different stress factors
(Urao et ai, 1994). A CDPK from Vigna radiata
is highly inducible by wounding, CaCh, IAA and
NaCI treatments (Botella et ai, 1996). AtCOPKl
and AtCDPKla are involved in regulating the expression of stress-inducible genes (Sheen, 1996).
The Ca2+ regulated phosphorylation has been
found to be necessary for stress-induced gene expression as phosphatases have been found to
counteract these responses. An immunohomologue of this Ca 2+/CaM dependent kinase has
been identified in Pisum sativum, which is involved in light and stress mediated signalling
(Pandey & Sopory, unpublished) .
•
•
•
•
•
AtCBL 1 mRNA expressed prefere ntially in stems
and roots and its level increases in response to
specific stress signals such as drought, cold, and
wounding, whereas AtCBL2 and AtCBL3 are
constitutively expressed (Kudla et ai, 1999).
Calcineurin B-like (CBL) -interacting protein kinases (CIPK 1 to 4) belong to the serine/threonine
class of kinases and show high homology to protein kinases, SNFI and AMPK from yeast and
mammalian systems, respectively. SOS2 (CBL4)
and other CBEs activate CIPKISIPK in a Ca 2+_
dependent manner, suggesting that CBLlSIPK
complex is likely to regulate Na+ and K+ homeostasis through phosphorylation. Chips/Spikes represent a novel family of CaBP kinases in plants.
The interaction of CBL proteins with protein kinases is very surprising since the CBL proteins
are known to activate a protein phosphatase in
animals . In addition to CaBP kinase, there is some
evidence for the role of a Ci+-regulated phosphatase in salt tolerance.
Ectopic expression of constitutively active yeast
calcineurin has been shown to confer salt tolerance in tobacco (Bressan et ai, 1998; Pardo et ai,
1998).
The authors have found the presence of homologue(s) of a Entamoeba histoiytica CaBP (EhCaBP) and EhCaBP stimulated kinase(s) in
plants, which may be involved in alternate calcium signalling pathway regulating development
and adaptation under unfavorable environmental
conditions (Pandey et ai, 2002).
PhospholipaseD (PLD) and its product phosphatidic acid have a role in stress signalling. PLD has
a putative catalytic domain and a C2 domain that
2
is involved in Ca +/phospholipid binding. It has
been found that the antisense expression of
Arabidopsis thaliana PLDa in transgenic Arabidopsis plants leads to retardation of senescence,
which is promoted by ethylene and ABA (Fan et
ai, 1997). The retardation of the senescence was
demonstrated by delayed leaf yellowing, lower
ion leakage, greater photosynthetic activity, and
higher content of chlorophyll and phospholipids
in the PLDa antisense plants than in those of the
wild type. Another PLO, AtPLDo, cloned from
the cDNA library prepared from dehydrated
Arabidopsis thaliana, has been found to accumulate in response to dehydration and high salt stress
(Katagiri et ai, 2001).
PANDEY el ai: ABIOTIC STRESS TOLERANCE IN PLANTS
Manipulation of CaBPs for Developing Abiotic
Stress Tolerance
Studies have focussed towards the over and under
expression of the genes encoding CaBPs employing
the contemporary tools and techniques of genetic engineering. Ttransgenic plants for CaBPs like calcineurin, Ca 2+/H+ anti porter, CDPK, glyoxalase I, and EhCaBP have been raised with improved stress tolerance
as follows:
(a) Calcineurin (CaN)
The altered expression of a calcium stresssignalling component, Ca 2+/CaM-dependent protein
phosphatase calcineurin (PP2B), has successfully
worked for raising salt tolerant plants in Arabidopsis.
CaN has been suggested to be an integral component
of a salt stress signal transduction pathway which has
been found to be regulating the influx and efflux of
2
Na + that ultimately affects salt tolerance.
Pardo et al (1998), have co-expressed a truncated
form of catalytic subunit and the regulatory subunit of
yeast CaN in transgenic tobacco plants to reconstitute
a constitutively activated phosphatase in vivo. Several
different transgenic lines expressing CaN, exhibited
substantial tolerance to NaCI. It has also been suggested that the CaN functions mainly in roots to
regulate the movement of ions from the apoplast to
the symplast, which controls the ion content of the
xylem sap, which in turn , is transported to the shoots
by the strength of the transpirational sink. Root
growth was found to be less perturbed than shoot
growth by NaCI in plants expressing CaN. Also, NaCI
stress survival of control shoots was enhanced substantially when grafted onto roots of plants expressing
CaN, further implicating a significant function of the
phosphatase in the preservation of root integrity during salt shock.
(b) Glyoxalase / (Gly /)
Besides phosphatases, there are enzymes that are
directly regulated by calcium and CaBP. One such
enzyme that authors have identified is glyoxalase I
which is involved in methylglyoxal detoxification and
maintaining glutathione homeostasis. The authors
have earlier purified Gly I (Deswal el al, 1993) and
shown that it is a calcium-binding enzyme and its activity is modulated by calcium/CaM (Deswal & Sopory, 1999).
To determine the functional significance of Gly I in
plants, authors cloned its gene (Veena et al, 1999) and
149
rai sed transgenic plants via Agrobacterium mediated
transformation. The transgenic plants over-expressing
Gly I showed tolerance to salt and heavy metals
(zinc). The wild type plants showed an early bleaching as compared to the Gly I overexpressors. The tolerance level of transgenic plants to zinc was dependent on the expression level of Gly I protein. The biochemical basis of this tolerance mechanism is not
clear. However, during stress, the levels of methyl
glyoxal that is a toxic compound increases and that
leads to decreased cell division (Szent-Gyorgi &
Egyud, 1966; Scaife, 1969) and causes cell death
(Kalapos et al, 1991). A system producing more glyoxalase could, therefore, convert methyl glyoxal into
non-toxic form, thus preventing the system from its
cytotoxic effects during stress condition. Besides detoxification of methylglyoxal, the glyoxalase system
could also play a role in providing tolerance under
stress by recycling glutathione that would be trapped
spontaneously by methyl glyoxal to form hemi thioacetal (Creighton et ai, 1988; Thornalley, 1990),
thereby maintaining the glutathione homeostasis.
(c) Ca 2+IJr Antiporter (CAX 1 or Calcium Exchanger 1)
2
The changes in the cytosolic Ca +, which are tightly
regulated in plants, have been implicated in convert2
ing numerous signals into adapted responses. Ca +
2
efflux from the cytosol modulates Ca + concentrations
in the cytosol, loads Ca2+ into intracellular compartments and supplies Ca2+ to organelles for supporting
biochemical functions. Vacuolar ion transporter,
CAX 1, is thought to be a key mediator of these processes.
CAX 1 transcript was found to be highly induced
in response to exogenous Ca2+. Transgenic tobacco
plants expressing CAX 1 displayed symptoms of Ca2+
deficiencies, including hypersensitivity to ion imbalances, such as increased magnesium and potassium
ions and to cold shock, but increasing the Ca2+ in the
media abrogated these sensitivities (Hirschi, 1999).
Tobacco plants expressing CAX I were found to
show an increased Ca2+ accumulation and altered activity of the CAX l. In a similar study, yeast vacuolar
Ca 2+'H+ anti porter (VCX 1) was expressed in Arabidopsis plants, which displayed increased sensitivity to
sodium and other ions, which could be suppressed by
addition of calcium to the media (Hirschi et al, 2001).
These results emphasize that regulated expression of
Ca 2+'H+ antiport activity is critical for normal growth
150
INDIAN J BIOTECHNOL, APRIL 2002
and adaptation to certain stresses and could be a valu abl e tool to experimentally dissect the role of Ca 2+
transport around the plant vacuole.
Further, expression of another calcium exchanger,
CAX 2 from Arabidopsis in tobacco, has been found
to regulate metal transport from the cytosol to the
vacuole. Tobacco plants expressing CAX 2 accumulated more Ca2+. Cd 2+ and Mn2+ and were more tolerant to elevated Mn 2+ levels. Expression of CAX 2 in
2
tobacco increased Cd + and Mn 2+ transport in isolated
root tonoplast vesicles (Hirschi et ai, 2000). These
results suggest that CAX 2 has a broad substrate range
and its modul ation may be an important component of
future strategies to improve plant ion tolerance and of
plant's potential use in phytoremediation.
(d) CDPK
As discussed earlier, the cytoplasmic Ca2+ levels in
plant cells increase rapidly in response to multiple
stress stimuli, including cold, salt and drought. Following this Ca 2+ influx, relay of signals is likely to be
mediated by combinations of protein phosphorylation/dephosphorylation cascades. It is presumed that
members of the CDPK family in plants perform the
majority of Ca2+ stimulated protein phosphorylation
predominantly . Despite the potential importance of
CDPKs, the physiological function of a specific
pathway has not been elucidated so far.
The selected members of CDPK family are involved in activation of stress/ABA-responsive promoter (Sheen, 1996). Transgenic rice plants have been
generated with altered levels of this protein. The extent of tolerance to cold and salt/drought stresses of
such plants correlated well with the level of
OsCDPK7 expression (Saijo et ai, 2000).
Overexpression of this protein in the transgenic
rice enhanced induction of so me-stress responsive
genes in response to salinity/drought, but not to cold.
Thus, the downstream pathways leading to cold and
salt/drought tolerance are different from each other. It
has also been suggested that at least two distinct
pathways commonly use a single CDPK, maintaining
the signalling specificity through unknown posttranslational regulation mechanisms. Thus, simple
manipulation of CDPK activity has great potential
with regard to stress tolerance.
(e) EhCaBP Kinase
Apart from CaM, there are several other CaBPs,
CBPs and kinases in plants that are performing vari-
ous functions. Authors have identified a different
class of protein kinase activity in plants e.g., a protein
kinase activity from Brassicajuncea that is stimulated
by a structural, but not funetional homologue of CaM
purified from E. histolytica [EhCaBP - a CaBP with
4 calcium binding sites (EF-hand motif) like CaM].
2
The Ca +/EhCaBP stimulated protein kin ase,
BjCCaBPK, gets stimulated over 6-fold by EhCaBP
(l0.5 nM) but not by CaM when used at equimolar
concentration. Moreover, this kinase also did not bind
CaM-Sepharose. There was no inhibition of the kinase
activity in presence of W-7 (a CaM antagonist), KN-62
(a specific calcium/CaM kinase inhibitor) and antiCaM antibodies. Even staurosporine (a protein kinase
C inhibitor) had no effect on the BjCCaBPK activity.
Furthermore, a CaM-kinase specific substrate, Syntide2, proved to be a poor substrate fo r the BjCCaBPK
compared with histone III-S. The phosphorylation of
histone III-S involved serine residues. Based on the
differences in the properties of various previously reported kinases, authors have suggested that BjCCaBPK
may be a novel protein kinase which does not fall in
the CDPKs, CaM kinases, and PKCs categories and
has an affinity towards a CaBP like EhCaBP (Deswal
et ai, 2000).
To elaborate the role of EhCaBP homologue(s) in
plants, transgenic approach has been followed. The
EhCaBP expressing plants were found to grow under
high salt concentration (200 mM NaCI), suggesting
that the expression of EhCaBP leads to tolerance under higher salinity level (Pandey et ai, 2002). Whether
the similar CaBPs are involved in stress tolerance
mechanism in tolerant varieties needs to be looked
into in future studies.
Conclusions
In living organisms, the cellular events following
stimulus perception and culminating in physio logical
respon se are referred to, broadly, as signal transduction. As sessile organisms, plants cope with abiotic
(cold, drought, etc.) and biotic (pathogen attack)
stress through cellular adaptations that are coordinated by an array of signal transduction pathways.
Plants, like other eukaryotes, use spatially and temporally complex patterns of calcium ion fluxes to communicate, intracellularly, information on the nature of
a stimulus. How cells interpret the information encoded in calcium signals remains unclear but is
known to involve CaBPs such as CaM. CaM, in response to calcium, binds to and regulates the activities
PANDEY et a/: ABIOTIC STRESS TOLERANCE IN PLANTS
of a wide assortment of proteins (enzy mes, ion channels and pumps, cytoskeletal components, etc.),
thereby orchestrating signalling pathways. Pl ants are
unique among eukaryotes in possessi ng numerous
CaM isoforms and evidence is emerging that CaMs
play important roles during stress-responsive signalling. The current focus of research involves identifying and characterizing the downstream protein targets
of CaMs and delineating the si gnalling pathways under their con trol. Though a plethora of other CaBPs
have been identified and characterized, transgenic
studies for only selective CaBPs have been attempted
till date. Certaining the use of specific mutants to ascertain the role of other CaBPs in enhancing stress
tolerance can also be of great advantage. Such work
will provide a basic knowledge of how plant cells
cope with stress and will lay the foundation for engineerin g specific pathways to improve stress tolerance
in crop plants.
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