The Role of SIAMESE in G2 Checkpoint Regulation in Arabidopsis thaliana

Abstract

In Arabidopsis, SIAMESE (SIM) is a cyclin-dependent kinase inhibitor that restricts progression through mitosis. SIM is well known as a regulator of endoreplication in trichomes and roots. Mathematical modeling of the cell cycle has indicated that SIM may also modulate the length of G2 during mitotic cycles, potentially replacing the WEE1/CDC25 circuit that regulates G2 timing in animals and fungi, which is absent in plants. The predictions of this model were tested in several ways. First, the root meristem is longer in sim mutant roots than in wild-type (WT) roots. Second, two independent methods of measuring cell cycle phases, long-term live-cell imaging using fluorescent protein cell cycle markers and 5-ethynyl-2-deoxyuridine (EdU) pulse-chase imaging, showed that the length of G2 in root meristem cortex cells is shorter in sim mutant plants compared to WT, consistent with the view that these changes in G2 length are due to greater G2 CDK activity in sim mutants. Additionally, the fluorescence of a CYCB:GFP fusion both rose and declined more sharply in the mutant than in wild-type. Because both transcription and degradation of this fusion are directly affected by G2 CDK activity, this result is consistent with sim mutants having greater G2 CDK activity. Taken together, these results suggest that, in addition to its known role in inducing endoreplication, SIM may play a role in regulating the length of G2 during mitotic cycles, at least partially replacing the absence of WEE1/CDC25 regulation of the mitotic cell cycle.