Maintenance of DNA methylation during the Arabidopsis life cycle is essential for parental imprinting

Abstract

Imprinted genes are expressed predominantly from either their paternal or their maternal allele. To date, all imprinted genes identified in plants are expressed in the endosperm. In Arabidopsis thaliana, maternal imprinting has been clearly demonstrated for the Polycomb group gene MEDEA (MEA) and for FWA. Direct repeats upstream of FWA are subject to DNA methylation. However, it is still not clear to what extent similar cis-acting elements may be part of a conserved molecular mechanism controlling maternally imprinted genes. In this work, we show that the Polycomb group gene FERTILIZATION-INDEPENDENT SEED2 (FIS2) is imprinted. Maintenance of FIS2 imprinting depends on DNA methylation, whereas loss of DNA methylation does not affect MEA imprinting. DNA methylation targets a small region upstream of FIS2 distinct from the target of DNA methylation associated with FWA. We show that FWA and FIS2 imprinting requires the maintenance of DNA methylation throughout the plant life cycle, including male gametogenesis and endosperm development. Our data thus demonstrate that parental genomic imprinting in plants depends on diverse cis-elements and mechanisms dependent or independent of DNA methylation. We propose that imprinting has evolved under constraints linked to the evolution of plant reproduction and not by the selection of a specific molecular mechanism.

Figure 1.

FIS2 Imprinting Is Controlled by DNA Methylation. (A) Allele-specific RT-PCR on RNAs extracted from siliques obtained at 0.5, 1.5, and 3 DAP after crosses were made between wild-type Col and wild-type C24. A 180-bp deletion in the FIS2 coding sequence of the wild-type accession Col allows the distinction of the Col FIS2 transcript from the FIS2 transcript in accession C24. Only the FIS2 maternal allele is expressed in developing seeds. By contrast, MINI3 is expressed from both parental alleles. (B) Allele-specific RT-PCR on RNAs extracted from siliques obtained at 4 DAP after crosses between wild-type ovules and pollen from met1-3/met1-3 plants (Col) show reduced activity of the maintenance DNA MET1 during the entire life cycle. In contrast with wild-type crosses, the FIS2 paternal allele is expressed in developing seeds, which inherit a paternal allele from met1-3/met1-3 plants. (C) Allele-specific RT-PCR on RNAs extracted from siliques obtained at 5 DAP after reciprocal crosses between wild-type plants and MET1as plants. The FIS2 paternal allele is expressed in developing seeds when pollen is provided by MET1as plants, which show reduced activity of the maintenance DNA MET1 during the vegetative developmental phase (Finnegan et al., 1996). (D) Allele-specific RT-PCR on RNAs extracted from siliques obtained at 5 DAP after crosses made between wild-type plants and different mutants affected for histone methylation or for DNA methylation. A 180-bp deletion in the FIS2 coding sequence of the wild-type accession Col allows the distinction of the Col FIS2 transcript from the FIS2 transcript in accessions C24, Landsberg erecta (Ler), and Wassilewskija. C24 is presented as a control. Only the maternal allele of FIS2 is expressed in seeds resulting from reciprocal crosses between the wild type and homozygous mutants for other pathways controlling DNA methylation in Arabidopsis cmt3 (Lindroth et al., 2001) and drm1 and drm2 (Cao and Jacobsen, 2002a). KYP methylates Histone3 Lys-9 residues (Jackson et al., 2002; Tariq et al., 2003), whereas the Polycomb group complex containing FIE methylates Histone3 Lys-27 residues (Bastow et al., 2004). The paternal (p) and maternal (m) alleles are indicated. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. No-RT control assays were performed, but the data are not shown.

Figure 2.

MET1 Targets a CpG-Enriched Region at the FIS2 Locus. (A) RT-PCR analysis shows that imprinted genes FWA and FIS2 are not expressed in vegetative tissues of the wild-type accession Col. Reduced methylation of DNA in null met1-3 homozygous mutant plants causes ectopic expression of FWA but not of FIS2 in leaves. met1-3/+ plants, which show no ectopic expression of FWA, were used in the experiments presented in Figure 5A. GAPDH was used as a loading control. (B) Analysis of DNA methylation using the methylation-sensitive restriction enzyme McrBC in wild-type Col leaves identifies a methylated region spanning −3104 to −1867 in the 5′ region of FIS2. The sensitivity to McrBC is decreased in the met1/met1 background, which indicates that MET1 controls DNA methylation in the −3104 to −1867 region. FWA repeats, which are methylated by MET1, were used as controls (Kinoshita et al., 2004). (C) Bisulfite sequencing of the FIS2 5′ region, which contains a 200-bp domain enriched in methylated cytosine residues predominantly on CpGs (see Supplemental Figure 2 online for the full sequence). Analysis was done with genomic DNA from rosette leaves. The structure of the FIS2 genomic sequence is depicted, with the start codon indicated by 0. Exons are indicated by black boxes, and the 5′ and 3′ untranslated regions are indicated by white boxes. The extent of the partial translational fusion FIS2-GUS is indicated below the schematic genomic structure.

Figure 3.

Maternal Control of FIS2 Expression by DME. (A) RT-PCR analyses performed on mRNA from buds (stages 11 and 12) and flowers (stage 13) (Smyth et al., 1990) show a lack of FWA expression and reduced FIS2 expression in the dme-4 homozygous mutant compared with the wild-type C24 accession. GAPDH was used as a control. (B) to (E) Effects of dme on FIS2-GUS expression. (B) Percentage of ovules expressing the FIS2-GUS reporter construct in dme-2/+ and dme-4/+ before pollination (BP) and at 2 DAP. Three different classes are observed: no expression (white), little expression (light blue), and wild-type expression (dark blue). Error bars correspond to the sd associated with each set of measurements. (C) to (E) Micrographs showing the three categories of FIS2-GUS expression in the dme-4/+ mutant background: no expression (white; [C]), little expression (light blue; [D]), and wild-type expression (dark blue; [E]). Bars = 20 μm.

Figure 4.

Maintenance of FIS2 and FWA Silencing during Male Gametogenesis. (A) During pollen development in met1-3/+ plants, half of haploid microspores produced by meiosis inherit the met1-3 mutation, and the other half inherit the wild-type MET1 allele. All microspores inherit a methylated copy of the genome (gray rectangles). DNA replication in met1-3 microspores produces hemimethylated DNA from the methylated template (gray/white rectangles). Hemimethylated DNA is inherited after mitosis by the vegetative nucleus (V) and the nucleus of the generative cell (G), which divides a second time and produces the two sperm cells (S). After this division, one sperm cell inherits a fully demethylated DNA copy (white rectangles) and the other sperm cell inherits hemimethylated DNA. The vegetative nucleus does not participate in fertilization. During sperm cell maturation, DNA replicates (Durbarry et al., 2005), and at fertilization, all sperm from met1-3 microspores is expected to contain at least one hemimethylated copy. (B) Segregating population of pollen grains from FWA-GFP/FWA-GFP, met1-3/+ plants (confocal section). The inset represents a confocal section of a pollen grain expressing FWA-GFP in the vegetative cell (V) and in the two sperm cells (s). Bar = 5 μm. (C) Ectopic expression of FWA and FIS2 observed by RT-PCR in RNA purified from stamens. GAPDH was used as a control to assess equal amounts of RNA loaded. DUO1 is expressed specifically in pollen (Rotman et al., 2005) and was used as a quality control for pollen mRNA extraction.

Figure 5.

Imprinting of FWA and FIS2 in Endosperm Relies on the Maintenance of Their Silencing during Male Gametogenesis. The imprinting status of FWA, MEA, and FIS2 was analyzed in developing seeds resulting from crosses between wild-type ovules and pollen from met1-3/+ plants. (A) Allele-specific RT-PCR detecting expression of the paternal and maternal alleles of FWA in seeds resulting from crosses between wild-type ovules (Ler accession) and pollen from met1-3/+ plants. (For the Col accession, two bands were obtained because digestion of the restriction polymorphism was partial; the lowest band was Col-specific [3 DAP].) We used met1-3/+ plants, which did not show expression of FWA in vegetative tissues (see Figure 2A). Although crosses between wild-type Ler and Col showed only expression of the maternal FWA allele, the FWA paternal allele was expressed in crosses with met1-3/+ pollen. (B) and (C) Accordingly, at 2 DAP, the FWA-GFP reporter was expressed paternally in endosperm when provided by pollen from met1-3/+ plants (C) but not from wild-type plants (B). (D) Allele-specific RT-PCR performed on crosses between wild-type accessions RLD and Col detected expression of the maternal MEA allele, typical of MEA imprinting (1 DAP). MEA imprinting was not altered if met1-3/+ plants provided the MEA paternal allele. (E) Crosses between wild-type accessions C24 and Col showed expression only from the maternal FIS2 allele typical of FIS2 imprinted status in wild-type endosperm. By contrast, allele-specific RT-PCR showed expression of the paternal and maternal alleles of FIS2 in seeds resulting from crosses between ovules from wild-type C24 and pollen from met1-3/+ plants (Col accession; 3 DAP). (F) to (J) Rescue of endosperm development upon removal of the FIS2 paternal allele imprint in met1-3 pollen. (F) and (H) GFP fluorescence of the enhancer trap marker KS117 was increased uniformly in endosperm of the fis2 homozygous mutant (F) compared with the wild type (H), in which it was restricted to the endosperm posterior pole at 5 DAP. (G) After pollination of fis2/fis2, KS117/KS117 plants by pollen from met1-3/+ plants, half of the seeds showed a pattern of KS117 expression similar to the KS117 pattern in wild-type endosperm (asterisks). Such seeds also showed a rescue of cytological alteration in fis2 endosperm ([I] and [J]). (I) The phenotype conferred by fis2 is characterized by the absence of cellularization of the syncytial endosperm and the enlargement of the posterior pole opposite the embryo (Guitton et al., 2004). (J) Rescue of such phenotypic traits was obtained in 19% of seeds resulting from crosses between fis2/fis2 plants and met1-3/+ pollen. Bars = 50 μm ([B], [C], [I], and [J]) and 200 μm ([F] to [H]). GAPDH was used as a control for the RT-PCR analyses ([A], [D], and [E]).

Figure 6.

Maintenance of FWA Imprinting in Endosperm Depends on MET1. (A) At 2 DAP, after fertilization of wild-type ovules with pollen from met1-3/+, FWA-GFP/FWA-GFP plants, expression of the paternally provided allele of FWA-GFP is observed in endosperm in half of the seeds. (B) and (C) The intensity of FWA-GFP expression decreases during endosperm development from 2 DAP (B) to 4 DAP (C). (D) In contrast with the gradual silencing of FWA-GFP paternal expression in met1-3/+ endosperm, one-quarter of the seeds show expression of the FWA-GFP paternal allele in crosses between ovules from met1-3/+ plants and pollen carrying met1-3/+ and FWA-GFP/FWA-GFP. (E) and (F) One-quarter of the seeds express low levels of FWA-GFP (E) and are presumed to be heterozygous for met1-3. Another one-quarter of the seeds show higher sustained expression of FWA-GFP (F) and are presumably homozygous for met1-3 in endosperm. Bars = 80 μm ([B], [C], [E], and [F]). The number of seeds observed for each cross is indicated above each bar. sd is indicated for each measurement. HO, homozygous.

Figure 7.

Model for the Dual Control of Parental Genomic Imprinting by DNA Methylation in Plants. The genes FIS2 and FWA are imprinted in endosperm and silenced by the continuous action of MET1, which targets methylation (pink circles) to an element in the promoter specific for each gene. MET1-dependent silencing of FIS2 and FWA is required during the vegetative phase, male gametogenesis, and endosperm development, when it maintains silencing of the paternal (p) allele. FIS2 and FWA expression is activated during female gametogenesis by the DNA glycosylase DME, leading to the expression of their maternal (m) allele in endosperm after fertilization. In endosperm, the paternal copy remains silenced through the continuous action of MET1, whereas the maternal copy is active, leading to an imprinted expression.