together referred to as regulon [33]. Interestingly, genes in this family possesses a common cis-regulatory
DNA sequence motif, CRT/DRE, in their promoter region. Further, the trans-acting protein factors
(CBF1, DREB1A) that bind to the CRT/DREmotif and up-regulate their gene expression have also been
identified [63,321,347]. Because the expression of a battery of stress-inducible corgenes is regulated
through a common ciselement (CRT/DRE) by a single transcriptional factor (CBF1 or DREB1A),
Thomashow and Yamaguchi-Shinozaki and their coworkers have used a transgenic approach to overex-
press transcriptional factors (CBF1 or DREB1A) from either constitutive promoter, CaMV35S-CBF1
[321] and CaMV35S-DREB1A[344], or stress-inducible promoter (rd29A-DREB1A) [344]. Although
growth retardation has been a common phenomenon observed in the plants expressing transcriptional fac-
tors from constitutive promoters, the corgene expression is hyperinducible in response to cold, drought,
and salt stresses. However, the plants expressing transcriptional factor (DREB1A) from the rd29Apro-
moter did not show negative effects on plant growth but the corgene expression is super up-regulated
only when stress conditions prevail. Both kinds of transgenics showed significant resistance to cold,
drought, and salt stress responses [321,344].
To dissect the role of specific Ca^2 -activated CaM isoforms and their target enzymes and/or proteins
in plant defense mechanisms [29,220,232] or in heavy metal tolerance [258], transgenic tobacco plants
expressing genes encoding specific CaM isoforms or mutated forms of CaM or CaM-binding proteins
were generated. Although preliminary experiments with a pharmacological approach indicated a role for
CaM isoforms in plant defense responses, the specificity of CaM antagonists is questionable in unequiv-
ocally elucidating Ca^2 -mediated signal pathways. These pharmacological agents can also influence
other cellular activities not related to Ca^2 /CaM signaling [16,19]. Therefore, the sense transgenic plants
proved to be valuable tools in studying the function of CaM isoforms and CaM-target proteins in planta.
Transgenic plants (VU-3) harboring a mutated form of CaM (amino acid, K115R in the CaM se-
quence abolished posttranslational modification of trimethylation at 115R) showed increased levels of re-
sistance to a variety of stimuli such as treatment with cellulase, bacteria-derived elicitor harpin, mechan-
ical stress, and osmotic stress [29]. Transgenic VU-3 cell lines treated with cellulase or mechanical stress
produced severalfold higher levels of AOSs compared with control cell lines. Diphenyleneiodonium, an
inhibitor of AOSs production, abolishes AOS synthesis in VU-3 cell lines. Production of AOSs has been
shown to initiate a battery of defense responses against invading pathogens [66,68,69,146]. The enhanced
resistance activity of VU-3 plants is primarily attributed to their ability to show hyperactivation of NAD
kinase (a Ca^2 /CaM-regulated enzyme) [29]. The only molecular difference between VU-3 and control
NC lines is a mutation at a single amino acid residue (K115R) in the CaM protein sequence. This modi-
fication showed hyperactivation of its target NAD kinase. This elegant study provided evidence that NAD
kinase may be one of the targets of CaM in plant defense responses. Certain CaM isoforms contain a 34-
amino-acid Caax box motif (prenylation domain) at their C-termini with CTIL (PhCaM53) [203] or CVIL
(OsCaM63) [348] amino acid residues. Although most CaM isoforms are soluble proteins (based on pro-
tein sequence) [349], transient expression of GFP fused with the full-length CaM53 or mutated CaM53
(without the CaaX box motif) in tobacco and petunia protoplasts showed functional significance of the
prenylation domain (CaaX-box) [203,350]. The cells harboring GFP-CaM53 with an intact prenylation
domain localized to plasma membrane, whereas the GFP mutant CaM53 (without a CaaX box) localized
to the nucleus. Although CaM53 does not contain NLS motifs, its localization to nucleus suggested a dual
role played by the Caax box in localizing CaM53. Further, the pattern of distribution of CaM53 may de-
pend on the prenylation status of the cell or the requirement and/or availability of other NLS protein to
interact and move into the nucleus to exert its different cellular activities [203]. Ectopically expressed
prenylated CaM53 in tobacco showed stunted growth and a necrotic phenotype. However, ectopic ex-
pression of neither the nonprenylated form nor the Caax box motif alone did not result in such altered
morphogenic alterations, indicating that high levels CaM could perturb specific cellular machinery in-
volved in growth and development [203]. Together, these studies suggest functional diversity of CaM iso-
forms in coordinating Ca^2 signaling pathways in higher plants.
Using a similar transgenic approach, another Ca^2 /CaM target enzyme, glutamate decarboxylase
(GAD), was expressed in tobacco. The tobacco transgenic plants expressing GAD or GAD devoid of
CBD (27-amino-acid region in the C-terminus) (GAD-CBD) (Figure 3) revealed different phenotypes and
levels of GAD’s enzymatic product and substrate, GABA and Glu, respectively. Transgenic tobacco
plants expressing GAD showed a normal wild-type phenotype with moderately increased GABA and re-
duced Glu. However, the transgenic plants bearing GAD-CBD exhibit severe morphological disorders in
CALCIUM IN STRESS SIGNAL TRANSDUCTION 719