Gametophyte differentiation and imprinting control in plants

Abstract

The Retinoblastoma (pRb) pathway has been implicated as a convergent regulatory unit in the control of cell cycle and disease. We have shown that a crosstalk between RETINOBLASTOMA RELATED (RBR), the Arabidopsis homologue of pRb, and the genes encoding proteins of the chromatin complexes involved in DNA or histone methylation, controls gametophytic and post-fertilization differentiation events and a subset of imprinting effects. We describe here a plausible model that incorporates several components of the plant Retinoblastoma pathway, thus offering a novel paradigm that merges the traditional cell cycle and the chromatin components in the control of cell ­differentiation and imprinting.

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The short-lived male and female gametophytes are the two haploid phases of the plant life cycle that are independently derived from the dominant diploid sporophyte as a consequence of meiosis.1 Gametophytes produce gametes and accessory cell types through a series of nuclear divisions, cell specification and differentiation events. At maturity, plant gametes are represented by an egg and a central cell in the female gametophyte, and two sperm cells in the male gametophyte (Fig. 1A). Fertilization of an egg by a sperm cell marks the completion of the gametophytic phase. It constitutes the maternal to embryonic transition by forming a diploid embryo, the sporophyte. At the same time the central cell is fertilized by a second sperm in all angiosperms. This gives rise to the endosperm, which is an extra-embryonic annex that nourishes the embryo. Both the gametophytic (central cell and sperm cell) and endosperm developmental phases are essential reproductive platforms in plants, in which imprinting effects that epigenetically distinguish parent-of-origin specific gene expression are established and/or maintained primarily by DNA and/or histone methylation.2

ReTINoBLAsToMA ReLATeD (RBR) is a single copy plant homologue of Retinoblastoma (pRb), a key cell cycle regulatory protein and also a tumor suppressor in animals.3 In our recent work, we have shown that in Arabidopsis thaliana RBR is essential for appropriate differentiation of all gametic and accessory cell types of the gametophytes (Fig. 1A).4 In our attempt to identify convergent mechanisms controlling cell differentiation, we observed that loss of RBR derepresses several genes encoding proteins of the chromatin-associated Polycomb Repressive Complex 2 (PRC2) in both gametophytes. For example, an Arabidopsis PRC2 histone methyltransferase, CURLY LeAF,5 which is a homologue of the Drosophila gene enhancer of zeste [E(z)] that has a canonical E2F binding motif in its promoter and a predicted RBR binding motif (LXCXD) within its protein sequence, was strongly upregulated in rbr male and female gametophytes. Similarly, the Arabidopsis METHYL TRANSFERASE 1 (MET1), an evolutionary homologue of the mammalian gene DNA Methyltransferase 1 (Dnmt1)6 and a direct target of the RBR/E2F/MSI1 (RbAP46/48) complex,4,7 is derepressed in rbr gametophytic cells. It must be noted that in the both cases described above, similar transcriptional repression was demonstrated in animal cells for the convergent homologues of E(z) and Dnmt1,8,9 however, if similar mechanism operates in animal gametes is unknown. Considering that the evolutionary homologues of pRb, E(z) and Dnmt1 play critical roles in cell differentiation,9,10 our results reiterate the synergism between the cell cycle and members of the chromatin-bound repressive complexes in gene silencing and cellular differentiation.

The interplay of key cell cycle regulators and chromatin associated proteins is expected to control the epigenetic differentiation of the endosperm from the fertilized central cells. Precocious activation of cell divisions in central cells is prevented until fertilization occurs, and presumably the chromatin structure is altered as part of the epigenetic reprogramming.4,11 Considering that rbr central cells initiate endosperm-like proliferation and heterochromatin status in the absence of fertilization similar to the maternal-effect mutants of the MEDEA (MEA)-PRC2 Fertilization-Independent-Seed (FIS) complex,2 we asked if the PRC2 members would cross-talk with RBR. Although we were unable to examine how RBR is regulated in the female tissues of the maternal fis mutants due to technical limitations, we observed that the maternal PRC2, and most likely a paternal PRC2 as well, target the paternal RBR allele in endosperm or pollen tissues. This particular finding of a regulatory circuit between RBR and PRC2 is novel and interesting. In mammals, cell cycle exit is controlled by pRb partly by regulating the PRC2 specific histone mark H3K27me3 on cell cycle genes.12 However, it remains to be demonstrated if the PRC2 and/or similar chromatin complexes would directly control pRb as well. It will therefore be intriguing to understand if parent-of-origin dependent gene targeting of pRb has operated in evolution.

Imprinting is a fascinating epigenetic process that evolved in mammals and plants for their placental behaviour.2 Although imprinting mechanisms are conserved in evolution, expression of sex-specific alleles in plants is established by differential erasure of silencing marks during gametophyte development. silencing marks can vary from DNA methylation, which is maintained by MET1 in the case of paternally imprinted FIS2 or FLOWERING LOCUS A (FWA), to histone methylation in case of MEA, or both DNA and histone methylation for PHERES1 (PHE1).2,13 It has also been shown that demethylation in central cells is achieved by a base-excision DNA repair mechanism involving DNA glycosylase, DEMETER (DME).2 DME erases the silencing marks from FIS2 and FWA locus by removing methylated cytosines, thus activating the corresponding maternal alleles in the central cells. While we could not obtain convincing evidence for the control of DME by RBR, our recent findings4 and the work of Julliene and co-workers7 suggest that erasure of the maternal FIS2 (and FWA) imprint mediated by MET1 could be directly controlled by a RBR (and MSI1) complex via RBR/E2F mediated direct repression of S-phase specific MET1 expression during each G₁-phase. Therefore, we propose that repression of MET1 activity by the RBR pathway likely initiates global DNA demethylation during gametogenesis, which is required for the activation of a subset of imprinted genes. While we are currently developing conditional rbr mutants to understand how RBR regulates PRC2, MET1 and ultimately the imprinting effects during endosperm development, our working hypothesis predicts that the maternal activity of PRC2 suppresses RBR in the endosperm tissues, whereas MET1-dependent expression of the imprinted FIS2, FWA and likely PHERES1 genes is maintained in a dynamic fashion. Together, our work provides the first insight that the evolutionarily ancient RBR, PRC2 and MET1 networks could predate the separation of animals and plants, and their regulatory interaction would play a significant role in mediating certain imprinting effects during gametic and post-fertilization differentiation events.

Abbreviations

RBR	=	retinoblastoma related
PRC2	=	polycomb repressive complex 2
MET1	=	DNA methyltransferase 1
FIS	=	fertilization-independent-seed

Figures and Tables

Figure 1 Epigenetic interaction between RBR and chromatin-associated regulatory proteins and complexes is critical for gametophyte development. (A) Sketch of fully differentiated Arabidopsis male (MG) and female (FG) gametophytes in the presence of RBR. Gametophytic cells such as sperm, egg and central cells are marked in blue, red and yellow, respectively. (B and C) Models illustrating the regulatory interactions of RBR, MET1, PRC2 and its targets. For simplicity, only the mature male (MG) and female gametophyte (MG) specific gene regulation prior to fertilization is shown. Post-fertilization interaction between RBR and the PRC2 genes in the embryo and endosperm could likely be similar.

Acknowledgements

The research was funded by the swiss National science Foundation grant to Wilhelm Gruissemm. Amal J. Johnston was supported by a post-doctoral fellowship from the Roche Research Foundation. We thank olga Kirioukhova for help with the figure and comments on the manuscript.

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