The oxidative burst in plants as a result of compatible plant-pathogen interaction leads to a hyper-
sensitive response and cell death at the infection site in order to build up resistance in the neighboring
cells against the invading microorganisms. Lamb and his colleagues [66] have provided evidence for the
involvement of [Ca^2 ]cytin these events. Pseudomonas syringaepv.glycinea(Psg), carrying the avrA
gene, causes an oxidative burst, hypersensitive response, and cell death in soybean cv. William 82, har-
boring its corresponding resistance gene Rpg2[67]. The early event in the Psgpathogen–mediated infec-
tion process in soybean is the production of AOSs including H 2 O 2. Levine et al. [66] have taken advan-
tage of soybean (Psgpv.glycineaand H 2 O 2 ) and tobacco (cryptogein elicitor derived from Phytophthora
cryptogea) cell culture systems as well as Arabidopsisseedlings (Psgpv.tomato) and pharmacological
agents to show that H 2 O 2 -mediated influx of Ca^2 is necessary and sufficient to induce cell death in re-
sponse to their compatible avirulent pathogen strains or fungal-derived peptides. Such a pathogen-medi-
ated cell death process can be initiated in the absence of either Psg(avrA) or H 2 O 2 by Ca^2 and ionophore
A23187 but not ionophore alone and inhibited by Ca^2 -depleted medium as well as treatment with Ca^2
channel blockers (La^3 ), indicating the role of Ca^2 in developing HR and cellular death in soybean cells
[66]. Furthermore, these authors have shown that the H 2 O 2 -induced cell death process in soybean cell cul-
tures is strongly inhibited by treatment with specific protein kinase inhibitors, staurosporine, K252A and
AEBSF; partially inhibited by leupeptin; and not inhibited by inhibitors such as H7, H89, TPCK, TLCK,
ANLM, and YVAD-CMK. These results indicate the role of specific CDPKs and phosphorylation events
mediated by Ca^2 . However, Ca^2 influx does not stimulate glutathione-S-transeferase induction as re-
vealed by the fact that the treatment of soybean cells with A23187 did not induce GST transcripts and that
H 2 O 2 -induced GST was not inhibited by La^3 , indicating that Ca^2 elicits a specific signal pathway prior
to the onset of the cellular death process in soybean cells.
Cho and his colleagues have used biochemical, cell biological, and transgenic approaches to address
the functional differences and significance of the conserved (SCaM1, -2, and -3) and divergent (SCaM4
and -5) CaM isoforms from soybean [219,220,250,316]. They have analyzed these isoforms to address
their interaction with the putative CaM-binding proteins isolated from plant protein extracts at different
developmental stages on gel overlay assay, differences in in vivo distribution of target proteins using in
situ hybridization with specific antibodies raised against conserved and divergent SCaM isoforms, abil-
ity to activate NAD kinase and phosphodiesterase, and finally their participation in the disease resistance
mechanism using a transgenic approach (see later under Transgenic Approaches). Although SCaM iso-
forms show similar patterns, they differ in their relative affinity in interacting with CaM-binding proteins.
Further, the isoforms show differences in their relative abundance in vivo. The conserved isoforms are
relatively abundant in their expression compared with divergent forms. All CaM isoforms activate phos-
phodiesterase (PDE) but they differ in their activation of NAD kinase, calcineurin, and nopaline synthase,
indicating Ca^2 /CaM specificity between CaM isoforms and target proteins. Recently, we have provided
evidence for the differential interaction of CaM isoforms with a kinesin-like microtubule-associated pro-
tein from Arabidopsis[218]. These results also suggested that all CaM isoforms may bind to CaMBPs but
with different affinity. Taken together, these findings suggest the existence of reciprocal regulatory mech-
anisms between Ca^2 /CaM and CBPs.
The studies just presented provide strong evidence for the role of Ca^2 , CDPKs, phosphatases,
CaMs, and CaMBPs in stress-induced signal transduction cascades. However, further experiments are
necessary to identify the intermediate components that induce the expression of target genes such as
HVA1and cellular responses such as physiological cell death and to identify and functionally character-
ize CaMBPs.
B. Molecular Genetic Approaches
Increases in free [Ca^2 ]cytlevels in response to a variety of abiotic and biotic stresses initiate a complex
signal network ranging from sensing to activation of a number of stress-responsive genes to an ultimately
altered physiological process. To address the downstream signal components in the stress-induced Ca^2 -
mediated signal cascades, a number of mutants with increased sensitivity or tolerance to stresses have
been isolated. For example, cold acclimation is a process in which plant species show enhanced resistance
to freezing after they are exposed to low-temperature conditions [33]. Molecular and genetic approaches
led to identification of a common set of genes that respond to cold, drought, and ABA [33,317], which
are well-known stress factors that elevate [Ca^2 ]cyt[3].
CALCIUM IN STRESS SIGNAL TRANSDUCTION 715