Mitotic progression is timely regulated by the accumulation and degradation of A- and B-type cyclins. In plants, there are three classes of A-, and two classes of B-type cyclins, but their specific roles are not known. We have generated transgenic tobacco plants in which the ectopic expression of a plant cyclin B2 gene is under the control of a tetracycline-inducible promoter. We show that the induction of cyclin B2 expression in cultured cells during G2 phase accelerates the entry into mitosis and allows cells to override the replication checkpoint induced by hydroxyurea in the simultaneous presence of caffeine or okadaic acid, drugs that are known to alleviate checkpoint control. These results indicate that in plants, a B2-type cyclin is a rate-limiting regulator for the entry into mitosis and a cyclin B2-CDK complex might be a target for checkpoint control pathways. The cyclin B2 localization and the timing of its degradation during mitosis corroborate these conclusions: cyclin B2 protein is confined to the nucleus and during mitosis it is only present during a short time window until mid prophase, but it is effectively degraded from this timepoint onwards. Although cyclin B2 is not present in cells arrested by the spindle checkpoint in metaphase, cyclin B1 is accumulating in these cells. Ectopic expression of cyclin B2 in developing plants interferes with differentiation events and specifically blocks root regeneration, indicating the importance of control mechanisms at the G2- to M-phase transition during plant developmental processes.

Under long hydroxyurea treatments, evidence was obtained for the sequential activation of four checkpoints located between the onset of S phase and mitosis in Allium cepa L. root meristems. Biparametric flow cytometry (Br-DNA/total DNA) showed that cells initially accumulated at early S phase but, after a delay, they resumed replication and paused again at mid S phase. Cells not only overrode this second replication block but also any G2 checkpoint they encountered. Thus, a late mitotic wave was produced in the presence of hydroxyurea. The wave was formed by cells that had apparently completed their replication (normal mitoses), while others displayed anaphases/telophases with less than the expected DNA content and with chromosomal breaks (aberrant mitoses). The presence of aberrant mitoses is direct evidence for the undue override of the two G2 checkpoints responsible for surveillance of completion of DNA synthesis and repair, respectively. Caffeine selectively abrogated the G2 block produced by the checkpoint that controls post-replication DNA repair, as it advanced the entry of cells into an aberrant mitosis. However, caffeine proved not to be the universal checkpoint-evading agent as postulated. Caffeine did not modify the spontaneous override of the replication checkpoints. Moreover, it seems to enforce the checkpoint that controls the completion of DNA synthesis, as the appearance of the late wave of normal mitoses produced in the presence of hydroxyurea was prevented by the use of caffeine.

No abstract

Allium cepa L. meristems were used as a plant model to study the p53-independent control of S and G2 phases by checkpoint pathways, in eukaryotic cells. Checkpoint blocks were induced at early and mid S by hydroxyurea. After their spontaneous override, cells became accumulated in G2-prophase, giving rise later on to a delayed mitotic wave. Cell growth was maintained during the checkpoint blocks, as the delayed mitoses were larger in size than the control ones. Under continuous hydroxyurea treatment, the delayed mitotic was formed by two subpopulations: normal mitoses corresponding to cells having properly recovered from the checkpoint block, and abnormal ones resulting from checkpoint adaptation. These latter cells displayed broken chromatids as they had unduly overriden the G2 checkpoint block, without completing DNA repair. The frequency of the checkpointadapted mitoses increased with the hydroxyurea concentration from 0.25 to 1.0 mM. However, from 1 mM hydroxyurea upwards, some of the cells lost their competence for checkpoint adaptation. Therefore, the dose of a genotoxic agent that still allows G2 checkpoint adaptation should always be applied in order to get rid of uncontrolled proliferating cells. This is specially suitable for cells lacking a functional p53 protein.