divergent and showed differences in 32 amino acids with the conserved group [219]. Surprisingly, these
divergent CaM isoforms are specifically induced by fungal elicitors or pathogen [220]. These results pro-
vided evidence for the differential regulation of CaM isoforms in plants. The Vigna radiata Camgenes
also differentially respond to a touch stimulus, Cam1being more responsive than Cam2[105]. In
hexaploid bread wheat, 10 distinct genes encode three CaM isoforms, some of which are differentially ex-
pressed in response to different stimuli [221,222].
- Calmodulin-Binding Proteins
Calmodulin itself has no enzymatic activity. It controls various cellular activities by modulating the ac-
tivity or function of a number of proteins. Hence, the role of CaM in a given cell or a tissue is determined
by the presence of its target proteins. Calmodulin is multifunctional because of its ability to interact with
and control the activity of a variety of target proteins (also called CaM-binding proteins). Therefore, char-
acterization of CaM-binding proteins (CBPs) is the prerequisite in dissecting Ca^2 /CaM-mediated sig-
naling pathways in plants. In animal systems, the activity of over 30 enzymes has been shown to be reg-
ulated by CaM in a Ca^2 -dependent manner [168,210,223,224]. These include several protein kinases, a
protein phosphatase (also known as calcineurin), a plasma membrane Ca^2 -ATPase, adenyl cyclases,
cyclic 3 ,5 -nucleotide phosphodiesterase, motor proteins, inositol triphosphate kinase, transcriptional
factors, nitric oxide synthase, and some structural proteins. Identification and characterization of CaM tar-
get proteins in animal cells have helped in elucidating the mechanisms by which Ca^2 /CaM regulate var-
ious biochemical and molecular events leading to a physiological response. The amino acid sequence of
the CaM-binding domain in different CaM target proteins is not conserved [208]. However, CaM-bind-
ing motifs from different CaM-binding proteins form characteristic basic amphipathic -helices
[209,223]. The amino acids in the CaM-binding domain, when arranged in a helical wheel, form an am-
phipathic helix with several positive residues on one side and a number of hydrophobic residues on the
other side. In most cases, binding of CaM to its target proteins requires Ca^2 . However, CaM binds to
some proteins (e.g., neuromodulin, myosins) in the absence of Ca^2 [225]. Furthermore, a CaM with no
Ca^2 -binding activity has been shown to rescue a mutation in the only Camgene in budding yeast [226],
suggesting that CaM may perform some functions in the absence of Ca^2 .
Gel overlay studies with plant proteins indicate the presence of a number of CaM-binding proteins
in plants [227,228]. Some enzymes and proteins that are activated by CaM have been identified in
plants. These include NAD kinase [29], Ca^2 -ATPase [229,230], nuclear NTPases [231], glutamate de-
carboxylase [232,233], transporter-like proteins [234], Ca^2 /CaM kinases [235], kinesin-like protein
[218,236–242], elongation factor-1[111], transcription factor [243], glyoxalase I [244,245], and heat
shock–inducible proteins TCB48 and TCB60 [246,247]. In fact, CaM was first discovered in plants as
an activator of NAD kinase [248,249]. New approaches to isolating CaM-binding proteins by screen-
ing expression libraries with labeled CaM [^35 S-labeled, biotinylated, or horseradish peroxidase (HRP)-
conjugated CaM] as probes [250–253] have greatly aided in isolating and characterizing cDNAs en-
coding CaM-binding proteins. Several cDNAs have been isolated with this approach [106,254–256]
and one of the isolated clones was found to have significant similarity to E. coliglutamate decarboxy-
lase (GAD) [256]. Using a gel overlay assay with SCaM4 and -5, Lee et al [250] have shown that the
two CaM isoforms compete for several CaM-binding proteins of total protein extracts prepared from
various soybean tissues. However, the molecular identity and function of these CBPs from soybean are
not known [250].
The expression of some of the CaM-binding proteins is regulated by heat and wind [246,254–257].
The plant GAD is unique in having a CaM-binding domain at its C-terminus [256] (Figure 3). Members
of this protein from bacteria do not contain a CaM-binding domain (CBD). Although the catalytic core
that catalyzes the conversion of glutamate in to -aminobutyric acid (GABA) and CO 2 is conserved
across bacteria, plants, and animals, the Ca^2 /CaM regulation of GAD appears to be unique to plants
[233]. At least three forms of GADs were identified in mammals [259], and the product GABA serves as
an inhibitory neurotransmitter in animal systems. GAD has now been cloned and characterized from a
number of plant species including petunia [256], Arabidopsis[260], rice [261], soybean [262], and as-
paragus [80]. Although the CBD is not conserved, all plant GADs isolated so far possess CBD and are
regulated by Ca^2 /CaM [233]. Particularly interesting is the presence of two GAD isoforms (GAD1 and
GAD2) in Arabidopsis. The CBDs of these two isoforms are different, raising the possibility of functional
diversity and regulation by Ca^2 /CaM [260].
CALCIUM IN STRESS SIGNAL TRANSDUCTION 709