Plant E2Fs and RBR Proteins Regulators of Cell Division and Differentiation

In contrast to DELs, manipulating E2F activities by gain or loss of function mutations strongly affects cell proliferation and the pattern of cell differentiation, strongly supporting the idea that E2Fs are regulators of the G1-S control point (De Veylder et al. 2002; del Pozo et al. 2002, 2006; Magyar et al. 2005; Sozzani et al. 2006). In agreement with their expected G1-S function Arabidopsis E2Fs are up-regulated at an early stage during re-entry into cell division (de Jager et al. 2001). On the basis of their cell cycle-dependent expression, E2FA function was linked to S-phase control, while both E2FB and E2FC might have overlapping or distinct roles outside of S-phase. Arabidopsis E2Fs work similarly to their animal counterparts; they must form heterodimers with one of the two DP proteins to efficiently bind to E2F-targeted promoters (Magyar et al. 2000; Mariconti et al. 2002; Kosugi and Ohashi 2002b). Furthermore, heterodimer-ization with DPA, but not with DPB, results in nuclear localization of E2FA and E2FB, suggesting that E2F-DPA and E2F-DPB heterodimers are functionally different (Kosugi and Ohashi 2002b). Interestingly, neither DPA nor DPB could enhance the nuclear translocation of E2FC, probably because another post-translational modification is required to fully activate E2FC-dependent transcription (Kosugi and Ohashi 2002b).

In Arabidopsis all the three E2Fs are controlled by the single retinoblas-toma related protein (RBR1). Interestingly, cereals such as maize and rice contain more than one RB-related gene and on the basis of their expression patterns it was suggested that they have different regulatory roles; maize RBR1 controlling cell differentiation and RBR3 regulating mitosis (Sabelli and Larkins 2006). Genetic analysis suggests that the Arabidopsis RBR1 has essential functions early in plant development since the knock out rbr1 mutant is gametophytic lethal. Inactivation of the RBR1 gene in Arabidopsis endosperm of female megagametophyte results in over-proliferation due to failure in blocking mitosis, indicating a negative regulatory role for RBR1 in the mitotic cell division cycle (Ebel et al. 2004). In addition, decreasing the level or activity of RBR1, either by virus-induced gene silencing (VIGS) in tobacco, or by inducible expression of a viral RBR-binding protein (gemi-nivirus RepA) in Arabidopsis, caused abnormal leaf development, probably due to prolonged cell proliferation (Desvoyes et al. 2006; Park et al. 2005). Interestingly, in both cases, leaf cells show increased ploidy levels later in development. Further studies revealed that RBR1 could control differentiation as well; ectopic expression in the shoot or root apical meristems stimulates in early differentiation (Wildwater et al. 2005; Wyrzykowska et al. 2006). Suppressing its expression in the root apical meristem by using RBR1-RNAi resulted in the production of several extra layers of undifferentiated cells in the columella root cap (Wildwater et al. 2005). Moreover, Arabidopsis RBR1 could work according to the canonical CycD/RBR/E2F pathway model to regulate stem cell maintenance: increasing RBR1 phosphorylation by the ectopic expression of cyclin D led to supernumerous stem cell layers while hypophos-phorylation of RBR1 by overexpression of KRP2, a cyclin-dependent kinase inhibitor from Arabidopsis, resulted in the loss of stem cells similar to that seen with RBR overexpression (Wildwater et al. 2006). In agreement with the model, increasing the amount of RB-free E2F complexes by ectopic co-expression of E2FA and DPA resulted in an excess of stem cells. However, these results raise several questions, e.g. how the single Arabidopsis RBR1 could regulate mitosis and differentiation, and how and which E2Fs are part of these RBR1-regulated processes during plant development?

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