Tobacco BY-2 Cells Expressing Fission Yeast cdc25 Bypass a\ud G2/M Block on the Cell Cycle

The mitotic inducer gene from Schizosaccharomyces pombe, Spcdc25, was used as a tool to investigate\ud regulation of G2/M in higher plants using the BY-2 (Nicotiana tabacum) cell line as a model. Spcdc25-\ud expressing BY-2 cells exhibited a reduced mitotic cell size through a shortening of the G2 phase. The cells often\ud formed isodiametric double files both in BY-2 cells and in cell suspensions derived from 35S::Spcdc25 tobacco\ud plants. In Spcdc25-expressing cells, the tobacco cyclin-dependent kinase, NtCDKB1, showed high activity in\ud early S phase, S/G2 and early M phase, whereas in empty vector cells CDKB1 activity was transiently high in\ud early S phase but thereafter remained lower. Spcdc25-expressing cells also bypassed a block on G2/M imposed\ud by the cytokinin biosynthetic inhibitor lovastatin (LVS). Surprisingly, cytokinins were at remarkably low levels\ud in Spcdc25-expressing cells compared with the empty vector, explaining why these cells retained mitotic\ud competence despite the presence of LVS. In conclusion, synchronised Spcdc25-expressing BY-2 cells divided\ud prematurely at a small cell size, and they exhibited premature, but sustained, CDKB1 activity even though\ud endogenous cytokinins were virtually undetectable