The genes that are involved in the cold acclimatization process are called COR(cold responsive) genes
and encode hydrophilic polypeptides with similar properties [33]. These include COR6.6(KIN),COR15a,
COR78(LT178),WCS19,CORa,CAS15a, COR47, and HVA1, and these gene products potentially pro-
mote tolerance to freezing. The expression of some of these genes is regulated by CBF (CRT/DRE bind-
ing factor) transcriptional activators through binding of CRT/DRE motifs present in their promoter region
[318,319]. The HVA1gene has been extensively used to test whether its expression is inducible by Ca^2 -
mediated stress signals [190]. Thomashow and his colleagues have studied extensively the functions of the
COR15aandCBF1genes in the cold acclimation process. Constitutive expression of COR15a[320] and
CBF1[321] independently in nonacclimated Arabidopsisled to improved tolerance to low temperatures.
Acclimation involves changes in the membrane lipid profile including phospholipids, sterols, cerebrosides,
and accumulation of sucrose, other simple sugars, and proline, which stabilize membranes against freeze-
induced damage [322]. Although the exact mechanism of action of the COR regulon in freeze tolerance is
not known, the COR polypeptides are probably involved in the regulation of the enzymes that produce
lipids, sugars, and other membrane-stabilizing components during acclimation process.
Mutants that show an altered response to a particular stress are invaluable resources in identifying
components of the signal transduction pathway(s). Arabidopsismutants sensitive to freezing (sfr) even
after cold acclimation have been identified [323]. One of these mutants, srf6, showed reduced transcript
levels for the CORgenes containing a CRT/DREsequence motif including KIN1,COR15a, and LTI78
[34]. However, the sfr6mutant is not defective in the expression of CBFgenes (CBF1toCBF3) that ac-
tivate the expression of KIN1,COR15a, and LTI78(CRT/DRE cis-containing genes) and AtP5CS1(which
does not contain the CRT/DREelement and is involved in proline biosynthesis). Further, the Ca^2 -sens-
ing mechanism in sfr6is intact, which means that upon cold treatment the [Ca^2 ]cytlevels are similar to
those in the wild type. These authors considered SFR6 an essential component that promotes the interac-
tion between CBF and CRT/DREresponsive genes, which are activated in response to cold, drought, and
ABA stress events [34]. These studies indicate the involvement of other components in Ca^2 -mediated
signal transduction pathways in plants.
Zhu and his colleagues [324] have screened ethyl methanesulfonate (EMS)-treated transgenic Ara-
bidopsislines (RD29A-LUC) expressing bioluminescence in response to cold and osmotic stress (NaCl
and ABA). The LUC coding sequence is under the regulation of the RD29A(COR78orLTI78) gene pro-
moter, which harbors both CRT/DREandABRE cis-regulatory DNA elements, and thus responds to cold
and osmotic stresses. Using this genetic approach, they isolated many genetic mutants that are defective
in one or a combination of cold or osmotic stress (NaCl or ABA), which will be invaluable in dissecting
the complex network of stress signaling in plants. Characterization of these mutants and cloning of mu-
tant genes would provide valuable insights into the stress signal transduction.
The plant stress–responsive hormone ABA modulates several cellular activities such as guard cell
closure and activation or repression of several genes in response to drought, cold, and high salt [325,326].
ABA is also involved in seed maturation, desiccation, dormancy, and germination. Unequivocally, Ca^2
has been implicated in ABA-mediated guard cell closure in response to environmental stresses
[53,141,327]. Genetic approaches have been employed to isolate mutants that are defective in ABA re-
sponses in several plant species [278,328–331]. Molecular and genetic approaches in Arabidopsisled to
identification of many ABA-insensitive mutants including abi1andab12mutants, which encode protein
phophatases of type 2C (PP2Cs). Furthermore, a site-directed mutant of PP2C together with ABA-re-
sponsive gene expression in maize protoplast has also shown the role of PP2C in ABA signal transduc-
tion [332]. Accumulated research investigations indicate that elevated [Ca^2 ]cytlevels act as a second
messenger in ABA-mediated cellular responses [52,190,332,333]. Recently, Allen et al. [334] provided
direct evidence for the role of Ca^2 in ABA-mediated stomatal closure using ABA-insensitive mutants
(abi1andabi2) of Arabidopsis. These mutants are unable to elevate sufficient [Ca^2 ]cytlevels in response
to ABA treatment as measured by fluorescent Ca^2 -binding dyes and stomata remain open. However,
treatment of abimutants with extracellular Ca^2 restored ABA-induced stomatal closure, indicating the
role of Ca^2 in ABA-mediated stomatal closure. The downstream events following [Ca^2 ]cytelevation,
such as kinase-activated S-type anion channels, membrane depolarization, and loss of turgor pressure, re-
main functional in abimutants [334]. Further research would provide the identity of the missing compo-
nents in this pathway.
About one third of arable lands contains high levels of NaCl in their soils [335]. Such high-salinity
soils cause accumulation of Naions and less uptake of K. This situation in plant cells causes severe
716 REDDY AND REDDY