A role for CHROMOMETHYLASE3 in mediating transposon and euchromatin silencing during egg cell reprogramming in Arabidopsis

Abstract

During embryogenesis there is a major switch from dependence upon maternally-deposited products to reliance on products of the zygotic genome. In animals, this so-called maternal-to-zygotic transition occurs following a period of transcriptional quiescence. Recently, we have shown that the early embryo in Arabidopsis is also quiescent, a state inherited from the female gamete and linked to specific patterns of H3K9 dimethylation and TERMINAL FLOWER2 (TFL2) localization. We also demonstrated that CHROMOMETHYLASE 3 (CMT3) is required for H3K9 dimethylation in the egg cell and for normal embryogenesis during the first few divisions of the zygote. Subsequent analysis of CMT3 mutants points to a key role in egg cell reprogramming by controlling silencing in both transposon and euchromatic regions. A speculative model of the CMT3-induced egg cell silencing is presented here, based on these results and current data from the literature suggesting the potential involvement of small RNAs targeted to the egg cell, a process conceptually similar to the division of labor described in the male gametophyte for which we show that H3K9 modifications and TFL2 localization are reminiscent of the female gametophyte.

Figure 1

Transposon reactivation in cmt3-7 mature ovules. (A) Athila2 and Atlantys2 transcripts detection by RT-PCR in wild type (WT), heterozygous (cmt3/+) and homozygous (cmt3/cmt3) cmt3-7 mature ovules. (B) Histochemical detection of GUS reporter expression in mature ovules of the F1 progeny of cmt3-7 × ET10306 (enhancer-trap line monitoring Atlantys transcription) crosses. Bar: 10 µm.

Figure 2

Effect of CMT3 loss-of-function on TFL2 localization in the mature ovule. The F1 progeny of both WT (Ler) × pTFL2:TFL2-GFP [Col(0)] and cmt3-7 (Ler) × pTFL2:TFL2-GFP [Col(0)] crosses was observed and segregated as expected for TFL2 signal in the gametophyte and cmt3 phenotype. (A) Wild type (WT) and cmt3-7 mature gametophytes expressing a pTFL2:TFL2-GFP transgene monitoring LHP1/TFL2 distribution; projection of consecutive optical sections showing the nuclei of the embryo sac and the ovule integuments; ec, egg cell; cc, central cell; sy, synergids; Bar, 10 µm. (B) 3-D reconstruction of WT and cmt3-7 synergids, central cell and egg cell nuclei with statistic coloration corresponding to the intensity mean of each cell type; ec:, egg cell; cc, central cell; sy, synergids. (C) Quantification of the relative TFL2 fluorescence intensity (yellow bars) between the egg cell and central cell (EC/CC) in WT and cmt3-7 gametophytes. The relative intensity is expressed as the ratio EC/CC of the mean intensity per voxel. The error bars represent standard deviation. EC, egg cell; CC, central cell.

Figure 3

H3K9 acetylation/dimethylation and TFL2 localization in wild-type pollen. (A) Wild-type pollen grain stained with DAPI (marking chromatin, white) and H3K9ac (green). (B) Wild-type pollen grain stained with DAPI (white) and H3K9me2 (green). (C) Wild-type pollen grain expressing a pTFL2:TFL2-GFP transgene monitoring LHP1/TFL2 distribution. vcn, vegetative cell nucleus; gcn, generative cell nucleus; scn, sperm cells nuclei; Bar, 10 µm.

Figure 4

Speculative model of the CMT3-induced egg cell silencing in the female gametophyte. The small interfering RNAs (siRNAs) are produced in transcriptionally active cells (central cell, antipodal cells or somatic cells), then targeted to the egg cell, the other cells being eventually protected by enzymes such as DEMETER-LIKE (DML) in the central cell. Finally, CMT3 is recruited for non-CG methylation, further triggering H3K9 dimethylation and leading to transposon and gene silencing.