A New Cold-lnduced Alfalfa Gene 1s Associated with Enhanced Hardening at Subzero Temperature'

When alfalfa (Medicago sativa 1. cv Apica) plants grown at room temperature are transferred to 2'C, the temperature at which 50% of the plants fail to survive (LISo) decreases from -6 to -14'C during the first 2 weeks but then increases to -9'C during the subsequent 2 weeks. However, when plants are kept for 2 weeks at 2°C and then transferred to -2°C for another two weeks, the LTm declines to -16°C. lhese changes in freezing tolerance are paralleled by changes in transcript levels of casl.5 (cold acclimation-specific gene encoding a 14.5-kD protein), a cold-induced gene. Cold-activation of cas1.5 occurs even when protein synthesis i s inhibited by more than 90%, suggesting that cold-initiated events up to and including the accumulation of cas1.5 transcripts depend on preexisting gene produds. a s 1 5 shows little homology to any known gene at the nucleotide or amino acid level. l h e deduced polypeptide (CAS15) of 14.5 kD contains four repeats of a decapeptide motif and possesses a bipartite sequence domain at the carboxy terminus with homology to the reported nuclear-targeting signal sequences. Although the relative amount of casl.5 DNA as a fradion of the total genomic DNA is similar in cultivars with different degrees of freezing tolerance, i ts organization in the genome i s different. The possible role of casl.5 in the development of cold-induced freezing tolerance is discussed.