SummaryPrevious studies on cold-triggered events leading to Ca 2+ in¯ux during cold acclimatization have been conducted on either unicellular cyanobacterium Synechocystis or plant cell suspensions, and used transcript levels of cold-induced genes as end-point markers. Whether the results of these studies are valid for intact plants or their organs is not known. Here we examine cold signaling in transgenic Brassica napus seedlings carrying, in addition to the endogenous cold-inducible BN115 gene, the b-glucuronidase (GUS) gene placed under control of the BN115 promoter. The activity of BN115 promoter was monitored at the transcriptional and translational levels by determining accumulation of BN115 transcripts and by histochemical assay of GUS activity. Cold-activation of BN115 was strongly inhibited by the membrane¯uidizer benzyl alcohol, but mimicked at 25°C by the membrane rigidi®er dimethylsulfoxide (DMSO). The cold induction of BN115 was also inhibited by stabilizers of microtubules and actin micro®laments, taxol and jasplakinolide, respectively, but was mimicked at 25°C by microtubule destabilizer oryzalin or colchicine, or by micro®lament destabilizer latrunculin B. Gd 3+ or ruthenium red prevented the cold activation of BN115, but Ca 2+ ionophore A23187 or cyclic ADP-ribose activated it at 25°C. Inhibitors of tyrosine kinases, protein kinase C and phosphoinositide kinases prevented the cold activation of BN115, but inhibitors of protein phosphatases (PP) 1 and 2 A activated BN115 at 25°C. Constitutively expressed GUS activity in another transgenic line of the same cultivar of B. napus, was not affected by cold or any of the chemical treatments used in the experimentation. Activation of BN115 at 25°C by DMSO, Ca 2+ ionophore, cADPR, and by inhibitors of PP1 and 2A was accompanied by an increased freezing tolerance. It was concluded that the cold-activation of BN115 requires membrane rigidi®cation, cytoskeleton reorganization, Ca 2+ in¯ux and action of several types of protein kinases.