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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.

Low-temperature signal transduction: induction of cold acclimation-specific genes of alfalfa by calcium at 25 degrees C.