Embryo and endosperm inherit distinct chromatin and transcriptional states from the female gametes in Arabidopsis

Abstract

Whether deposited maternal products are important during early seed development in flowering plants remains controversial. Here, we show that RNA interference-mediated downregulation of transcription is deleterious to endosperm development but does not block zygotic divisions. Furthermore, we show that RNA POLYMERASE II is less active in the embryo than in the endosperm. This dimorphic pattern is established late during female gametogenesis and is inherited by the two products of fertilization. This juxtaposition of distinct transcriptional activities correlates with differential patterns of histone H3 lysine 9 dimethylation, LIKE HETEROCHROMATIN PROTEIN1 localization, and Histone H2B turnover in the egg cell versus the central cell. Thus, distinct epigenetic and transcriptional patterns in the embryo and endosperm are already established in their gametic progenitors. We further demonstrate that the non-CG DNA methyltransferase CHROMOMETHYLASE3 (CMT3) and DEMETER-LIKE DNA glycosylases are required for the correct distribution of H3K9 dimethylation in the egg and central cells, respectively, and that plants defective for CMT3 activity show abnormal embryo development. Our results provide evidence that cell-specific mechanisms lead to the differentiation of epigenetically distinct female gametes in Arabidopsis thaliana. They also suggest that the establishment of a quiescent state in the zygote may play a role in the reprogramming of the young plant embryo.

Figure 1.

Transcriptional Requirements in Embryo and Endosperm. (A) Whole-mount clearing of a wild-type seed at the one-cell embryo stage. Emb, embryo; End, endosperm. (B) Whole-mount clearing of developmental abnormalities induced by downregulating POLII in pNG-POLII RNAi lines at the one-cell (top) or 4/8-cell (center) embryo stages with arrested central cell development and seed at 2 DAP with a slow-growing endosperm (bottom). (C) Distribution of embryo and endosperm developmental stages in RNAi lines with arrested central cell development and wild-type seeds 2 to 4 DAP. (D) Whole-mount seeds counterstained with DAPI (marking chromatin) after immunostaining of the active form of RNA POLII (H5 antibody) or RNA POLII independently of its transcriptional engagement (4H8 antibody), as indicated. Projections of consecutive sections are shown. The seeds are at the zygotic stage, and the endosperm has undergone two cleavages. The box corresponds to the close-up. The arrow indicates the zygote nucleus; endosperm nuclei are indicated by stars. Bars = 10 μm. [See online article for color version of this figure.]

Figure 2.

Patterns of H3K9me2 in the Early Seed. (A) Wild-type (Columbia-0 ecotype) early seed following the first division of the primary endosperm nucleus; strong and well-defined H3K9me2 foci are visible in the zygote, while dispersed foci distributed in a polar fashion are detected in endosperm nuclei. Top: DAPI staining (white) of the whole early seed. Bottom: close-up (indicated by the yellow rectangle) showing overlay of DAPI (blue) and H3K9me2 (green). Zyg, zygote; End, endosperm. (B) Additional example, early seed with primary endosperm nucleus. DAPI, blue; H3K9me2, green. (C) Seed after three rounds of divisions in the endosperm showing well-defined chromocentric H3K9me2 foci in the endosperm, while the distribution in the zygote remains similar to the previous stage (A). DAPI, white; overlay of DAPI, blue; H3K9me2 signals, green. Endosperm nuclei are indicated by stars. (D) Somatic cells from ovule integuments showing well-defined H3K9me2 foci corresponding to the heterochromatic chromocenters. DAPI, white, left; H3K9me2 signals, green, right. All images are projections of consecutive optical sections. Bars = 10 μm.

Figure 3.

POLII Activity and H3K9 Dimethylation in the Ovule. (A) Ovule following polar nuclei fusion. Top: DAPI staining of the whole ovule. Bottom: Close-up showing overlay of DAPI (blue) and H3K9me2 (green) and projection of consecutive optical sections. EC, egg cell; CC, central cell. (B) Ovule at maturity prior to fertilization, overlay of DAPI (blue) and H3K9me2 (green), and projection of consecutive optical sections. (C) Quantification of the relative H3K9me2 fluorescence intensity (green bars) between the egg cell and central cell (EC/CC) at two developmental stages (25 each) as shown in (A) and (B), respectively. The relative intensity is expressed as the ratio EC/CC of the mean intensity per pixel. Quantification of DAPI signals (blue bar) controls the accuracy of the measurements: despite differences between the egg and central cells in nuclear size, ploidy level and DNA compaction, the DAPI signal EC/CC ratio is equal to 1. The error bars represent sd. (D) Mature ovule prior to fertilization stained with DAPI (white) and H5 (green) showing different levels of active POLII in the egg and central cell nuclei; projection of consecutive optical sections. (E) Immature ovule prior to gametophyte cellularization showing comparable H3K9me2 distribution in all gametophytic nuclei; single optical section of the whole ovule stained with DAPI (left) reveals three gametophyte nuclei. Overlay of DAPI (blue) and H3K9me2 (green) signals (right) following projection of consecutive optical sections shows six out of the eight gametophytic nuclei. Bars = 10 μm.

Figure 4.

TFL2 and Histone H2B Distribution in the Ovule. (A) and (B) GFP or YFP signals are displayed as green, and chlorophyll autofluorescence is displayed in red. ANT, antipodal cells; EC, egg cell; PN, polar nuclei; CC, central cell; SYN, synergids. Bars = 10 μm. (A) Mature ovule prior to fertilization expressing a pTFL2:TFL2-GFP transgene monitoring LHP1 distribution. Left: projection of consecutive optical sections showing the nuclei of the embryo sac and the ovule integuments. Right: Close-up of a three-dimensional reconstruction of central cell and egg cell nuclei, as used for quantification of fluorescence intensity signals (see Methods) shown in the inset (n = 20 ovules). The error bars represent sd. (B) Immature (left) and mature (right) ovules after cellularization of the female gametophyte; projection of consecutive optical sections of a transgenic line expressing a pAKV:H2B-YFP transgene monitoring H2B dynamics.

Figure 5.

Effect of CMT3 and DML Loss of Function on H3K9me2 in the Ovule. (A) to (C) Ovules after fusion of the polar nuclei, as in Figure 3A, counterstained with DAPI (white). Left: Single optical sections; right: close-ups showing DAPI (blue) and H3K9me2 (green) overlays; projections of consecutive optical sections. Patterns shown below were observed in at least 30 ovules of each wild-type or mutant line. Two examples for each line are shown. EC, egg cell; CC, central cell. Bars = 10 μm. (A) Wild-type ovule. (B) dml1 dml2 dml3 mutant ovule showing an egg cell-like pattern of H3K9me2 in the central cell nucleus. (C) cmt3 mutant ovule showing altered H3K9me2 signal specifically in the egg cell nucleus.

Figure 6.

Phenotypic Characterization of cmt3-Defective Plants. Whole-mount clearing of wild-type (Ler) and homozygous mutant (cmt3^−/−) seeds obtained from self-pollinated plants. All images are at the same magnification. (A) cmt3 embryos display a significant proportion of abnormal divisions (P < 0.01, t test) observed from the two- to four-cell to the 32-cell stages, prominently at the transition zone between the suspensor and the embryo (arrow). The frequency of wild-type and defective embryos is indicated as a percentage of total seed counts at the bottom (Ler self, n = 451, and cmt3^−/− self, n = 216). (B) At the globular stage, cmt3 embryos recover a wild-type phenotype, similar to Ler. Bar = 10 μm.