Apoptosis in heat shock-treated tobacco protoplasts was evidenced by DNA fragmentation, flow cytometric analysis and activation of caspase 3-like protease, Furthermore, an in vitro apoptosis system was established which reproduced the apoptotic events, Western blotting analysis using an antibody against lamin A and C showed that in both in vivo and in vitro systems lamin-like proteins mere cleaved into a 35-kDa fragment, and that lamin-like protein degradation precedes DNA fragmentation. Moreover, we found a 22.8-fold increase in caspase 6-like activity in cytosol of heat-treated protoplasts as compared with the control. (C) 2000 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.