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. 2006 Apr;18(4):805–814. doi: 10.1105/tpc.105.038836

DNA Methylation Is Critical for Arabidopsis Embryogenesis and Seed Viability

Wenyan Xiao a, Kendra D Custard a, Roy C Brown b, Betty E Lemmon b, John J Harada c, Robert B Goldberg d, Robert L Fischer a,1
PMCID: PMC1425851  PMID: 16531498

Abstract

DNA methylation (5-methylcytosine) in mammalian genomes predominantly occurs at CpG dinucleotides, is maintained by DNA methyltransferase1 (Dnmt1), and is essential for embryo viability. The plant genome also has 5-methylcytosine at CpG dinucleotides, which is maintained by METHYLTRANSFERASE1 (MET1), a homolog of Dnmt1. In addition, plants have DNA methylation at CpNpG and CpNpN sites, maintained, in part, by the CHROMOMETHYLASE3 (CMT3) DNA methyltransferase. Here, we show that Arabidopsis thaliana embryos with loss-of-function mutations in MET1 and CMT3 develop improperly, display altered planes and numbers of cell division, and have reduced viability. Genes that specify embryo cell identity are misexpressed, and auxin hormone gradients are not properly formed in abnormal met1 embryos. Thus, DNA methylation is critical for the regulation of plant embryogenesis and for seed viability.

INTRODUCTION

Early embryogenesis in Arabidopsis thaliana is distinguished by a predictable pattern of cell divisions (Bowman and Mansfield, 1994). The zygote divides asymmetrically to give rise to a small apical cell and a large basal cell, which have distinct developmental fates (Figures 1A to 1F) (Goldberg et al., 1994; Scheres and Benfey, 1999). The apical cell develops into the embryo proper, whereas the basal cell elongates and divides transversely to generate the suspensor, an ephemeral organ that supports the development of the embryo proper (Berleth and Chatfield, 2002). The apical cell undergoes two rounds of longitudinal cell divisions (two- and four-cell stage) and one round of transverse divisions (eight-cell stage). Each of the eight cells derived from the apical cell of the octant embryo divides periclinally to produce a 16-cell embryo. During early embryogenesis, a shoot-root axis of polarity is fixed, shoot and root meristems are formed, cotyledon storage organs are generated, and tissue layers are specified. The embryo passes through a series of stages that are defined morphologically as globular, heart, torpedo, and walking stick stages (Goldberg et al., 1994; Jurgens, 2001). By contrast, the basal cell elongates and divides transversely to form a structure of seven to nine cells. The uppermost cell of the basal lineage, the hypophysis, becomes integrated into the embryo proper and becomes the quiescent center of the root meristem (Berleth and Jurgens, 1993; Hamann et al., 1999). The remaining cells in the basal lineage follow an extraembryonic cell fate and form the suspensor.

Figure 1.

Figure 1.

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met1-6 Mutation Affects Suspensor and Embryo Development.

(A) to (X) Nomarski photographs of wild-type and homozygous met1-6 mutant embryos at 1 to 6 DAP.

(Y) to (JJ) Histological section photographs of wild-type and met1-6 mutant embryos at 4 to 6 DAP.

Photographs of the wild type (A) to (F) at 1, 2, 3, 4, 5, and 6 DAP, respectively; the met1 mutant embryos at 1 DAP ([G], [M], and [S]), 2 DAP ([H], [N], and [T]), 3 DAP ([I], [O], and [U]), 4 DAP ([J], [P], and [V]), 5 DAP ([K], [Q], and [W]), and 6 DAP ([L], [R], and [X]). Histological sections of wild-type embryos (A) to (C) at 4, 5, and 6 DAP, respectively; the met1 mutant embryos at 4 DAP ([BB], [EE], and [HH]), 5 DAP ([CC], [FF], and [II]), and 6 DAP ([DD], [GG], and [JJ]). (A) to (D), (G) to (J), (M) to (P), (S) to (V), (Y), (BB), (CC), (EE), (HH), and (II) are the same scale, and (E), (F), (K), (L), (Q), (R), (W), (X), (Z), (AA), (DD), (FF), (GG), and (JJ) are the same scale. Bars = 20 μm in (A) and 50 μm in (E). Arrowheads indicate the plane of the first zygotic cell division ([A], [G], [M], and [S]), the boundary between the apical and basal lineage-derived cells ([B] to [L]), the hypophysis (Y), the apical cell nucleus (BB), or cell planes of the first two longitudinal cell divisions of the apical cell ([EE] and [HH]).

Early embryogenesis is regulated by transcription factors, signal transduction pathways mediated by kinases, and proteins that establish and maintain auxin hormone gradients (Willemsen and Scheres, 2004). For example, the YODA (YDA) gene, which encodes a mitogen-activated protein kinase kinase kinase, regulates embryo and suspensor cell identity. In yda mutant plants, the suspensor cells adopt an embryonic cell fate, divide longitudinally, and are integrated into the embryo proper instead of forming the suspensor (Lukowitz et al., 2004). WUSCHEL-related homeobox (WOX) transcription factor genes mark cell fate decisions during early embryogenesis. WOX2 and WOX8 are expressed in the egg cell and zygote, and their expression is limited to the apical and basal cell lineages, respectively. WOX2 is necessary for cell divisions that form the apical embryo domain. Auxin hormone gradients help form the embryonic apical-basal axis, the shoot and root meristems, and the cotyledon organs (Jenik and Barton, 2005; Friml et al., 2006). PIN-formed (PIN) genes encode transporter-like membrane proteins that are important for regulating auxin transport (Friml, 2003; Weijers et al., 2005). PIN proteins display asymmetric subcellular localization at the plasma membrane, regulate polar auxin transport, and establish auxin gradients during embryogenesis. Mutations in PIN1 and PIN7 disrupt the establishment of the embryonic apical-basal axis (Steinmann et al., 1999; Friml et al., 2003). PINOID (PID), encoding a Ser-Thr protein kinase (Christensen et al., 2000), regulates PIN localization and apical-basal axis formation (Friml et al., 2004). Overexpression of PID causes a basal-to-apical shift in PIN localization, resulting in the loss of auxin gradients and defects in embryogenesis.

DNA methyltransferases covalently methylate cytosine at the 5-position. DNA methylation is a heritable epigenetic process that regulates developmental processes in animals and plants (Martienssen and Colot, 2001; Reik et al., 2001; Li, 2002). DNA methylation plays an important role in genome stabilization, X chromosome inactivation, silencing of transposons and endogenous retrovirus, gene expression, and imprinting (Bird and Wolffe, 1999; Bestor, 2000; Reik and Walter, 2001; Bender, 2004; Gehring et al., 2004; Chan et al., 2005).

In mammals, 5-methylcytosine is at CpG dinucleotides. During gametogenesis and embryogenesis, DNA methylation is lost and subsequently reestablished by DNA methyltransferase3a (Dnmt3a) and Dnmt3b (Okano et al., 1999). The patterns of DNA methylation are maintained during somatic development by the Dnmt1 DNA methyltransferase (Li et al., 1992). DNA methylation is essential for mammalian embryonic development, and targeted mutations of the Dnmt1 or Dnmt3 genes result in embryonic lethality (Li et al., 1992; Okano et al., 1999). In Arabidopsis, CpG DNA methylation is maintained by METHYLTRANSFERASE1 (MET1), an ortholog of DNA methyltransferase Dnmt1 (Finnegan and Dennis, 1993; Finnegan and Kovac, 2000). In addition to CpG methylation, Arabidopsis has CpNpG and CpNpN methylation. CHROMOMETHYLASE3 (CMT3) and DOMAINS REARRANGED METHYLASE1 (DRM1) and DRM2 (a homolog of Dnmt3 for de novo DNA methylation) maintain patterns of CpNpG and CpNpN methylation (Henikoff and Comai, 1998; Bartee et al., 2001; Lindroth et al., 2001; Cao and Jacobsen, 2002a, 2002b).

Arabidopsis plants with an antisense MET1 transgene, partial-loss-of-function met1 mutations, or cmt3 drm1 drm2 mutations revealed that reduced DNA methylation results in abnormal postembryonic plant development (Finnegan et al., 1996; Kakutani et al., 1996, 2004; Ronemus et al., 1996; Cao, 2003; Kankel et al., 2003; Kato et al., 2003; Saze et al., 2003). Here, we show that a significant fraction of met1 and met1 cmt3 mutant embryos show reduced viability. met1 and met1 cmt3 embryos often have incorrect patterns of cell divisions, polarity, and auxin gradients and misexpress genes that specify embryo cell identity. Thus, DNA methylation is necessary for proper embryo development and viability in Arabidopsis.

RESULTS

Previously, we isolated four met1 mutant alleles (met1-5 to met1-8) (Xiao et al., 2003). The met1-6 allele is likely to be a null allele because, due to a premature translation stop codon, it encodes a truncated DNA methyltransferase that lacks a catalytic domain. The met1-6 mutation leads to late flowering (W. Xiao and R.L. Fischer, unpublished results), results in genomic hypomethylation, and reduces DNA methylation in the promoter of an imprinted gene, MEDEA (Xiao et al., 2003). Plants heterozygous for the met1-6 mutant allele, which were derived from mutagenized plants that were never homozygous for this mutation (Xiao et al., 2003), were used to generate homozygous met1-6 plants used in these studies. We found that siliques from homozygous met1-6 plants contained aborted seeds (12%) at ∼40-fold higher frequency than wild-type controls (0.3%) (Table 1). Observation of embryogenesis using cleared seeds and histological sections revealed that 33% of the met1-6 embryos developed abnormally (Table 1). As described below, these homozygous met1-6 mutant embryos displayed a wide range of developmental abnormalities that were consistently observed.

Table 1.

Effect of Mutations in DNA Methyltransferase Genes on Embryogenesis and Seed Viability

Genetic Cross
Abnormal F1 Embryosa
F1 Seed Abortion
Self-Pollinated Female Male % n % n
Wild type 0 967 0.3 678
met1-6/met1-6 33 568 12.2 986
cmt3-7/cmt3-7 3 578 0.5 875
MET1/met1-6 10 562 7.8 539
cmt3-7/cmt3-7 23 816 16.3 1096
MET1/met1-6
met1-6/met1-6 Wild type 16 550 7.2 690
Wild type met1-6/met1-6 8 822 1.2 982
MET1/met1-6 Wild type 8 420 2.2 683
Wild type MET1/met1-6 5 403 1.0 853
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a

Embryos at 1 to 6 d after pollination were examined by whole-mount seed clearing.

Asymmetric First Cell Division

Abnormalities in F2 homozygous met1 embryo development were apparent after the first zygotic division, 1 d after pollination (DAP). We detected met1 mutant zygotes (Figures 1G, 1M, and 1S) that divided more symmetrically than wild-type control embryos (Figure 1A). Approximately 13% (n = 266) of the embryos displayed basal cells that failed to elongate and undergo transverse divisions (Figures 1N and 1T). These results show that genome-wide changes in DNA methylation affect the earliest stages of embryogenesis in Arabidopsis.

Suspensor Development

In wild-type embryos, the basal cell elongates and divides transversely to form a suspensor with seven to nine cells (Figures 1B to 1E). In wild-type suspensors, longitudinal divisions do not occur (Figure 1Y). By contrast, longitudinal cell divisions in the basal cell lineage were detected in ∼27% (n = 266) of homozygous met1-6 embryos at 2 (Figures 1H and 1N) and 3 DAP (Figures 1I and 1O). In wild-type embryos, there is a clear boundary between the spherical proembryo and the linear file of suspensor cells (Figure 1C), and at 4 to 6 DAP, the hypophysis, the uppermost cell of the basal lineage, becomes prominent (Figures 1D to 1F). This clear demarcation between embryo and suspensor is often not detected in met1-6 mutant embryos because of the many longitudinal cell divisions in the suspensor (Figures 1J, 1K, and 1U). Thus, DNA methylation is necessary for proper development of the suspensor during Arabidopsis embryogenesis.

Embryo Development

In wild-type embryos, the apical cell undergoes two rounds of symmetrical divisions with the division planes perpendicular to each other to form a four-cell proembryo (Goldberg et al., 1994). In certain abnormal met1 embryos, the apical cell divided longitudinally, but the subsequent division was not in the correct orientation (Figures 1EE and 1HH). This sometimes generated asymmetric embryos with two cells on one side of the hypophysis and no cells on the other side (Figures 1DD and 1GG). Moreover, these embryos were significantly delayed in their development compared with wild-type control embryos. These results reveal that early planes of cell division in homozygous met1-6 mutant embryos are sometimes not properly specified.

Abnormalities in numbers and planes of cell division persisted throughout met1 embryogenesis. In wild-type Arabidopsis, at 4 to 6 DAP, the embryo passes through a series of stages that are defined morphologically as globular, heart, and torpedo (Figures 1D to 1F and 1AA). Among the abnormal met1 embryos, we observed abnormal embryos without a clear boundary between apical and basal cell lineages due to longitudinal cell divisions in the basal cells of suspensor (Figures 1J, 1K, 1P, and 1U) or embryos that did not display an apical-basal axis and whose basal cell lineage resembled the apical cell lineage (Figures 1Q and 1FF). We also observed abnormal structures in met1-6 embryos (Figures 1V and 1W).

At the transition and early heart stages of wild-type embryogenesis, two symmetrical cotyledons are initiated from lateral domains of the embryo, and an embryonic shoot apical meristem is differentiated from the medial domain between the two cotyledons (Figure 1F) (Berleth and Chatfield, 2002; Prigge et al., 2005). In homozygous met1-6 embryos, the embryo sometimes failed to differentiate two cotyledons (Figures 1L and 1JJ) or initiated three cotyledons (Figure 1R). Mutant embryos having one cotyledon also lacked a medial domain where the embryonic shoot meristem is generated. This phenotype was also observed in F2 homozygous met1-6 seedlings, which had a single cotyledon lacking the apical shoot meristem (Figures 2B and 2C), two abnormal cotyledons (Figure 2D), three cotyledons (Figures 2F and 2G), or four cotyledons (Figure 2H). Taken together, these results show that loss of DNA methylation alters the number and planes of cell division required for generating the embryo proper, apical-basal axis, cotyledons, and meristems.

Figure 2.

Figure 2.

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Effect of the met1-6 Mutation on Cotyledons and the Shoot Apical Meristem.

Seedlings were photographed at the same magnification at 5 d after germination. Bar = 2 mm.

(A) and (E) Wild-type seedlings.

(B) to (D) and (F) to (H) met1-6 seedlings.

Partial Dominant Effect of met1-6 Mutations on Embryogenesis

To examine whether a paternal or maternal hypomethylated genome can affect embryogenesis, we reciprocally crossed homozygous met1-6 plants with wild-type plants and examined embryogenesis within the F1 seeds. We found that either maternal- or paternal-derived hypomethylated genomes were sufficient to cause abnormal embryogenesis (Figure 3) and seed abortion (Table 1). Inheritance of a maternal hypomethylated genome resulted in 16% abnormal embryos, whereas 8% of embryos developed improperly when a paternal-derived genome was hypomethylated. These results suggest that hypomethylated genomes have a partial dominant effect on embryogenesis and seed abortion.

Figure 3.

Figure 3.

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A Hypomethylated Maternal or Paternal Genome Influences Embryo Development.

Histological sections of wild-type embryos and the F1 seeds of reciprocal crosses with wild-type and met1-6 plants at 3 to 6 DAP. Bars = 20 and 50 μm at 3 and 5 DAP, respectively.

DNA Hypomethylation during Gametogenesis Affects Embryogenesis

Plants heterozygous for met1 mutations produce gametes that are hypomethylated during meiosis (Kankel et al., 2003; Saze et al., 2003). To determine if loss of DNA methylation during gametogenesis is sufficient to influence embryo and seed development, we self-pollinated heterozygous met1-6/MET1 plants and analyzed the F1 seed. Approximately 10% of the F1 embryos displayed developmental abnormalities (e.g., unusual numbers and planes of cell division) similar to the embryos shown in Figure 1, and ∼8% of the F1 seed aborted (Table 1). To determine if the loss of DNA methylation in the female or male gametophyte is sufficient to perturb seed development, we performed reciprocal crosses with met1-6/MET1 heterozygotes and wild-type plants. When the maternal parent was heterozygous, ∼8% of F1 embryos displayed abnormalities in the number and planes of cell division, and 2% of the F1 embryos aborted (Table 1). When the paternal parent was heterozygous, the effect was diminished, with 5 and 1% of the F1 embryos showing developmental abnormalities and aborting, respectively (Table 1). In control crosses with wild-type plants, we did not detect any abnormal F1 embryos, and only 0.3% aborted (Table 1). These results show that loss of DNA methylation during female or male gametogenesis is sufficient to influence embryogenesis and seed viability.

Synergy between Mutations in the MET1 and CMT3 DNA Methyltransferase Genes

One hypothesis to explain how met1-6 plants produce viable seeds is that both MET1 and CMT3 are biologically redundant; mutating one methyltransferase does not cause 100% lethality because the other methyltransferase is still present. To test this hypothesis, we self-pollinated MET1/met1-6 cmt3-7/cmt3-7 plants and analyzed the F1 progeny. In these experiments, the parent plants were never homozygous for the met1-6 mutation, so that the CpG hypomethylation was initiated in the female and male gametophytes. We found that 23% of the F1 embryos developed abnormally compared with 10% of self-pollinated MET1/met1-6 and 4% of cmt3-7/cmt3-7 controls (Table 1). Based upon visual inspection of seeds, ∼16% of the F1 progeny from self-pollinated MET1/met1-6 cmt3-7/cmt3-7 plants had aborted (Table 1). We determined the genotype of 276 seedlings from a self-pollinated MET1/met1-6 cmt3-7/cmt3-7 plant. All progeny were homozygous for cmt3-7, 36% were homozygous for MET1, 60% were heterozygous for MET1/met1-6, and 4% were homozygous for met1-6. The 2:1 ratio of homozygous MET1 and heterozygous MET1/met1-6 progeny (χ2 = 2.8, P > 0.09) demonstrates Mendelian segregation of the met1-6 allele during meiosis and is consistent with most of the homozygous met1-6 seeds not producing viable seedlings. Rare met1-6 cmt3-7 homozygous plants showed a dramatic reduction in stature compared with met1-6 and cmt3-7 control plants (Figure 4) and were sterile (data not shown). Thus, reduction of both CpG and non-CpG DNA methylation caused by met1-6 and cmt3-7 mutations results in a synergistic decrease in seed viability and plant robustness.

Figure 4.

Figure 4.

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met1-6 and cmt3-7 Mutations Have a Synergistic Effect on Arabidopsis Growth and Development.

Representative 45-d-old plants were photographed. Bars = 2.5 cm in (A) to (D) and 0.4 cm in (E).

(A) Wild type.

(B) Homozygous cmt3-7.

(C) Homozygous met1-6.

(D) and (E) The same homozygous cmt3-7 met1-6 plant.

Specification of Cell Identity

Longitudinal cell divisions in the suspensor (Figures 1H, 1I, 1N, and 1O) suggest that the suspensor cells are adopting an embryonic fate (Lukowitz et al., 2004). To investigate whether DNA methylation might influence cell fate decision during early embryogenesis, we analyzed expression of genes important for cell fate specification. As shown in Figure 5, expression of YDA, a mitogen-activated protein kinase kinase kinase gene (Lukowitz et al., 2004), is elevated in homozygous met1-6 seeds at 4 DAP. By contrast, expression of the WOX2 and WOX8 homeodomain transcription factor genes is reduced in homozygous met1-6 seeds at 4 DAP. Thus, DNA methylation, either directly or indirectly, influences transcription of genes that regulate cell identity during early embryogenesis.

Figure 5.

Figure 5.

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Effect of the met1-6 Mutation on Expression of Genes That Regulate Embryo Cell Identity.

RNA was isolated from wild-type and homozygous met1-6 seeds. WOX2, WOX8, YDA, and ACTIN RNAs were amplified by RT-PCR as described in Methods.

Auxin Gradients

Abnormal met1-6 mutant embryos (Figure 1) resembled those with defects in establishing auxin gradients (Friml et al., 2003). To understand the relationship between DNA methylation and auxin gradients during embryogenesis, we compared DR5:green fluorescent protein (GFP) transgene expression in wild-type embryos and homozygous met1-6 aborted embryos. DR5:GFP is a synthetic auxin-responsive promoter (DR5) ligated to the GFP that has been used to reveal auxin gradients in the early stages of wild-type embryogenesis (Sabatini et al., 1999; Friml et al., 2002a, 2002b). As has been reported previously (Friml et al., 2003), DR5:GFP expression is primarily in the basal lineage cells 3 to 4 DAP, especially in the hypophysis and upper suspensor cells (Figure 6). By 5 to 6 DAP, maximum DR5:GFP activity is detected in the quiescent center of the root meristem, the future columella and its initials, and weak expression in the tips of the developing cotyledons and provascular strands (Figure 5). We found that auxin gradients, as revealed by the pattern of DR5:GFP promoter activity, were not properly specified in abnormal homozygous met1-6 embryos (Figure 6). DR5:GFP expression was relatively evenly distributed in cells derived from either the apical or basal cells in abnormal embryos at 4, 5, and 6 DAP. This result reveals that normal DNA methylation patterns, either directly or indirectly, are required for generating auxin gradients consistently during embryogenesis.

Figure 6.

Figure 6.

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Expression of the DR5:GFP Transgene in Abnormal met1-6 Mutant Embryos.

Micrographs of DR5:GFP transgene expression in wild-type and abnormal met1-6 embryos at 3, 4, 5, and 6 DAP. For each embryo, two micrographs were taken, one in bright field (top) and the other using epifluorescence with blue light excitation (bottom). Arrowheads point to the boundary between the apical and basal lineage-derived cells.

PIN1 encodes an auxin efflux carrier and is responsible for establishing auxin gradients in early embryogenesis (Friml, 2003; Weijers et al., 2005). To determine whether PIN1 promoter activity, either directly or indirectly, is affected by DNA methylation, we introduced the PIN1:GFP transgene into met1-6/MET1 heterozygous plants. As shown in Figure 7, in control wild-type plants at 3 and 4 DAP, the PIN1:GFP transgene was primarily expressed in the globular embryo proper, especially within the top half of the embryo proper (Friml et al., 2003). However, in abnormal met1-6 embryos, PIN1:GFP was expressed throughout the entire embryo proper and was evenly distributed in both apical- and basal-derived cells (Figure 7). This result suggests that DNA methylation, either directly or indirectly, regulates PIN1 gene expression that, in turn, is necessary for establishing auxin gradients.

Figure 7.

Figure 7.

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Expression of the PIN1:GFP Transgene in Abnormal met1-6 Mutant Embryos.

Micrographs of PIN1:GFP transgene expression in wild-type and abnormal met1-6 embryos at 3 and 4 DAP. For each embryo, two micrographs were taken, one in bright field (top) and the other using epifluorescence with blue light excitation (bottom). Arrowheads point to the boundary between the apical and basal lineage-derived cells taken in bright field, and the plane between the top half of the embryo proper where PIN1:GFP was mainly expressed and the bottom half in wild-type embryos.

DNA Methylation Status of Genes That Regulate Embryogenesis

DNA methylation usually represses gene expression (Bender, 2004). Embryogenesis may be affected in met1-6 mutants because genes are misexpressed or overexpressed due to the absence of repressive DNA methylation. Alternatively, embryogenesis in met1-6 mutants may be defective because of ectopic de novo methylation and gene silencing, a process that has been previously documented in DNA methylation mutant backgrounds (Chan et al., 2005).

To investigate whether DNA methylation could directly influence embryonic regulatory genes, we performed gel blot analyses on DNA isolated from wild-type or homozygous met1-6 seedlings. Genomic DNA was digested with endonucleases HpaII (CCGG) and HpyCH4 IV (ACGT), both of which are inhibited by the presence of 5-methylcytosine within their recognition sequences. Digested DNAs were blotted and hybridized to labeled probes complementary to the 5′-region, coding region, and 3′-region of PIN1 and YDA. These genes were examined because of their integral roles in embryogenesis and possibility of being directly regulated by MET1.

We detected no DNA methylation at PIN1. Both wild-type and met1 mutant DNA had the same size HpaII and HpyCH4 IV restriction fragments, indicating that 5-methylcytosine was not present at these restriction enzyme sites (data not shown). Thus, it is likely that MET1 indirectly affects the expression of the PIN1 gene. By contrast, we found the methylation status for the YDA gene was affected in the met1-6 mutant background. In the 5′-region, coding region, and 3′-region of YDA, HpaII and HpyCH4 IV sites were not digested in wild-type DNA, whereas these sites were digested in met1-6 DNA (Figure 8). This indicates that these sites are methylated in wild-type plants, and this methylation is dependent on MET1 activity. Thus, the met1-6 mutation directly affects DNA methylation at the YDA locus. This may account for the higher YDA expression detected in met1-6 mutant seeds (Figure 5).

Figure 8.

Figure 8.

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DNA Methylation of the YDA Gene.

Wild-type and met1-6 DNAs were digested with HpaII or HpyCH4 IV, blotted, and hybridized to probes that hybridize to the YDA 5′-region, gene, and 3′-region that were prepared as described in Methods.

(A) YDA 5′-region.

(B) YDA gene.

(C) YDA 3′-region

DISCUSSION

DNA Methylation Is Critical for Arabidopsis Embryo Development

Arabidopsis embryo development involves a predictable pattern of planes and numbers of cell division (Bowman and Mansfield, 1994). We found this pattern was not maintained in a significant fraction of embryos with mutations in the MET1 and CMT3 DNA methyltransferase genes (Table 1, Figure 1). We observed defects in the plane of the first asymmetric division that produces the apical and basal cell lineages, as well as those divisions in the embryo proper that form distinct cell layers and partition the globular stage embryo (Mayer et al., 1993). Later-stage embryos sometimes displayed massive cell proliferation of the basal cell lineage and thereby lost their apical-basal axis of polarity (Figure 1). This may be attributed to a failure of the embryo to suppress the embryonic potential of the suspensor, which has been previously observed (Yeung and Meinke, 1993; Yadegari et al., 1994). Many of the abnormal embryos likely abort their development, resulting in nonviable seed (Figure 1, Table 1).

Pleiotropic phenotypes were observed in both met1-6 and met1-6 cmt3-7 developing embryos. This fact makes it difficult to pinpoint the developmental processes and genes directly affected in these backgrounds. We were however able to determine that embryonic auxin gradients (Figure 6) and PIN1 promoter activity (Figure 7) were highly perturbed in abnormal met1-6 embryos. Mutants defective in auxin transport and signaling also exhibit pleiotropic phenotypes (Friml, 2003), some of which are similar to ones described here (Figure 1).

Mechanism of DNA Methylation in Regulating Embryogenesis

How does DNA hypomethylation affect embryogenesis and reduce seed viability in Arabidopsis? Loss of DNA methylation derepresses silenced transposons (Kakutani et al., 2004), and these could insert into genes necessary for early embryogenesis; however, the low frequency of transposition events in a single generation (Miura et al., 2001) cannot account for the high level of abnormal embryos in met1-6 and met1-6 cmt3-7 mutants (Table 1). Hypomethylation in general is more likely to cause phenotypic defects due to improper gene expression (Bender, 2004), such as the case of ectopic FWA expression and delayed flowering in met1 mutant backgrounds (Soppe et al., 2000). It is also possible that ectopic hypermethylation and gene silencing, a phenomenon that occurs at the SUPERMAN and AGAMOUS loci in methylation mutants (Jacobsen et al., 2000), may be responsible for some met1-6 embryonic phenotypes. Thus, met1-6 embryogenesis may be perturbed because hypomethylation and ectopic hypermethylation cause changes in gene transcription.

We found subtle yet reproducible differences in the mRNA levels of WOX2, WOX8, and YDA between wild-type and met1-6 developing seeds (Figure 5). We also found that PIN1:GFP is improperly expressed in abnormal met1-6 embryos (Figure 7). If DNA methylation directly affects the establishment of auxin gradients, it is likely not through the regulation of PIN1 gene transcription, as there was no DNA methylation detected at the HpyCH4 IV and HpaII sites of the PIN1 gene in wild-type or met1-6 plants. In contrast with PIN1, we found that the YDA locus was methylated in a wild-type genome and that this methylation was dependent on MET1 (Figure 8). This loss of DNA methylation correlates with an increase in YDA expression in met1-6 developing seeds (Figure 5). Whether this methylation directly influences YDA expression is unknown. YDA regulates extraembryonic cell fates in the basal cells (Lukowitz et al., 2004). It is possible that some of the met1-6 embryonic phenotypes are attributable to ectopic expression of YDA. However, we do not know the number or identity of all the genes that are directly regulated by DNA methylation during embryogenesis. It is likely that they encode both regulatory proteins and enzymes involved in cell metabolism.

Parent-of-Origin Effects of DNA Hypomethylation on Embryogenesis, Viability, and Seed Size

Reciprocal crosses between a wild-type parent and a hypomethylated parent due to expression of an antisense MET1 transgene result in F1 seeds with altered embryo and endosperm size (Adams et al., 2000). Inheritance of a hypomethylated maternal genome produced larger embryo and endosperm, whereas inheritance of a hypomethylated paternal genome produced smaller embryo and endosperm. It is thought that hypomethylation of one parental genome allows for expression of normally silenced, imprinted alleles that influence seed size (Adams et al., 2000). We observed parent-of-origin effects on seed size in progeny from reciprocal crosses between wild-type and homozygous met1-6 plants (W. Xiao and R.L. Fischer, unpublished results) similar to those observed with MET1 antisense plants (Adams et al., 2000). Reciprocal crosses between homozygous met1-6 and wild-type parents also produced a significant fraction of F1 aborted embryos (Figure 3, Table 1). Thus, a hypomethylated maternal or paternal genome is sufficient to have an adverse influence on embryogenesis. This partial dominant effect is probably due to the fact that regions that have lost their DNA methylation due to mutations in DNA methyltransferases are very inefficiently remethylated (Chan et al., 2004), allowing the hypomethylated state of the maternal- or paternal-derived genome to persist in the F1 heterozygous progeny.

Reciprocal crosses with met1 and wild-type plants revealed a higher percentage of abnormal F1 embryos among progeny with a hypomethylated maternal genome than those with a paternal hypomethylated genome (Table 1). These results suggest that embryogenesis is particularly sensitive to hypomethylation of the maternally derived genome and support the hypothesis that the maternal genome plays the predominant role in controlling early seed development (Vielle-Calzada et al., 2000).

Redundancy of DNA Methylation in the Plant Genome

We found that mutations in both the MET1 and CMT3 genes had a much more dramatic effect on embryogenesis, seed viability (Table 1), and plant development (Figure 4) compared with mutations in just one of these genes. This suggests that the level of DNA methylation must be reduced below a critical threshold level before its role in seed viability and plant development is evident. In mammals, 5-methylcytosine is mainly present at a single sequence context, CpG dinucleotides, that is maintained by the DNA methyltransferase Dnmt1. Loss-of-function mutations in the Dnmt1 gene remove most DNA methylation and result in embryo lethality (Li et al., 1992). By contrast, plant genomes have 5-methylcytosine in multiple sequence contexts (CpG, CpNpG, and CpNpN) that is maintained by multiple DNA methyltransferases (Bender, 2004; Chan et al., 2005). Our results suggest that the plant genome is epigenetically modified by MET1 and CMT3 in a partially redundant fashion.

METHODS

Plant Materials and Growth Conditions

Arabidopsis thaliana plants were grown in greenhouses under continuous light at 23°C. cmt3-7 mutant plants were crossed with MET1/met1-6 heterozygous plants, then MET1/met1-6 CMT3/cmt3-7 plants were selected in the F1 progeny, and MET1/met1-6 cmt3-7/cmt3-7 plants were obtained in the F2 progeny. Genotyping plants for met1-6 and cmt3-7 was performed as described (Lindroth et al., 2001; Xiao et al., 2003).

Whole-Mount Seed Clearing, Histology, and Microscopy

Whole-mount immature seeds 1 to 6 DAP were cleared in Hoyer's fluid (70% chloral hydrate, 4% glycerol, and 5% gum arabic) and observed with a Zeiss Axioskop 50 microscope using Nomarski optics. Thin section studies of seeds were performed using methods as described (Brown et al., 1999). Briefly, seeds were fixed in 4% glutaraldehyde in 0.1 M phosphate buffer, pH 6.9, postfixed in osmium ferricyanide, dehydrated through a graded acetone series, and infiltrated with Spurr's resin (EMS). Ovules were sectioned sagittally in the plane of the micropyle and stalk with an LKB historange microtome equipped with glass knives. The 2.0- to 5.0-μm sections were mounted on glass slides (Brown and Lemmon, 1995) and stained with polychrome stain (Fox, 1997). GFP fluorescence microscopy was conducted as described (Yadegari et al., 2000).

RT-PCR Analysis

RT-PCR analysis was performed as described (Kinoshita et al., 1999). Total RNA was isolated from wild-type and met1-6 mutant seeds at 4 DAP. Primers used in the experiment were as follows: for WOX2, WOX2F (5′-CGTTTCTTCTACCCCCCTCC-3′) and WOX2R (5′-ATCACGGAGGGCAAATCTGT-3′); for WOX8, WOX8F (5′-CCTATCATCTTCCTTTTCCTCA-3′) and WOX8R (5′-TTGTGATGAACACGAAGCTTG-3′); for YDA, YDA-F (5′-ATACCGGTGCTGAGCCTGAT-3′) and YDA-R (5′-GTCCAGATCCAAGCAAGGAA-3′). All primer pairs spanned intron sequences so that amplification of RNA could be distinguished from amplification of any contaminating DNA.

DNA Gel Blot Analysis

Genomic DNA was isolated from 10-d-old wild-type (Columbia gl1) and met1-6 seedlings grown in culture (Tai and Tanksley, 1990). DNAs were cleaved by methylation-sensitive restriction endonucleases HpaII and HpyCH4 IV for 4 h at 37°C, run on 1.2% agarose gels, and blotted to a positively charged nylon membrane (Amersham Pharmacia Biotech). Membranes were hybridized with probes randomly labeled using Prime-It II (random primer labeling kit) from Stratagene. Primers used to amplify DNA for radioactive labeling were as follows: for the PIN1 promoter region, PIN1m_F1 (5′-CAAGGCGGCACGAATTTTAGT-3′) and PIN1m_R4 (5′-ATAGCTACGTATAACGGAACC-3′); for the PIN1 gene, PIN1m_F4 (5′-CGAGCGATTTTGTTAACTAGTG-3′) and PIN1m_R2 (5′-TGAAGGAAATGAGGGACCAG-3′); for the PIN1 3′-intergenic region, PIN1m3-F1 (5′-GAGATATTACCAAAACACAGGG-3′) and PIN1m3-R4 (5′-AAGAATCGGTAAAAGGATACAC-3′); for the YDA promoter region, pYDA-f1 (5′-TTTTTCACTTTTTAAATATTTTGC-3′) and pYDA-r (5′-GATCTTCTTCCCACAAACCA-3′); for the YDA gene, YDAm-F1 (5′-ATGCCTTGGTGGAGTAAATCA-3′) and YDAm-R1 (5′-GGGTCCTCTGTTTGTTGATC-3′); for the YDA 3′-intergenic region, YDAm-F2 (5′-CCCGTTCGAGTCAAATGATTC-3′) and YDAm-R2 (5′-GTTGTTCTCACTTGCTCGATT-3′).

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers AT1G73590 (PIN1) and AT1G63700 (YDA).

Acknowledgments

We thank J. Penterman for stimulating discussions and for help with the preparation of this manuscript. We also thank M. Gehring, T.-F. Hsieh, J.H. Huh, and D. Michaeli for critically reading this manuscript and J. Friml and the ABRC (Ohio State University, Columbus, OH) for providing the DR5:GFP and PIN1:GFP transgenic seeds. This work was supported by National Institutes of Health Grant GM069415 and USDA Grant 2005-02355 to R.L.F.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Robert L. Fischer (rfischer@berkeley.edu).

Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.105.038836.

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