idence for the elevation of Ca^2 levels in epidermal cells of cowpea plant infected with cowpea rust
fungus,Uromyces vignae. These authors observed the elevation of [Ca^2 ]cytby use of the green-1-dex-
tran Ca^2 reporter dye at the site of infection before fungus penetrates into the cell wall and the onset
of HR [146]. During the interaction of ArabidopsisandPseudomonas syringae, the resistance gene
product, RPM1, functions immediately and elevates the Ca^2 levels as monitored by the aequorin trans-
genic method [147,148]. Similar results were obtained for the C. fulvum–tomato interaction [149]. Iden-
tification and characterization of the Arabidopsisand rice gp91phoxhomologues,RbohA(for respiratory
burst oxidase homologue A), provided evidence for the downstream target for the elevated levels of
Ca^2 in oxidative burst [150]. The RbohAshows high similarity to human gp91phox(phoxfor phago-
cyte oxidase). It has been found that the human gp91phoxis a plasma membrane–bound neutrophil
phagocyte oxidase and is involved in the generation of superoxide radicles via its NADPH oxidase ac-
tivity. In addition to the gp91phoxregion, the plant gp91phox(RbohA) also contains two EF hand motifs
that are not present in human gp91phox, suggesting that Ca^2 modulates the formation of superoxide
radicals through gp91phoxEF hands during the oxidative burst in plants [150].
Systemin has been shown to be an important mediator in wound-induced activation of defense genes
in tomato [151,152]. Addition of systemin to the cell cultures of tomato causes rapid alkalinization of the
medium [153]. However, the Ca^2 channel inhibitor lanthanum and protein kinase inhibitors K252a and
staurosporine inhibit the systemin-induced signal process, suggesting the involvement of a Ca^2 -depen-
dent protein kinase in systemin-induced signal transduction in tomato [153]. Tomato plants expressing
prosystemin showed higher levels of CaM transcripts, indicating the role of Ca^2 -dependent CaM in the
defense response [151]. Further, Flego et al. [154] showed a correlation between increased Ca^2 concen-
tration in plants and increased resistance to the bacterial pathogen Erwinia carotovora. Using a dextran-
linked Ca^2 indicator dye, elevated Ca^2 spikes were measured in developing nodules of alfalfa induced
byRhizobium melilotinodulation factors, suggesting the participation of Ca^2 in nodule formation [70].
Accumulating evidence indicates the involvement of a Ca^2 signal in plant defense responses such as
phytoalexin biosynthesis, induction of defense-related genes, and hypersensitive cell death. However, the
exact mechanisms by which Ca^2 regulates these processes are poorly understood [18,26,27,66,
69,144,155,164]. Various components of the Ca^2 -mediated signal transduction pathways are discussed
later in this chapter. Influx of Ca^2 ions from extracellular or intracellular Ca^2 stores seems to contribute
to signal-induced changes in [Ca^2 ]cyt. Based on the type of signal or cell type, both processes could be
involved in raising [Ca^2 ]cyt. The elevated Ca^2 interacts with other proteins and signal-transducing com-
ponents located downstream of the signal cascades. The presence of the several of the downstream Ca^2 -
based signal components has been reported in plants.
III. CALCIUM-SENSING MECHANISMS
Changes in free Ca^2 concentration in the cytoplasm are believed to regulate various cellular processes
at the biochemical and molecular level, eventually leading to a physiological response. A transient Ca^2
increase in the cytoplasm in response to abiotic and biotic stress factors is sensed by an array of Ca^2 -
binding proteins. Once Ca^2 sensors decode the stress signal, [Ca^2 ]cytlevels are restored to the resting
level by Ca^2 efflux into cellular organelles such as vacuoles, ER, and mitochondria or the cell exterior.
Decoding of the Ca^2 signal to the metabolic machinery is accomplished through intracellular Ca^2 re-
ceptors or Ca^2 -binding proteins. All Ca^2 -binding proteins, except annexins, contain a 29-residue he-
lix-loop-helix structure called an EF hand that binds to Ca^2 with high affinity [18,165,166]. However,
different Ca^2 -binding proteins differ in the number of EF hand motifs and their affinity for Ca^2 with
dissociation constants (Kds) ranging from 10^5 to 10^9 M. Binding of Ca^2 to the receptor results in a
conformational change in the receptor that enables it to interact with other proteins and modulate their
function and/or activity. A number (over 150) of Ca^2 -binding proteins have been identified and charac-
terized in animals [16,166]. Of these, only a few are present in all eukaryotic cells and are believed to be
involved in mediating Ca^2 action, whereas the majority of them (e.g., troponin C and parvalbumin) are
found in specific tissues and play restricted roles. Studies in plants have identified several Ca^2 -binding
proteins including CaM, CaM-related proteins [19,106,167], protein kinases, phosphatases, phospholi-
pases, proteinases, and other proteins [3,6,13,16,18,159]. In the following sections we discuss Ca^2 -bind-
ing proteins involved in stress signal transduction pathways in plants.
CALCIUM IN STRESS SIGNAL TRANSDUCTION 703