The switch from phase I to phase II growth mode involves two known mechanisms: (i) the suppression of mitosis and (ii) the stimulation of cell expansion, during which repeated rounds of DNA synthesis persist until cells are fully expanded. CDK-cyclin complexes control the commitment to S phase, but the execution of S-phase is enabled by a CDK-controlled hierarchy of enforcers that include the plant homolog of the retinoblastoma gene (Rb), and a family of related transcription factors that include E2F, DP, and DEL genes (Gutierrez et al. 2002; Inze and Veylder 2006). E2F and DP gene products heterodimerize to bind their canonical target sites, while DEL proteins can bind these as monomers and lack conspicuous activation domains. Rb keeps E2F proteins in check by binding them through a so-called A/B pocket, but upon hyperphosphorylation by CDKs, releases these so they can directly activate expression of S phase genes. Over-expression of Arabidopsis E2F3 (also known as E2Fa) stimulates expression of S phase genes, and enhances proliferation and endoreplication. This phenotype is exacerbated when a DP gene is co-expressed (De Veylder et al. 2002). However, not all E2F genes promote S phase: E2F2 (also known as E2Fc) lacks an apparent activation domain and hence can suppress S-phase associated gene expression (e.g., CDC6) (del Pozo et al. 2002). Reduced E2F2/c expression results in increased expression of S-phase gene markers, enhanced cell production and plants with more, but smaller cells with reduced levels of endoreplication (del Pozo et al. 2006). Likewise, enhanced E2Fd (also known as DEL1) expression reduces ploidy levels, while reduced E2Fd/DEL1 activity increases endoreplication (Vlieghe et al. 2005). In contrast, altered levels of E2Ff/DEL3 had a modest impact on endoreplication, but elevated expression promoted precocious differentiation in roots, possibly because several expansins and xyloglucan endotransgly-colase/hydrolase involved in cell wall extension during phase II growth are E2Ff/DEL3 targets (Ramirez-Parra et al. 2004).
Taken together, these results suggest that E2F/DEL genes are involved in regulation of S-phase-specific gene expression as well as promoting phase I/II transition. Therefore, they are likely targets of regulatory pathways that control the suppression of mitosis and the stimulation of cell expansion. However, how the activity of different E2F-like factors, in some cases possibly on common target genes, is regulated is still not well understood, but at least one of them, E2F2c, is unstable (del Pozo et al. 2002). Further evidence for a possibly pivotal role for regulated protein degradation in controlling the phase I/II transition comes from the observation that CCS52, a regulatory component of the anaphase promoting complex (APC) orthologous to CDH1 and fizzy-related (which functions as an inhibitor of mitosis), is required for endoreplication (Cebolla et al. 1999).
Although the experimental evidence clearly points to a complex involvement of Rb/E2F/DP/DEL proteins, as well as regulated proteolysis in controlling the phase I/II transition, the regulators that orchestrate the deployment of these enforcers, specifically their order of action, have not yet been identified.
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