There is some evidence that Ca^2 -regulated protein phosphorylation is involved in cold stress and in
host-pathogen interaction. In a freezing-tolerant cultivar of Medicago sativa, elevated cytosolic [Ca^2 ]cyt
levels in response to low temperature stimulated phosphorylation of several proteins including protein ki-
nases as revealed by a phosphoprotein profile [195]. Treatment of parsley suspension cultures with fun-
gal elicitors resulted in rapid and transient phosphorylation of specific proteins. These fungal elicitor–in-
duced changes in phosphorylation have been shown to be dependent on the presence of Ca^2 in the
medium [196]. Fungal elicitor–induced protein phosphorylation, phytoalexin production, and mRNAs for
phenylalanine ammonia lyase and 4-coumarate:CoA ligase were greatly reduced in Ca^2 -deprived cells
[196]. Furthermore, addition of Ca^2 to the cultures restored the inhibitory effects of Ca^2 deprival, sug-
gesting the participation of Ca^2 -dependent protein phosphorylation in fungal elicitor–induced re-
sponses.
The UV-B–regulated CHSgene expression in Arabidopsiscell cultures is inhibited in the presence
of pharmacological agents such as W7 (inhibitor of CaM or CDPKs), K252a and staurosporine (inhibitors
of protein kinases), and okadaic acid (inhibitor of protein phosphatases 1 and 2A). These results suggest
a role for kinases and phosphatases in UV-B–mediated CHSgene expression in Arabidopsiscell cultures
[88]. UV-B light also causes tremendous induction of anthocyanin pigment biosynthesis in rice [197]. It
would be interesting to test the involvement of a Ca^2 -dependent signal cascade in monocots in response
to UV-B radiation.
Calcium-dependent protein kinases and phosphatases and Ca^2 /CaM-dependent protein kinases
have been shown to be involved in the response to thigmotropism in soybean cells. In this process, ki-
nases and phosphatases play a crucial role in alterations of the actin and microtubule network that influ-
ence cell shape. These findings have been obtained using pharmacological inhibitors such as W7, calmi-
dazolium, okadaic acid, or inhibitors specific to CaM-dependent phosphatase 2B [198]. Mastoparan- and
hypoosmotic stress–induced cytosolic free Ca^2 levels are elicited through the IP 3 system in tobacco cell
cultures expressing apoaequorin [49,50]. The elevated [Ca^2 ]cytlevels activate three different protein ki-
nases (50, 75, and 80 kDa) whose activities are inhibited by neomycin and staurosporine treatments. In
both processes, elevated levels of free [Ca^2 ]cytare essential to establish the phosphorylation and de-
phosphorylation process [49,50]. These studies together suggest the involvement of Ca^2 -dependent pro-
tein kinases in a variety of stress-responsive mechanisms.
Dephosphorylation of specific proteins involved in the signal transduction cascade is found to be im-
portant in Ca^2 signaling. Wheat aleurone cells treated with gibberellic acid (GA) induce [Ca^2 ]cytlev-
els as well as activation of cellular hydrolases. Okadaic acid (OA), a protein phosphatase inhibitor, par-
tially inhibits ABA action and does not inhibit hypoxia-related stress responses. JA-dependent and
-independent would-induced gene expression in Arabidopsishas also shown involvement of reversible
phosphorylation events. Okadaic acid and staurosporine-sensitive protein phosphatase (type 2A) and ki-
nases positively regulate the genes expressed through the JA-dependent pathway. However, JA-indepen-
dent wound-induced gene expression relies on a phosphoprotein as this pathway is inhibited by stau-
rosporine and activated by okadaic acid [58]. The existence of Ca^2 /CaM-dependent protein phosphatase
(PP2B) and its involvement in stress signal transduction pathway have been reported in plants [116].
These findings indicate that the phosphorylation and dephosphorylation events are important in Ca^2 me-
diated signaling in plants.
B. Calmodulin and Calmodulin-Related Proteins
Calmodulin is found in all eukaryotic organisms and is a well-characterized Ca^2 receptor in both animal
and plant cells. In plants, CaM, CaM-related proteins, Ca^2 -dependent protein kinases, and other Ca^2 -
binding proteins are believed to sense the changes in [Ca^2 ]cyt. All these proteins are called Ca^2 sensors
or receptors. Calmodulin was first discovered in animals as an activator of cyclic nucleotide phosphodi-
esterase [199] and subsequently isolated and characterized from all eukaryotic organisms. It is, in fact, the
discovery of CaM in plants that led plant scientists to propose a messenger role for Ca^2 in plant cells.
Calmodulin has been isolated and characterized from many different plants [13,16,19,106,167]. Gene
structure and expression of CaMs from a number of plants have been analyzed [106].
Molecular cloning of CaM genes from plants indicates the presence of a small gene family that en-
codes different CaM isoforms. CaM is a low-molecular-weight protein of 148 amino acids that is highly
conserved between plants and animals. CaMs from all eukaryotes, except from budding yeast [200], have
706 REDDY AND REDDY