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Calmodulin(CaM),a multifunctional Ca2+sensor,mediates multiple reactions involved in plant growth and environmental stress regulation.In this study,we found that AtCaM4 plays a negative role in freezing tolerance in Arabidopsis.The deletion of AtCaM4 resulted in enhanced freezing tolerance in cam4 mutant plants.Although AtCaM4 and AtCaM1 were cold-induced isoforms,cam4/cam1Ri double-mutant and cam4 single-mutant plants exhibited similar improvements in freezing tolerance indicating that AtCaM4 plays major role in freezing tolerance.