To study the role of calcium in cold acclimation, we examined the relationship between calcium influx and accumulation of transcripts of two cas (cold acclimation-specific) genes of alfalfa, cas15 and cas18. Whereas a decline in temperature from 25 to 15 degrees C had little effect on the influx of extracellular 45Ca2+, an increasing influx was observed when the temperature was lowered further. The influx of 45Ca2+ at 4 degrees C was nearly 15 times greater than at 25 degrees C. The addition of calcium chelators or of calcium channel blockers, which have been shown to prevent cold acclimation, inhibited the influx of extracellular 45Ca2+ as well as the expression of cas genes at 4 degrees C. The addition of a calcium ionophore or a calcium channel agonist to nonacclimated cells caused the influx of extracellular 45Ca2+ and induced the expression of cas genes at 25 degrees C. These results suggest that a cold-induced calcium influx plays an essential role in cold acclimation. To further study the role of calcium, we isolated two sequences corresponding to calcium-dependent protein kinases. The transcript level of one of them was markedly upregulated at 4 degrees C. We propose a sequence of signaling events that is likely to occur early during cold acclimation and leads to the expression of cas genes and the development of freezing tolerance.

l h e role of Ca'+ in cold-induced changes i n protein phosphorylation, gene expression, and development of freezing tolerance has been studied in cell-suspension cultures of a freezing-tolerant cultivar of alfalfa (Medicago safiva spp. falcafa cv Anik). Chemical treatments to block Caz+ channels, antagonize calmodulin action, or inhibit protein kinases markedly inhibited the cellular capacity to develop cold-induced freezing tolerance but had little effect on cell viability. An analysis of phosphoprotein profile by two-dimensional polyacrylamide gel electrophoresis revealed that at low temperature the relative level of phosphorylation of several proteins increased, whereas that of several others decreased. When cold acclimation was carried out in the presence of M(6-aminohexyl)-5-chloro-l-naphthalene-sulfonamide hydrochloride, an antagonist of calmodulin and ca'+-dependent protein kinases, or the CaZ+ channel blocker La3+, the cold-induced changes i n protein phosphorylation were strongly inhibited, cells lost their capacity to develop freezing tolerance, and accumulation of transcripts of cold acclimation-specific genes was substantially reduced. An inhibitor of protein kinases, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, had less pronounced effects on the cold-induced protein phosphorylation and caused only a partia1 inhibition of the cold-induced development of freezing tolerance and accumulation of the transcripts. l h e level of phosphorylation of one protein, of about 15 kD, increased more than 10-fold at low temperature and showed a strong positive correlation with cold-induced freezing tolerance and gene expression even when the latter were altered with various chemical treatments. lhese results suggest that CaZ+ and protein phosphorylation, or perhaps a coupling of the two, play an important role during the acquisition of freezing tolerance during cold acclimation.

The involvement of calcium signaling during cold-induction of the kin genes of Arabidopsis thaliana (L.) Heynh. was examined. Treatments with chemicals which either chelate extracellular calcium (EGTA) or block the plasma-membrane calcium channels (La3+, Gd3+) inhibited cold acclimation as well as kin gene expression. Ruthenium red, an inhibitor of calcium release from intracellular stores partially inhibited kin gene expression and development of freezing tolerance. An inhibitor of calcium-dependent protein kinases (CDPKs) and calmodulin prevented cold acclimation as well as the cold induction of kin genes. Using restriction fragment length polymorphism-coupled domain-directed differential display, five CDPK clones were identified which showed differential regulation by cold. The amplified fragments showed homology to known plant CDPKs. The involvement of calcium and calcium-binding proteins in cold acclimation of A. thaliana is discussed.

To study the role of calcium in cold acclimation, we examined the relationship between calcium influx and accumulation of transcripts of two cas (cold acclimation-specific) genes of alfalfa, cas15 and cas18. Whereas a decline in temperature from 25 to 15 degrees C had little effect on the influx of extracellular 45Ca2+, an increasing influx was observed when the temperature was lowered further. The influx of 45Ca2+ at 4 degrees C was nearly 15 times greater than at 25 degrees C. The addition of calcium chelators or of calcium channel blockers, which have been shown to prevent cold acclimation, inhibited the influx of extracellular 45Ca2+ as well as the expression of cas genes at 4 degrees C. The addition of a calcium ionophore or a calcium channel agonist to nonacclimated cells caused the influx of extracellular 45Ca2+ and induced the expression of cas genes at 25 degrees C. These results suggest that a cold-induced calcium influx plays an essential role in cold acclimation. To further study the role of calcium, we isolated two sequences corresponding to calcium-dependent protein kinases. The transcript level of one of them was markedly upregulated at 4 degrees C. We propose a sequence of signaling events that is likely to occur early during cold acclimation and leads to the expression of cas genes and the development of freezing tolerance.

No abstract