An analysis was undertaken of the effects of a toxic metal, zinc, on plant cell suspension cultures of the TBY–2 cell line of tobacco (Nicotiana tabacum cv. Bright Yellow 2) in order to determine whether Zn acts in a cell cycle-specific manner. In the control treatment (0 Zn), following a 24 h synchronization with aphidicolin and 7 h after the release from the inhibitor, the mitotic index peaked at 45%. The inclusion of Zn in the 24 h aphidicolin treatment (100, 200 or 300 μM Zn) resulted in a concentration–dependent decrease in the mitotic peak to 30%, 22% and 10%, respectively, but did not affect the timing of the peak. Hence, despite high concentrations of Zn, cells traversed from S–phase to mitosis, albeit in smaller proportions, at the same rate as the controls. Cells treated with 0, 100 or 200 μM Zn during synchronization and then released into Zn–free media showed successive peaks in mitotic index at 7 h and 21 h following release, i.e. Zn-treated cells progressed through a complete cell cycle at the same rate as the controls. Synchronization and subsequent release into Zn–containing medium (100 μM) examined the effect of the metal on predominantly late G1 cells. In this treatment, the mitotic index peaked at 7 h and 19 h, indicating a slightly faster cell cycle (12 h) compared with the control (14 h). Continuous exposure to 100 μM Zn through both synchronization and release resulted in a cell cycle of 11 h and a differential effect on the component phases: M–phase lengthened (1.5 h to 3.5 h) and G1 shortened (6 h to 1 h) compared with the control treatment.
Vital staining (Evans Blue) revealed that cell mortality increased from 2.7% (0 Zn) to 6.1% and 6.5% at 100 and 200 μM Zn, respectively. The Zn content of cells increased 40–lfold from 0 to 100 μM Zn. The data are consistent with the effects of Zn reducing the cycling cell population primarily through cell arrest rather than cell death, but also reveal that a substantial population of TBY–2 cells progressed through the cell cycle despite accumulating Zn. In particular, the duration of G2 and S-phase was remarkably invariant, clearly indicating that once plant cells meet the requirements of late G1 check-points, they are committed to divide, even in the presence of toxic concentrations of Zn.
The synchronous TBY–2 cell suspension, which lacks the heterogeneity and developmental constraints of plant meristems, is an excellent system to study the effects of known toxic metals, and indeed other environmental factors, on the plant cell cycle.