CaM-binding assa ys with recombinant SODs or SODs purified from other sources.
Recently, Euphorbia latex peroxidase was shown to have two distinct amino acid
sequences recognized as CaM-binding sites. CaM-binding assays and the determi-
nation of steady-state parameters showed unequivocally that Euphorbia peroxidase
is a CaM-binding protein activated by Ca
2+
/CaM. These findings suggest that
peroxidase might be another node in the Ca
2+
/H
2
O
2
-mediated plant defense system,
having both positive and negative effects in regulat ing H
2
O
2
homeostasis (Mura
et al. 2005). Importantly, the expression of genes encoding calmodulin-binding
transcription factors is stimulated by H
2
O
2
, such as AtBTs and AtSR, which has
been discussed in other sections, suggesting the broader impact of calcium/calmod-
ulin on oxidative signaling.
6.2 Aminobutyric Acid and Glutamate Decarboxylase
Aminobutyric acid (GABA) is a four-carbon nonprotein amino acid. In human and
mammals, GABA acts as a major inhibitory neurotransmitter (Kazemi and Hoop
1991). The presence of GABA in plants was first described in potato tubers in 1949
(Steward et al. 1949). The production of GABA has been associa ted with stresses
such as anaerobic conditions (Streeter and Thompson 1972), low or high tempera-
tures and darkness, and mechanical manipulation (Wallace et al. 1984; Bouche
et al. 2004). The majority of scientific studies carried out with the aim of
deciphering the functional roles of GABA have concentrated on stress-response
and its signaling roles (Bouche and Fromm 2004), as well as its roles as a metabolite
(Bouche and Fromm 2004; Fait et al. 2008).
GABA is synthesized from glutamat e and this reaction is catalyzed by GAD
(Bouche and Fromm 2004). In plants, petunia GAD was first characterized as a
calmodulin-binding protein. GAD activity is essentially upregulated by calcium/
calmodulin in physiologica l pH (Baum et al. 1993, 1996; Snedden et al. 1996).
Later the GAD was cloned and characterized in many other plant species, including
Arabidopsis, tomato, Vicia faba, soybean, tobacco, rice, and asparagus (Bouche and
Fromm 2004). Almost all of plant GADs have a plant-specific calmodulin-binding
domain in the C-terminus, while this feature is not present in any mammalian or E.
coli GADs, suggesting that the regulation of GAD activity by calcium–calmodulin
is plant specific.
The physiological relevance of GAD activation by calmodulin was first
addressed in tobacco by using transgenic plants ecto pically expressing either
GAD or a truncated GAD that could not bind CaM. Removal of the C-terminal
calmodulin-binding domain of GAD resulted in constitutive GAD activity (i.e.,
calcium independent), abnormal steady-state levels of glutamate (low) and GABA
(high), and aberrant plant development. Therefore, calcium/calmodulin is critical in
controlling the activity of GAD in vivo and this regulation is necessary for normal
plant development. In Arabidopsis, disrupting the root-specific GAD1 gene using
T-DNA insertion mutagenesis revealed that it plays a major role in GABA synthesis
210 L. Du et al.