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First published online January 16, 2004; 10.1105/tpc.017780 © 2004 American Society of Plant Biologists Extensive Maternal DNA Hypomethylation in the Endosperm of Zea mays
a Plant Biochemistry Laboratory, Department of Plant Biology, Royal Veterinary and Agricultural University, DK-1871 Frederiksberg C, Denmark 2 To whom correspondence should be addressed. E-mail gel{at}kvl.dk; fax 45-35283333.
A PCR-based genomic scan has been undertaken to estimate the extent and ratio of maternally versus paternally methylated DNA regions in endosperm, embryo, and leaf of Zea mays (maize). Analysis of several inbred lines and their reciprocal crosses identified a large number of conserved, differentially methylated DNA regions (DMRs) that were specific to the endosperm. DMRs were hypomethylated at specific methylation-sensitive restriction sites upon maternal transmission, whereas upon paternal transmission, the methylation levels were similar to those observed in embryo and leaf. Maternal hypomethylation was extensive and offers a likely explanation for the 13% reduction in methyl-cytosine content of the endosperm compared with leaf tissue. DMRs showed identity to expressed genic regions, were observed early after fertilization, and maintained at a later stage of endosperm development. The implications of extensive maternal hypomethylation with respect to endosperm development and epigenetic reprogramming will be discussed.
Genomic DNA of many eukaryotes can be modified by the covalent attachment of a methyl group to the 5-carbon position of cytosine residues. In plants, DNA methylation is mainly restricted to the symmetrical sequences 5'-CpG-3' or 5'-CpNpG-3' but also can occur in a nonsymmetrical context (Gruenbaum et al., 1981  ; Meyer et al., 1994). It is widely recognized that DNA methylation has the ability to influence gene activity mainly by repressing gene expression (reviewed in Bird and Wolffe, 1999). Methylation can act directly by blocking or reducing the binding of transcription factors to target DNA sequences (Prendergast and Ziff, 1991) or indirectly by inducing a condensed chromatin structure (Kass et al., 1997). In the latter case, repression is mediated by proteins that specifically bind to methylated residues, which in turn recruit chromatin-remodeling factors (Jones et al., 1998; Nan et al., 1998). Both mechanisms are likely to occur in plants because methylation affects the accessibility of several plant proteins to their target sequences (Gierl et al., 1988; Stäiger et al., 1989; Inamdar et al., 1991; Sturaro and Viotti, 2001), and proteins with affinity for methylated sequences also have been isolated (Zhang et al., 1989; Ehrlich, 1993).
Imprinting refers to the differential expression of a gene depending on the sex of the parent that transmits it. The regulatory mechanism of imprinted gene expression implies that a cell can discriminate between genetically identical alleles and determine which is to be transcribed. Therefore, it is presumed that parental alleles are differentially marked by an epigenetic imprint, which can modulate gene activity without imposing an irreversible change on nucleotide sequence. The finding that the vast majority of mammalian imprinted genes exhibit parental differences in methylation (i.e., differentially methylated regions [DMRs]) supports this idea (reviewed in Sleutels and Barlow, 2002
To date, only genes that are preferentially expressed upon maternal transmission have been identified in Arabidopsis thaliana and Z. mays endosperm (reviewed in Baroux et al., 2002 Because methylation plays a role in the parent-of-origin effects observed in seed development, a PCR-based approach to identify DMRs in embryo, endosperm, and young leaf tissues of Z. mays was undertaken. The results showed that Z. mays endosperm is characterized by a high degree of maternal hypomethylation. The potential role of unidirectional hypomethylation in seed development is discussed.
Identification of DMRs in Z. mays Endosperm Methylation-sensitive amplified polymorphism (MSAP) was undertaken to estimate the extent of DMRs in Z. mays endosperm. This technique is a modification of amplified fragment length polymorphism (AFLP), a procedure that is based on random amplification of restriction fragments typically generated by digestion of genomic DNA with the EcoRI and MseI restriction enzymes (Vos et al., 1995). In MSAP, MseI is replaced by an enzyme sensitive to cytosine methylation, such as HpaII (Reina-Lopez et al., 1997 ). After digestion of genomic DNA, adaptors are attached to restriction sites that have been successfully digested. It follows that the products of the MSAP ligation reaction consist of DNA fragments that are flanked by hypomethylated HpaII and/or EcoRI sites. Thereafter, two consecutive PCR reactions, a preamplification and a selective amplification, are performed to enrich a subpopulation of the restriction fragments. The primers employed are complementary to the core sequence of adaptors and recognition sites of the restriction enzymes, and the number of nucleotides added to their 3' termini determines their selectivity. Typically, the number of selective nucleotides is increased in the selective amplification in which one of the two primers is radioactively labeled. This enables the visualization of a subset of restriction fragments by autoradiography after separation by acrylamide gel electrophoresis.
Because DMRs have been identified previously in the zein and tubulin genes by DNA gel blot analysis using the inbred lines W64A, A69Y, and their reciprocal crosses W64A/A69Y and A69Y/W64A (the seed parent of the cross is underlined) (Lund et al., 1995a
Although the most likely explanation for the parent-of-origin profiles was that they represented DMRs, similar profiles could result from lack of homozygosity of the inbred lines employed, contamination of pericarp tissue, or preferential elimination/recombination of specific paternal chromosomal fragments, or they could represent maternally inherited organelle DNA. To exclude such possibilities, samples also were analyzed by AFLP, substituting the HpaII restriction enzyme with MseI, which is insensitive to methylation. Twelve selective primer combinations were employed, and the EcoRI primer was labeled in the selective PCR reaction. In contrast with MSAP, only 0.2% of total profiles exhibited a parent-of-origin effect (data not shown), indicating that the parental profiles observed in Figures 1B and 1C probably represented DMRs. We define the profiles as maternal methylation profiles (MMPs) and paternal methylation profiles (PMPs), respectively.
DMRs Are Conserved in Different Genetic Backgrounds and during Endosperm Development Most DMRs proved to be genetic background independent. For example, 48 (22/46) and 51% (29/57) of MMPs present in the A69Y or W64A inbred lines also were found when either inbred line was crossed to the W23 inbred line (Figure 2A, top and bottom panels, respectively). Other MMPs, initially identified by comparing W64A with the A69Y inbred line also were evident for the W23 inbred line (Figure 2B, top and bottom panels, respectively). As expected, these conserved MMPs showed monomorphic profiles in the crosses between W23 and W64A or between W23 and A69Y inbred lines (Figure 2B, top and bottom panels, respectively). The frequencies of DMRs were found to vary from 12 to 14% in the different inbred lines studied (data not shown).
The stability of DMRs during endosperm development was studied to determine if endoreduplication affected the generation or stability of DMRs. Endoreduplication is a maternally controlled process by which DNA replication is not followed by cell division (Kowles et al., 1997 ; Dilkes et al., 2002; Leblanc et al., 2002). Because this process initiates at  9 DAP, DNA was extracted from endosperms harvested at 7 DAP and analyzed by MSAP. The restriction fragment profiles from 16 selective primer combinations were compared with those previously obtained from endosperm DNA harvested at 15 DAP (Figure 3). The majority (92%) of DMRs were maintained during endosperm development (Figures 3A and 3B), although 4% showed a decrease in signal from 7 to 15 DAP (Figure 3B). Only 8% of DMRs exhibited changes during endosperm development. These regions exhibited an MMP at 7 DAP but either a polymorphic or monomorphic profile at 15 DAP (Figures 3C and 3D, respectively). The results indicated that all DMRs were present early in endosperm development and that these methylation patterns were faithfully maintained during development.
DMRs Are Specifically Hypomethylated in the Endosperm Numerous lines of evidence suggest that imprinting is central to endosperm development, with mainly indirect consequences on embryo growth (Kermicle, 1970 ; Lin, 1984; Haig and Westoby, 1991). If methylation is a component of the imprinting process, DMRs would be expected to be endosperm-specific. To investigate this, embryo and leaf DNAs of the W64A and A69Y inbred lines and their reciprocal F1 hybrids were subjected to MSAP. Based on 24 selective primer combinations, the endosperm had the largest number of total and tissue-specific profiles (i.e., profiles that were absent in the two other tissues) compared with leaf and embryo tissues, and 91% of endosperm-specific polymorphic profiles represented DMRs (Table 1).
Comparisons of MSAP profiles between tissues showed that 82% of DMRs were completely absent in both embryo and leaf tissues (Figure 4A). Twelve percent of fragments that exhibited an MMP in the endosperm were detected in both leaf and embryo, but none showed an MMP in these tissues (Figure 4B). This indicated that the majority of endosperm-specific DMRs were methylated both maternally and paternally in leaf and embryo. To confirm this assumption, the total methyl-cytosine content of leaf and endosperm DNA was analyzed by high performance capillary electrophoresis (HPCE). Indeed, the results showed that the endosperm exhibited a 13% reduction in total methylation compared with leaf DNA (Table 2).
Although no DMRs specific to embryo or leaf tissues were detected, 6% (7/114) of MMPs were common to all three tissue types (Figure 4C). To elucidate if common MMPs represented genomic or organelle DNA, the seven fragments were sequenced and analyzed in reciprocal backcrosses of 18 individual endosperms by MSAP. In addition, 10 endosperm-specific DMRs (nine representative of an MMP and one representative of a PMP) were included as controls. Backcrosses were generated using the reciprocal F1 hybrids W64A/A69Y and A69Y/W64 as seed parents and the W64A inbred line as a pollen parent. Only three out of seven fragments with a common MMP had sequence identity to mitochondrial genes, but all fragments exhibited grandparental effects in the segregation analysis (Figure 5A). By contrast, endosperm-specific DMRs segregated as expected. For example, a restriction fragment that showed an endosperm-specific MMP specific to the A69Y inbred line segregated in both backcrosses (Figure 5B), whereas a fragment with a PMP specific to the A69Y inbred line was absent in both reciprocal backcrosses (Figure 5C). The results indicated that common MMPs did not represent DMRs but probably resulted from mitochondrial or chloroplast genomes.
Sequence Analysis of DMRs To characterize the nature of endosperm-specific DMRs, fragments showing an MMP or a PMP were excised from acrylamide gels, reamplified by PCR, cloned, and sequenced. The sequences of 31 fragments with an MMP and one fragment with a PMP were successfully obtained (Table 3). The fragments ranged from 162 to 985 bp in size, the largest representing PMP2. The low success rate of obtaining consistent sequence data of the four PMP fragments identified probably reflected their size because all of them were 1 kb or larger. Under the experimental conditions employed, such fragments were poorly resolved and often lead to the isolation of multiple bands upon excision from acrylamide gels. Approximately 53% of DMRs revealed identity to cDNA sequences from a variety of different tissues and almost all of them showed sequence identity to unmethylated sequences from methyl-filtered DNA libraries (Rabinowicz et al., 1999). Only two DMRs (MMP5 and MMPa3) showed homology to retrotransposons, indicating that the majority of highly repetitive DNA regions were methylated on both parental genomes in the endosperm.
All sequenced DMRs represented EcoRI/HpaII restriction fragments, indicating that a DMR can result from differential methylation of either an HpaII site or an EcoRI site (the EcoRI restriction enzyme is sensitive to methylation of the cytosine residue of the GAATTC restriction site). However, because few DMRs were revealed by AFLP when the EcoRI primer was labeled as opposed to the HpaII selective primer, it was concluded that DMRs resulted from parental differences in methylation of an HpaII site. Seventy-five percent of sequenced MMP fragments lacked internal HpaII sites. This implied that the failure to detect these fragments upon paternal transmission resulted from paternal methylation of the external HpaII site (Figure 6A). Because 25% of the remaining MMP fragments contained an internal HpaII site (Figure 6B, i and ii), this site must have been methylated upon maternal transmission, whereas the external HpaII site was hypomethylated (Figure 6B). The lack of these fragments upon paternal transmission resulted from either paternal hypomethylation or hypermethylation of one or both HpaII sites (Figure 6B, i, ii, and iii). Assuming that the internal HpaII site of these MMP fragments was paternally hypomethylated, HpaII/EcoRI restriction fragments ranging from 120 to 555 bp in size would have been generated in the initial MSAP restriction digest. Obviously, the probability of detecting these particular PMP fragments together with their corresponding MMPs using the same primer combinations is remote, but it illustrates that paternal hypomethylation of specific HpaII sites would produce fragments within the range of effective separation by MSAP (i.e., between 50 and 1000 bp). Because only four PMP fragments 1 kb or larger in size were detected (corresponding to 3.5% of total DMRs), this suggests that few, if any, DNA regions were specifically hypomethylated upon paternal transmission. It follows that a PMP fragment is predicted to contain an internal HpaII site that is maternally hypomethylated. This was found to be the case for PMP2 (see below).
DNA Gel Blot Analysis of DMRs Nine fragments showing an MMP and one showing a PMP were used as probes in DNA gel blot analysis (marked with asterisks in Table 3). Genomic DNA from endosperms harvested at 15 DAP or from 2-week-old leaf tissue was digested with the combination of EcoRI and HpaII (EH) restrictions enzymes used in MSAP or with HindIII, a restriction enzyme that is insensitive to 5'-CpG-3' methylation. For some probes, additional restriction digests were undertaken.
DMRs without Internal HpaII Sites
DMRs with Internal HpaII Sites We have argued previously that DMRs, with or without internal HpaII sites, were hypomethylated upon maternal transmission. To strengthen this conclusion, MMP43, a 665-bp W64A-specific fragment that contained an internal HpaII site, was hybridized to an EH digest of endosperm DNA. If both internal and external HpaII sites of MMP43 were hypomethylated paternally (Figure 6B, i), 555- and 110-bp fragments would be expected to show PMPs, whereas a 665-bp fragment should exhibit an MMP. Because the smallest hybridizing fragment was 600 bp (Figure 8, arrow in top panel), the internal HpaII site of MMP43 was methylated both paternally and maternally. Because of a lack of polymorphism between the W64A and A69Y inbred lines of the 600-bp fragment, the parental methylation status of the external HpaII site could not be determined. However, upon hybridization of MMP43 to an HpaII digest of endosperm DNA, it was confirmed that the external HpaII site of MMP43 was maternally hypomethylated (Figure 8, top middle panel). Likewise, PMP2, a 985-bp W64A-specific fragment, was found to be hypomethylated upon maternal transmission. In an EH digest of endosperm DNA, hybridization to an 1-kb fragment in both inbred lines impeded the identification of a PMP (Figure 8, arrow in bottom panel). However, two smaller comigrating fragments, which corresponded in size to those expected if the internal and external HpaII sites of PMP2 were maternally hypomethylated (i.e., 475 and 510 bp; Figure 8, open triangle in bottom panel at left), showed an MMP. It was concluded that the internal HpaII site of PMP2 was hypomethylated upon maternal transmission. Because of the poor resolution of the PMP in the EH digest, PMP2 was hybridized to endosperm DNA restricted with SstI and HpaII, confirming the presence of DMRs in both inbred lines (Figure 8, bottom middle panel). As observed previously, neither of the probes exhibited parental profiles in endosperm DNA digested with HindIII (Figure 8, top and bottom panels at right) nor in leaf DNA digested with EH (data not shown).
In summary, the 10 DMRs analyzed were all hypomethylated in the endosperm, and DMRs were specific to the endosperm. The majority of fragments were hypomethylated upon maternal transmission, but for 4/10 probes (MMP5, MMP9, MMP11, and MMP43), the parental origin of hypomethylation could not be determined because of the lack of polymorphism between the W64A and A69Y inbred lines in the EH digest. However, extensive analysis of MMP43 showed that the failure to detect an MMP resulted from the reduced sensitivity of DNA gel blotting compared with MSAP (Figure 8, top panel). Several DMRs, identified as being specific to a particular inbred line by MSAP, were found to be differentially methylated in both inbred lines by DNA gel blotting (MMP7, MMP21, MMP43, and PMP2). For MMP26, maternal hypomethylation was partial.
Characteristics of DMRs MSAP analysis of embryo, endosperm, and leaf DNA has provided clear evidence of an asymmetry in parental patterns of methylation that is specific to the endosperm. Approximately 96% of DMRs exhibited an MMP, whereas only 4% showed a PMP. Sequence and DNA gel blot analysis demonstrated that the origin of both profiles was identical; for example, DMRs resulted from maternal hypomethylation of specific HpaII sites, whereas the corresponding sites were methylated paternally. An almost identical situation was reported for specific alleles of the  -zein genes, the -tubulin genes, and the R locus in Z. mays (Lund et al., 1995a, 1995b; Alleman and Doctor, 2000). At least 50% of DMRs were conserved between genotypes. This percentage is presumably higher because the identification of a conserved DMR relies on polymorphism between the inbred lines studied. Indeed, the differences in MMP frequencies between the inbred lines studied most probably reflect differences in their degree of polymorphism. In addition, MMPs identified as being specific to one inbred line by MSAP exhibited DMRs in both inbred lines by DNA gel blotting. The high frequency and diverse nature of DMRs is indicative of a genome-wide process that leads to parental differences in methylation in the endosperm.
The majority of endosperm-specific maternally hypomethylated DNA regions were methylated to a similar extent in both embryo and leaf. Interestingly, a similar situation has been observed in the mammalian placenta, which like the endosperm, mediates the transfer of nutrients from mother to offspring. In the placenta, general hypomethylation of both genic and repetitive DNA sequences is observed, whereas the same sequences are methylated in embryonic and adult tissues (Chapman et al., 1984
Epigenetic Reprogramming during Seed Development
Methylation and Parent-of-Origin Effects in Endosperm Development
It is tempting to suggest that global hypomethylation plays a role in the genome-wide maternal expression observed early in seed development either directly by silencing paternally derived genes or by acting downstream of other chromatin remodeling factors such as histone modifications. Whatever the mechanism, several observations suggest that maternal hypomethylation is a hallmark of expressed genic regions in Z. mays endosperm. An inverse correlation between methylation and gene expression is evident for the imprinted R and
The parental conflict hypothesis proposed by Haig and Westoby (1989)
Plant Material Z. mays plants were grown in the field and under standard greenhouse conditions during the years 1993 to 2002. Immature seeds were collected at 7 and 15 DAP, frozen in liquid nitrogen, and stored at -80°C. Endosperms harvested at 7 DAP were dissected under a dissecting microscope and collected in a mannitol solution (750 mosM/kg H2O). The embryo and remaining sporophytic tissues were manually eliminated under an inverted microscope using glass needles.
MSAP Analysis The preamplification reaction was performed with primers complementary to the core of the adaptor sequences and to the target sequences of EcoRI and HpaII. The sequences of the EcoRI (EcoRI-00) and HpaII (HpaII-00) preselective primers were 5'-AGACTGCGTACCAATTC-3' and 5'-TCATGAGTCCTGCTCGG-3', respectively. Two microliters of the digestion-ligation mix (diluted 1:3) were added to the preamplification mix consisting of 1 x PCR buffer (100 mM Tris HCl, 15 mM MgCl2, and 500 mM KCl, pH 8.3), 0.1 mM deoxynucleotide triphosphate (dNTP), 50 ng of EcoRI-00 primer and HpaII-00 primer, and 1 unit of Taq polymerase (Roche Biochemicals, Indianapolis, IN). PCR conditions were 72°C for 2 min and 94°C for 3 min, followed by 25 cycles of 95°C for 30 s, 56°C for 30 s, and 72°C for 1 min. The final extension was at 72°C for 4 min.
Selective primers had identical sequences to the preselective primers but included the addition of a number of nucleotides at the 3' termini. Selective nucleotides of the EcoRI-00 primer were EcoRI-01 AGT, EcoRI-02 ACA, EcoRI-03 AGA, and EcoRI-04 ACC. The HpaII selective primers were HpaII-01 TCCA, HpaII-02 TAGC, HpaII-03 CGAA, HpaII-03A CGTT, HpaII-04 AATT, and HpaII-04A AACC. Selective PCR used 2 µL of preamplification mix (diluted 1:10) in a 10-µL reaction volume containing 1 x PCR buffer, 0.1 mM dNTP, 50 ng of EcoRI-00 primer, 50 ng of The PCR samples were mixed 1:1 (v/v) with denaturating buffer (98% formamide, 10 mM EDTA, 0.1% bromophenol blue, and 0.1% xylene cyanol) and separated on 6% polyacrylamide sequencing gel (Bio-Rad, Hercules, CA) for 3 h at 90 W. Gels were dried and exposed to BioMax MR film (Eastman Kodak, Rochester, NY) for 1 to 4 d at -80°C. For each sample, three independent MSAP reactions were performed. When the results were reproducible, one sample was used for further analysis.
AFLP Analysis Preamplification used 5 µL of the diluted restriction-ligation mixture in a 20-µL reaction volume containing 1 x PCR buffer, 0.1 mM dNTP, 50 ng preselective primers, and 1 unit of Taq polymerase. Preselective primers were complementary to the core sequences of EcoRI and Mse1 adaptors, including one selective nucleotide for both EcoRI (E-01, 5'-GACTGCGTACCAATTCA-3') and MseI (Mse-02, 5'-GATGAGTCCTGAGTAAC-3') primers. The preamplification consisted of 25 cycles at 94°C for 30 s, 56°C for 30 s, and 72°C for 1 min, with a final extension at 72°C for 5 min. The preamplification product was diluted to 200 µL and stored at -20°C before use. Selective amplification was as described for MSAP analysis. The selective primers were identical to the AFLP preselective primers but included two additional nucleotides at the 3' termini. The EcoRI selective primers were E31 AA and E32 AC. The selective MseI primers were M47 AA, M48 AC, M49 AG, M50 AT, M51 CA, M52 CC, M58 GT, M59 TA, and M60 TC.
Isolation, Cloning, and Sequence Analysis of DMRs
DNA Extraction and DNA Gel Blot Analysis
Measurement of Total Methyl-Cytosine Content Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession numbers AY224046–AY224077.
We thank Marlis Nissen and Petra von Wiegen for endosperm isolation, Hans Hartings for advice on AFLP techniques, Epiphysage Group, Oviedo University for the HPCE analysis, and Vincenzo Rossi, Alexandra Mant, Silvio Zaina, Birger L. Møller, and Franceso Salamini for critical reading of the manuscript. This work was supported by a grant from the Danish National Research Foundation and partially by a grant from Ministero dell'Istruzione, dell'Università e della Ricerca–ll Fondo per gli Investimenti della Ricerca di Base (RBNE01TYZF) to A.V.
1 Current address: Istituto Sperimentale per la Cerealicoltura, Sezione di Bergamo, Via Stezzano 24, 24126 Bergamo, Italy.  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: Gertrud Lund (gel{at}kvl.dk). Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.017780. Received October 1, 2003; accepted November 18, 2003.
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INTRODUCTION
-33P–labeled HpaII-00 primer, and 1 unit of Taq polymerase. The HpaII primer was end-labeled by incubating 50 ng of primer with 50 µCi of [