Cold-activation of Brassica napus BN115 promoter is mediated by structuralchanges in membranes and cytoskeleton, and requires Ca2+ influx

Previous studies on cold-triggered events leading to Ca2+, influx during co ld acclimatization have been conducted on either unicellular cyanobacterium Synechocystis or plant cell suspensions, and used transcript levels of col d-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 c old signaling in transgenic Brassica napus seedlings carrying, in addition to the endogenous cold-inducible BN115 gene, the beta -glucuronidase (GUS) gene placed under control of the BN115 promoter. The activity of BN115 prom oter was monitored at the transcriptional and translational levels by deter mining accumulation of BN115 transcripts and by histochemical assay of GUS activity. Cold-activation of BN115 was strongly inhibited by the membrane f luidizer benzyl alcohol, but mimicked at 25 degreesC by the membrane rigidi fier dimethylsulfoxide (DMSO). The cold induction of BN115 was also inhibit ed by stabilizers of microtubules and actin microfilaments, taxol and jaspl akinolide, respectively, but was mimicked at 25 degreesC by microtubule des tabilizer oryzalin or colchicine, or by microfilament destabilizer latruncu lin B. Gd3+, or ruthenium red prevented the cold activation of BN115, but C a2+ ionophore A23187 or cyclic ADP-ribose activated it at 25 degreesC. Inhi bitors of tyrosine kinases, protein kinase C and phosphoinositide kinases p revented the cold activation of BN115, but inhibitors of protein phosphatas es (PP) 1 and 2 A activated BN115 at 25 degreesC. Constitutively expressed GUS activity in another transgenic line of the same cultivar of B. napus, w as not affected by cold or any of the chemical treatments used in the exper imentation. Activation of BN115 at 25 degreesC by DMSO, Ca2+ ionophore, cAD PR, and by inhibitors of PP1 and 2A was accompanied by an increased freezin g tolerance. It was concluded that the cold-activation of BN115 requires me mbrane rigidification, cytoskeleton reorganization, Ca2+, influx and action of several types of protein kinases.