Abstract
The rapidly proliferating cells in plant meristems must be protected from genome damage. Here, we show that the regulatory role of the Arabidopsis RETINOBLASTOMA RELATED (RBR) in cell proliferation can be separated from a novel function in safeguarding genome integrity. Upon DNA damage, RBR and its binding partner E2FA are recruited to heterochromatic γH2AX‐labelled DNA damage foci in an ATM‐ and ATR‐dependent manner. These γH2AX‐labelled DNA lesions are more dispersedly occupied by the conserved repair protein, AtBRCA1, which can also co‐localise with RBR foci. RBR and AtBRCA1 physically interact in vitro and in planta. Genetic interaction between the RBR‐silenced amiRBR and Atbrca1 mutants suggests that RBR and AtBRCA1 may function together in maintaining genome integrity. Together with E2FA, RBR is directly involved in the transcriptional DNA damage response as well as in the cell death pathway that is independent of SOG1, the plant functional analogue of p53. Thus, plant homologs and analogues of major mammalian tumour suppressor proteins form a regulatory network that coordinates cell proliferation with cell and genome integrity.
Highlights
The continuous post-embryonic growth of plants is supported by rapidly proliferating cells in meristems
To investigate whether the observed cell death was associated with S-phase progression, we quantified DNA synthesis using 5-ethynyl20-deoxyuridine (EdU) incorporation and cell death in two Columbia 0 (Col-0) transgenic lines with reduced RETINOBLASTOMA RELATED (RBR) levels; the 35Spro:amiGORBR line, in which an artificial miRNA against RBR is expressed constitutively (Cruz-Ramirez et al, 2013), and the RCH1::RBR RNAi line, in which an antisense RNA is expressed locally in the root meristem (Wildwater et al, 2005)
To investigate whether cell death upon RBR silencing was due to a general deregulation of cell cycle entry, or reflected a specific role of RBR in cell viability, we analysed CYCD3.1 overexpression, which promotes cell cycle progression through RBR phosphorylation (Dewitte et al, 2003, 2007; Magyar et al, 2012; Nowack et al, 2012) and E2FA and E2FB overexpression, which act downstream of RBR (De Veylder et al, 2002; Magyar et al, 2005, 2012)
Summary
The continuous post-embryonic growth of plants is supported by rapidly proliferating cells in meristems. Mitogenic signals promote RBR phosphorylation by cyclin-dependent kinases (CDKs) in association with D-type cyclins, the best characterised being CYCLIN D3;1 (CYCD3;1) (Dewitte et al, 2003; Magyar et al, 2012). Upon this RBR phosphorylation, the E2FB transcription factor is released and promotes cell cycle gene expression and cell proliferation, while E2FA remains associated with RBR and maintains meristems through repression of differentiation (Harashima et al, 2013; Kuwabara & Gruissem, 2014; Magyar et al, 2012; Polyn et al, 2015). The low rate of cell division in the QC protects cells against DNA damage while surrounding stem cells are more sensitive
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