In higher plants, 5-methylcytosine (m5C) can constitute as much as one-third of the cytosine residues of nuclear DNA. Moreover, DNA methylation has been found to control transposable element activity, genomic imprinting, gene silencing, inhibit gene transcription, and be implicated in the inheritance of a variety of epigenetic phenomena. A recent method based upon chemical treatment of genomic DNA (Frommer et al., PNAS 89:1827-1831, 1992) has allowed detailed analysis of the methylation state of several genes in different organisms. In transgenic plants, the application of this method allowed observation of the occurrence of m5C outside the canonical symmetrical CpG and CpNpG sites, and revealed association between the high level of m5C observed within a silenced 35S promoter and the transcriptional inactivation of the transgene (Meyer et al., EMBO J. 13: 2084-2088, 1994; Park et al., Plant J. 9:183-194, 1996). Furthermore, analysis of the methylation pattern of the maize Ac transposable element suggested a model to explain the association of Ac transposition with replication (Wang et al., Plant Cell 8:747-758, 1996). However, at present little is known about the methylation state of endogenous plant promoters (Ronchi et al., EMBO J. 14:5318-5328, 1995). To address this question, we determined the methylation state of a 390 bp region of the Opaque2 (O2) gene promoter (from -436 to -46) in endosperm and leaf cells by means of the bisulfite treatment of maize genomic DNA. This method is based upon a chemical treatment that converts unmethylated C residues into U residues that appear as T residues after PCR amplification of specific, single stranded DNA. Conversely, all m5C residues remain unmodified. For each tissue analyzed, separate PCR amplifications were performed for the upper and lower strands to produce DNA fragments that were subsequently cloned and sequenced.

In immature endosperm, where the O2 gene is expressed, 13 upper strand and 16 lower strand cloned PCR products were sequenced, respectively (Figure 1A). All clones displayed different methylation patterns and thus were derived from individual genomic O2 sequences. The O2 promoter has a considerably high level of C-methylation, 84%, present in this tissue. Particularly, almost all the C residues embedded within the O2-protein binding site appeared methylated. Interestingly, the methylation pattern of the lower strand differs markedly from that of the upper strand. Statistical analysis (student t test) of the mean methylation values obtained from the collected data indicated that, with p=1%, the lower strand is significantly less methylated than the upper strand (80% m5C vs. 89%). Moreover, an unequal m5C distribution is observed in the lower strand, less methylated at the most distal part (-436; -370) of the region analyzed. Student t analysis was also used to compare the methylation level of each C residue within their sequence context. Accordingly, we found that in endosperm, for both strands, CpT and CpG dinucleotides were significantly less methylated than CpA and CpC (p=1% and p=5%, respectively).

The methylation pattern of the same O2 promoter region was also analyzed in leaf by sequencing 10 upper strand and 10 lower strand clones (Figure 1B). Again, different methylation patterns were observed, thus indicating that they derive from individual genomic O2 sequences. Student t analysis was also used to compare the mean methylation values obtained from leaf and endosperm. Data indicate that, with p=1%, the level of C-methylation in leaf is significantly higher than in endosperm (96% m5C vs. 84%). Similar to endosperm, in leaf an unequal m5C distribution is also observed in the lower strand of the most distal part of the region analyzed.

Figure 1. Methylation map of the -436, -46 O2 promoter region in endosperm and leaf. (A) Methylation map of the O2 promoter region in endosperm. Data from 13 and 16 clones have been compiled for the upper and lower strand, respectively. (B) Methylation map of the O2 promoter region in leaf. Data from 12 and 10 clones have been compiled for the upper and lower strand, respectively. Methylated C residues are indicated by black squares, whereas unmethylated C residues are indicated by white squares. CpG dinucleotides are underlined. The O2 consensus sequence is boxed. 

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