Ethylene induces cell death at particular phases of the cell cycle in the tobacco TBY-2 cell line

It was examined whether ethylene induces programmed cell death in a cell cy cle-specific manner. Following synchronization of the tobacco TBY-2 cell li ne with aphidicolin and its subsequent removal, ethylene was injected into the head space of 300 cm(3) culture flasks at 0 h or 3.5 h later and cells were sampled for 26 h. There were significant increases in cell mortality a t G(2)/M in both the 0 h and 3.5 h ethylene treatments, and for the latter treatment, another peak in S-phase. The effect at G(2)/M was greater in the 3.5 h treatment, but was ameliorated by the simultaneous addition of silve r nitrate (1.2 muM). In addition, the 3.5 h ethylene treatment resulted in a 1 h delay in the characteristic rise in the mitotic index following aphid icolin-induced synchrony. The addition of silver nitrate alone (1.2 muM), a lso delayed the entry of cells into mitosis but had no effect on cell cycle length compared with the controls (14 h throughout all treatments) but it induced a peak of mortality 2.5 h after its addition. Nuclear shrinkage was also a characteristic feature of dying cells at G(2)/M. Using Apoptag (R), an in situ apoptosis detection kit, nuclear DNA fragmentation was observed in the TBY-2 cells which were often isolated on the end of a filament of n ormal cells. In the 3.5 h ethylene treatment, a marked increase was noted i n the percentage of such cells at the G(2)/M transition compared with the c ontrols. Hence, the data show cell death occurring at a major phase transit ion of the cell cycle and the observations of nuclear shrinkage, isolation of dying cells and nuclear DNA fragmentation suggest a programmed mechanism of cell death exacerbated by ethylene treatment.