37:	In vitro apoptosis in tobacco cell free system was selectively inhibited by iodoacetamide.

Authors:	Zhou, Jun(A)Chen, Haoming(A)Dai, Yao-Ren(A)
Affiliations:	(A): Department of Biology, Tsinghua University, Beijing 100084, China
Presenter:	Dai, Yao-Ren , zhaizhh@tsinghua.edu.cn

To investigate the apoptotic pathway in plant cells we have developed a cell free system that consists of the isolated nuclei (2.5x107) from healthy tobacco cells and the cytosolic fraction (9x107 cell equivqlents) made from apoptotic tobacco cells as induced by heat shock treatment. After 4 h incubation at 28oC stereotypic hallmarks of apoptosis including nucleus condensation and formation of apoptotic-like bodies were observed in these nuclei which would otherwise maintain a normal morphology as shown by DAPI staining. These changes were accompanied by the internucleosomal DNA fragmentation known as DNA laddering. The apoptotic changes were sensitive to 5 mM iodoacetamide, a cysteine-alkylating agent, but resistant to 100mM TLCK, 1mM pepstatin A and 100mM leupeptin. Strikingly Ac-DEVD-CHO, the specific inhibitor of caspase-3 showed also very slight inhibition. At the same time, a very low activity of Ac-DEVD-pNA clevage was observed during apoptosis in the cell free system suggesting that in tobacco cells a different cysteine protease may function as the counterpart of caspase-3, the death enzyme in mamalian cells.