abnormalities in cellular activities including inhibition of photosynthesis, reduction in protein synthesis
and potassium content, and increase in Naand organic solutes such as proline, glycine betaine, and poly-
ols. Zhu and his colleagues [63,117,336–338] have made significant contribution to our understanding of
salt tolerance. They provided a molecular view of the relationship between Ca^2 , potassium, and sodium
cations in salt tolerance mechanisms in Arabidopsisusing a novel genetic approach [63,117,336,
337,338]. These authors screened a large number of EMS or fast neutron mutagenized M2 seed or T-DNA
insertionArabidopsislines (~267,000) to isolate salt overly sensitive (sos) mutants using a root-bending
assay on agar plates containing 50 mM NaCl [336]. Using this genetic screening approach, Zhu et al. dis-
covered three genes, namely SOS1,SOS2, and SOS3, and numerous alleles for each gene.
Construction of combinations of double sosmutants revealed that they function in a linear pathway
and exhibit similar phenotypes in that they are all hypersensitive to Naand Liand are unable to grow
on a low-Kculture medium. They differ in their sensitivity to 100 mM NaCl (sos1being the most sen-
sitive followed by sos2andsos3). The concentration of NaCl required to inhibit root growth 50% (I 50 )
was shown to be ~4 mM, ~10 mM, and ~40 mM for sos1,sos2, and sos3, respectively, as compared with
the wild type, which requires 100 mM [117,336]. Interestingly, the normal growth pattern of sosmutants
is restored by inclusion of Kin the growth medium along with 50 mM NaCl (50 mM Kforsos1and
sos2and 1 mM for sos3). These abnormal growth patterns in the presence of NaCl could be mitigated by
the addition of increased levels of Ca^2 in the same medium. Calcium restores NaCl-inhibited growth
partially in sos1(at 10 mM) and significantly in sos3(at 2 mM) mutant seedlings, indicating that Ca^2
plays a crucial role in selective uptake of Kto Na. These results provided a direct relationship between
three cations in a salt tolerance mechanism using the genetic approach.
On the basis of these results, Zhu and his colleagues predicted that these SOSgenes encode regula-
tory proteins that control Na/Kuptake and a Ca^2 sensor that controls Knutrition [337]. Indeed, the
SOS3gene encodes a Ca^2 sensor protein that shows high affinity to Ca^2 [63]. Its significant homology
to calcineurin (CaN) of yeast [339,340] and neural Ca^2 sensors (CNS) of animals [341] raises the pos-
sibility that the SOS3gene product might regulate the K/Natransport system via Ca-mediated acti-
vation of phosphatase and/or inhibition of a kinase signal cascade and confers salt tolerance. Using a
transgenic approach, Pardo et al. [116] were able to express constitutively both catalytic and regulatory
components of yeast CaN in tobacco with a 35S promoter and showed that the transgene enhances the salt
tolerance of tobacco. Complementation of plant Ca^2 pumps (ACA2 and ECA1) in yeast mutants also
confirmed the similar functional Ca^2 machinery between yeast and plants [229,342]. Together, these re-
sults provide direct molecular genetic evidence for the participation of Ca^2 -binding proteins in adapting
salt tolerance in higher plants.
C. Transgenic Approaches
To address the functions of Ca^2 and its interacting components at the whole plant level, researchers from
a number of laboratories have developed transgenic plants with the genes involved in Ca^2 -mediated
stress signaling cascades. The transgenes include the genes encoding stress-inducible transcriptional fac-
tors, CaM specific isoforms, Ca^2 antiporters, and CaM-binding proteins (Table 2). In this section, we
have presented some known examples of transgenic approaches that unravel the role of Ca^2 in initiating
stress-responsive mechanisms in plants.
Plants maintain an asymmetric distribution of Ca^2 (millimolar versus nanomolar levels in or-
ganelles and cytosol, respectively) to avoid its toxic effects on cellular metabolism. This kind of Ca^2
homeostasis is achieved by high-affinity Ca^2 pumps (Ca^2 -ATPases) and low-affinity Ca^2 /Han-
tiporters. These Ca^2 pumps and antiporters also play a crucial role in raising and restoring [Ca^2 ]cytlev-
els in response to various stimuli. A vacuolar localized Ca^2 /Hantiporter (calcium exchanger) has been
identified from Arabidopsis(CAX1andCAX2) [345] and mung bean [346]. The Arabidopsis CAX1gene
functionally complements yeast mutant defective in its antiporter activity [345]. Its transcripts are in-
ducible by extracellular Ca^2 levels, Na, K, Ni^2 , PEG, and Zn^2 but not by plant hormones. In order
to gain more insight into the action of the CAX1gene product on Ca^2 homeostasis, Hirschi [343] con-
stitutively expressed the Arabidopsis CAX1coding region under the control of the 35S promoter in to-
bacco plants. The transgenic plants expressing the CAX1gene in sense orientation showed stunted growth
with a poorly developed root system, necrosis of leaves and apical meristem, hypersensitivity to Kand
Mg^2 ions, and sensitivity to cold [343]. However, these abnormalities could be reversed by Ca^2 sup-
CALCIUM IN STRESS SIGNAL TRANSDUCTION 717