To investigate whether a change in membrane fluidity affects old signaling in Arabidopsis, seedlings were treated with either a membrane rigidifer, DMSO, or a membrane fluidizer, benzyl alcohol (BA). MPK4 was activated by DMSO without cold treatment whereas cold-induced MPK4 activation was suppressed by the BA pretreatment. Phosphorylation of MKK2 and MEKK1. the upstream components of MPK4 in the cold signaling pathway, was also induced by DMso treatments and decreased by BA pretreatments these data indicate that membrane rigidification occurs upstream of MEKK1 -MKK2-MPK4 cascade during cold acclimation. The cold-inducible gene,COR15a, was expressed by DMSO without cold treatment, whereas its expression was suppressed by the BA pretreatment. Moreover, we determined that the DMSO-treated seedlings acquired freezing tolerance without cold acclimation, and the