EGTA [195]. Furthermore, a CaM inhibitor (W7) but not its inactive analogue (W5) inhibited the devel-
opment of cold acclimation–induced freezing tolerance. These results suggest that an increase in cytoso-
lic [Ca^2 ]cytis necessary for developing cold-induced freezing tolerance and that CaM is involved in
freezing tolerance. Accumulation of cold-induced mRNAs in alfalfa was partially blocked by lanthanum,
a Ca^2 channel blocker, and a CaM inhibitor (W7) completely blocked the expression of cold-regulated
genes [195]. Lanthanum and W7 affect low temperature–induced changes in protein phosphorylation.
However, the effects of these antagonists on phosphorylation are more severe and are not restricted to
cold-induced changes.
As described earlier, there are several reports implicating Ca^2 in regulating the expression of spe-
cific genes, including its own receptors, in plant cells. Calcium-regulated protein phosphorylation is likely
to be involved in Ca^2 -regulated gene expression (see Sec. V) [106,190]. It is not yet known to what ex-
tent the changes in the [Ca^2 ]cytlevels are reflected in changes in free Ca^2 concentration in the nucleus.
Studies show that there is a Ca^2 gradient between the nucleus and cytoplasm indicating the presence of
regulatory mechanisms that control Ca^2 movement into and out of nucleus [313–315]. ATP stimulates
Ca^2 uptake into nuclei and studies implicate CaM involvement in this uptake process [314]. Currently,
little is known about the participation of nuclear Ca^2 stores in increasing cytosolic Ca^2 and vice versa.
V. APPROACHES TO DECIPHER CALCIUM SIGNALING PATHWAYS
IN STRESS SIGNAL TRANSDUCTIONThe availability of well-characterized stress-induced genes and reporter genes such as GUS and GFP is
facilitating plant biologists in the search for the intermediate components and mechanisms involved in
stress signal transduction. Furthermore, availability of mutant plants that show increased sensitivity or re-
sistance to stresses together with identification of these mutant genes is further advancing our quest to un-
derstand Ca^2 signaling pathways. In this section we summarize three main approaches (cell biological,
genetic, and transgenics) that have been used to elucidate stress signal transduction.
A. Cell Biological Approaches
Using isolated protoplast or cell culture systems, plant biologists have obtained important insights into
the role of Ca^2 in stress signal transduction. To study the effect of Ca^2 -activated CDPKs and phos-
phatases on the expression pattern of stress inducible genes, Sheen [190] used a cell biological approach.
In this elegant experiment, maize protoplasts were utilized to monitor the transient expression of reporter
and effector genes driven by stress-inducible promoter in the presence of elevated levels of CDPKs or
phosphatases. Constitutive promoter (CaMV 35S) was used to direct the expression of effector genes
(CDPKs and phosphatases), whereas the barley ABA (osmotic) stress–responsive gene promoter HVA1
was used to express reporter genes (either GFPorLUC). A reporter gene (GUSorGFP) driven by the
ubiquitin (UBI) promoter sequence, which is not inducible by any stress, was used as a control.
Initially, enhanced expression of GFP was observed in maize protoplasts harboring the HVA1-GFP
reporter construct under cold, salt, dark, and ABA stresses. The same extent of GFP expression was also
observed without stress treatments but in the presence of both Ca^2 and Ca^2 ionophore (Ca^2 -iono-
mycin or Ca^2 -A23187). However, in both experiments GFP expression was not observed in the maize
protoplasts harboring the UBI-GFPreporter construct, indicating that Ca^2 is involved in stress-induced
gene expression [190]. Because Ca^2 is able to induce the expression of a stress-inducible gene, Sheen
has tested the effect of CDPKs on the expression of the HVA1-LUCgene. The maize protoplasts were co-
transformed with reporter (HVA1-LUC) along with the truncated versions (containing the kinase domain)
of one of the eight CDPK (35S-PK) constructs and the effect of eight effector protein kinases was moni-
tored by quantifying luciferase activity. These results revealed that of the eight ArabidopsisCDPKs tested
(ATCDPK1, ATCDPK1a, AK1/ATCDPK, ATCDPK2, ATPKa, ATPKb, ASK1, and ASK2), ATCDPK1
and ATCDPK1a activated the expression of the LUCgene driven by HVA1promoter. Furthermore, co-
transformation of HVA1-LUCalong with the constitutively expressed ATCDPK1a and PP2C, a protein
phosphatase, or with the combination of CDPK and PP2C null mutants into maize protoplasts showed de-
creased or abolished LUC activity, indicating the involvement of phosphorylation and dephosphorylation
events in the signal transduction leading to the activation of HVA1-LUC[190].
714 REDDY AND REDDY