role of divalent calcium (Ca^2 ) cations as one of the key signaling molecules has been known for a long
time in animal systems. In plants also, Ca^2 has been implicated for decades in regulating various phys-
iological processes during growth and development. However, research during the last two decades
strongly indicates that Ca^2 plays an important messenger role in transducing a variety of hormonal and
environmental signals. Several comprehensive reviews on various aspects of Ca^2 messenger system in
plants have appeared [3–7,12–19]. In this chapter, we will mainly focus on the role of Ca^2 as a messen-
ger in stress signal transduction. A number of criteria have been used to consider a chemical or an ion as
a messenger in signal transduction in living cells. These include (1) quantitative changes in the concen-
tration of putative messenger in response to a signal prior to a response, (2) presence of receptors to sense
the changes in the level of messenger, (3) induction of a signal-induced response by changing the levels
of putative messenger in the absence of a primary signal, and (4) blocking signal-induced responses in the
presence of a primary signal by blocking the changes in the level of putative messenger. Evidence ob-
tained in recent years indicates that Ca^2 satisfies all these criteria as a messenger molecule in transduc-
ing various stress signals.
II. STRESS-INDUCED CHANGES IN CYTOSOLIC CALCIUM LEVELS
Despite initial technical problems in measuring cytoplasmic calcium ([Ca^2 ]cyt) in plant cells, tremen-
dous progress has been made in this area [3,13,19–23]. Different methods using Ca^2 -binding fluorescent
dyes (fura-2, indo-1); Ca^2 -selective electrodes; aequorin, a Ca^2 -binding photoprotein; and green fluo-
rescent protein–based cameleon are employed to measure signal-induced changes in [Ca^2 ]cyt
[20,24–29]. Transgenic plants expressing targeted aequorin to different organelles combined with the use
of different pharmacological agents that block or release Ca^2 have greatly facilitated measurement of
the Ca^2 concentration in different plant cell compartments in response to different stress stimuli [30,31].
Calcium measurement studies indicate that the concentration of Ca^2 in the cytoplasm of plant cells, as
in animal cells, is maintained low in the nanomolar range (100 to 200 nM) [3]. However, the Ca^2 con-
centration in the cell wall and in organelles is in the millimolar range [4,7,13,19].
TransgenicArabidopsisplants expressing cameleon have been used to measure Ca^2 transients in
guard cells in response to abscisic acid (ABA) and plasma membrane polarization and the Ca^2 concen-
tration [20]. Cameleon, a chimeric protein, consists of an enhanced cyan fluorescent protein (CFP), a
calmodulin (CaM), a CaM-binding domain (CBD) from myosin light chain kinase, and an enhanced yel-
low fluorescent protein (YFP). Increase in Ca^2 level activates CaM, which in turn interacts with CBD
and brings two GFPs closer by intramolecular arrangement and allows fluorescence resonance energy
transfer (FRET) from CFP to YFP. Hence, in the presence of elevated Ca^2 levels, excitation of CFP per-
mits emission by YFP at 535 nm. Using cameleon, Allen et al. [20] have measured [Ca^2 ]cytspikes in
guard cells of Arabidopsisin response to ABA and high levels of Ca^2 . Despite the existence of a large
electrochemical gradient for Ca^2 entry into the cytoplasm, plant cells maintain their [Ca^2 ]cytconcen-
tration between 0.1 to 1.5 M [20]. Maintenance of low [Ca^2 ]cytlevels requires active pumping of Ca^2
to the apoplast or organelles. Using different approaches, a number of signals including stress signals
have been shown to elevate [Ca^2 ]cyt. Table 1 shows various signals that have been shown to change the
level of [Ca^2 ]cyt. Here, we describe only stress-induced changes in [Ca^2 ]cytlevels. Elevation of
[Ca^2 ]cytin response to other signals has been discussed elsewhere [3,4,7,19,24].
A. Abiotic Stress Signals
There are several reports indicating that cold and salt stress affects Ca^2 homeostasis in plants
[60,61,71]. Increasing evidence obtained during the last several years suggests that abiotic stress sig-
nals rapidly elevate the level of [Ca^2 ]cyt. Knight et al. [26] for the first time used transgenic plants ex-
pressing apoaequorin to reconstitute aequorin and measure changes in [Ca^2 ]cytin response to various
signals in tobacco seedlings. These studies have shown that signals such as cold, touch, and wind that
are known to influence markedly plant growth and development [10,72], elevate the levels of [Ca^2 ]cyt
[26,37,55]. In vivo imaging of cold-induced changes in [Ca^2 ]cytindicated that cotyledons and roots of
a seedling are highly responsive whereas hypocotyls are relatively insensitive to cold shock [28]. Fur-
thermore, it has been shown that cold shock causes wavelike Ca^2 increases in the cells of cotyledons.
698 REDDY AND REDDY