documented that distinct plant CaM isoforms differ in their ability to bind and
activate known CaM-regulated enzymes in vitro (Reddy et al. 1999; Lee et al. 2000;
Choi et al. 2002). The differential CaM/CML and target interactions and
regulations have also been observed under in vivo conditions during the regulation
of AtNHX, a vacuolar Na
+
/H
+
exchanger from Arabidopsis (Yamaguchi et al.
2005). In addition, various CaMs and CaM-like proteins might also have differen-
tial affinities to Ca
2+
, due to the variation in the number of EF-hand motifs, or
mutations in the sequence of EF-hand motifs, the mos t conservative portion in
calmodulins and calmodulin-like proteins. Although most of the EF-hand motifs in
calmodulin-like proteins retain the canonical Ca
2+
-binding coordination compris-
ing of a loop structure with 12 reserved residues between the E and F helices,
several CMLs carry a loop structure slightly different from those of typical
calmodulins with an E to D substitution in position 12 of the Ca
2+
binding loop
(McCormack and Braam 2003). Research on the interaction of EF hands in
parvalbumin and d ivalent cations revealed that E to D substitution in this position
might lower its binding affinity to Ca
2+
and increase its affinity to Mg
2+
(Cates et al.
2002). Hence the E to D substitution in these CML might affect its interaction with
Ca
2+
and render the abili ty for these CMLs to bind to other divalent cations such as
Mg
2+
although it remains to be confirmed. Furthermore, variation in CaMs/CMLs
could even be correlated with their subcellular localization. Generally, CaM is a
cytosolic protein, but CaM and CML have also been found in the nucleus (van der
Luit et al. 1999), the peroxisome (Yang and Poovaiah 2002b), vacuole (Yamaguchi
et al. 2005), and even in the extrace llular matrix (Ma et al. 1999). The necessity of
multiple subcellular locales is understandable because the CaM-target proteins are
present in different subcellular locations. However, the manner in which CaM
targets these organelles is not clear. In at least one case, posttranslational modifica-
tion correlated with structural variations in CaM or CMLs could serve to control its
subcellular localization. CaM53 from petunia (PhCaM3) is a calmodulin-like
protein with an extra extension in its C-terminal region which is rich in basic
amino aci d and also carries a CTIL CaaX-box motif. It was shown to associate
with the plasma membrane when prenylated and target to the nucleus if prenylation
was inhibited (Rodriguez-Concepcion et al. 1999).
Why multiple CaM and CML genes encode for the same or similar proteins in
plants are not fully understood. Although the functional redundancy between
different members cannot be excluded, accumulated evidence suggests that each
CaM genes could have unique functional application and significance, revealing a
much more complicated signaling system required to support a sessile life style in
plants in which environmental changes bring forth endless challenges. This is
further supported by the differential expression patterns of CaMs/CMLs. In all
plants examined, CaM genes, even genes encoding the same isoform, are differen-
tially expressed in response to numerous external stimuli such as touch, heat shock,
cold, li ght, and pathogens, and to hormones such as auxin (Reddy 2001a; Yang and
Poovaiah 2003; Bouche et al. 2005). CaMs are also differentially expressed in
different stages, as well as in different tissue and cell types (Y ang et al. 1998;
Duval et al. 2002; Yang and Poovaiah 2003). The complexities in the interpretation
188 L. Du et al.