kinase II (CaM K II) catalytic domain. The region that joins the kinase domain to the CaM-like region
corresponds to the autoinhibitory/CaM-binding region of CaM K II and prevents kinase activity in the ab-
sence of Ca^2 [172]. The cDNAs that encode CDPKs have also been isolated from other systems
[178,179]. The activity of an ArabidopsisCDPK that is expressed in Escherichia coliis stimulated by
Ca^2 [178,179].
Immunological and cloning studies as well as Southern analyses of soybean and Arabidopsisgenomic
DNAs suggest that there are several isoforms of CDPK in plants [18]. Using a polymerase chain reaction
(PCR) strategy, Urao et al. [120] cloned two CDPKcDNA sequences, AtCDPK1andAtCDPK2. The tran-
scripts of these two genes 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. The E. coli
expressed AtCDPK2 protein phosphorylates casein and myelin basic protein in a Ca^2 -dependent man-
ner. Accumulating evidence suggests that there are more than 40 CDPKs in the Arabidopsisgenome [180]
and they are classified into seven groups on the basis of their sequence domain organization (myristoyla-
tion, PEST, and the number of EF hand motifs) [181]. Furthermore, these CDPKs differ in their affinity
for Ca^2 . For example, AtCDPK1 differs from AtCDPK2 in its Ca^2 -stimulated activity, although both of
them possess four EF hand motifs [181]. Studies indicate that, besides Ca^2 , lipids are involved in the reg-
ulation of CDPK activity [178,182,183]. A carrot calmodulin-like domain protein kinase, DcCPK1, re-
sembles animal protein kinase C (PKC) in its activation by Ca^2 and certain phospholipids, suggesting that
lipids regulate the activity of some CDPKs and perform specific biological functions in plants [184]. The
molecular weight of purified CDPKs from different plant systems ranges from 35,000 to 90,000 [18]. The
wide range in the size and differences in their substrate specificity suggest that there could be multiple iso-
forms and functions. It is also possible that some of the small enzymes are derived from larger ones by pro-
teolytic cleavages, which has been shown to be the case in oat [182]. A mutation in the CDPKgene did not
reveal a phenotype, suggesting functional complementation among CDPKs [181].
In vitro and in vivo protein phosphorylation studies have demonstrated Ca^2 -regulated protein phos-
phorylation in a number of plant systems. Arabidopsisand soybean CDPKs phosphorylate -TIP, a tono-
plast intrinsic protein [185], and nodulin-26, respectively, that is involved in the formation of nodule [186].
Another CDPK has been shown to phosphorylate a guard cell vacuolar chloride channel [187]. It has been
shown that alfalfa and Arabidopsisseedlings treated with W7 (a potent inhibitor of CaM and CDPKs) were
unable to acclimatize and tolerate cold and freezing temperatures. These results suggest the involvement
of Ca^2 - or CaM-dependent protein phosphorylation events in these process [188,189]. Using a PCR strat-
egy, Botella et al. [121] isolated a cDNA clone encoding CDPK from Vigna radiata. The corresponding
messenger RNAs (mRNAs) are highly inducible by wounding, CaCl 2 , indoleacetic acid (IAA), and NaCl
treatments [121]. These results provide evidence for the phosphorylation events in Ca^2 -mediated stress
signal transduction cascades in plants. In an elegant experimental system, Sheen [190] has shown that Ara-
bidopsisAtCDPK1 and AtCDPK1a are involved in regulating the expression of stress-inducible genes.
Furthermore, phosphatases counteract these responses, suggesting that involvement of Ca^2 -regulated
phosphorylation is necessary for stress-induced gene expression (also see Sec. V).
A cDNA that shows significant similarity to mammalian CaM K IIhas been isolated from plants by
screening an expression library with radiolabeled CaM [173,191] (Figure 1). However, the biochemical
properties of this plant CaM K II homologue are not known. A Ca^2 /CaM-dependent protein kinase
(CCaMK) was cloned and characterized from lily [174] and tobacco [192]. A comparison of the sequence
analysis revealed the presence of an N-terminal catalytic domain, a centrally located CaM-binding do-
main, and a C-terminus visinin-like domain containing three conserved EF hands (Figure 1). Biochemi-
cal studies of CCaMK established that Ca^2 /CaM stimulates CCaMK activity and in the absence of CaM,
Ca^2 promotes autophosphorylation of CCaMK. The phosphorylated form of CCaMK possesses more ki-
nase activity than the nonphosphorylated form [193]. These authors suggested involvement of CCaMK
in male gametophyte development. The same research group showed differential regulation of tobacco
CCaMKs by CaM isoforms [192]. These studies indicate the presence of CaM-regulated protein kinases
in plants, although how widely these kinases are distributed and their exact role are not clear.
Fungal elicitor–induced cytosolic Ca^2 has been implicated in changes in the phosphorylation status
of proteins in tomato suspension cultures [194]. The cell nuclei in cowpea plants infected with cowpea
rust fungus are shown to migrate to the fungal penetration site. Calcium chelators as well as protein ki-
nase inhibitors inhibit such nuclear movement, suggesting the involvement of a Ca^2 -dependent phos-
phorylation cascade in nuclear migration [146].
CALCIUM IN STRESS SIGNAL TRANSDUCTION 705