Using Southern analysis of genomic DNA, digested with methylation-sensitive restriction enzymes, we investigated the methylation state of three families of storage protein genes in the following tissues and organs of inbred line W64A: endosperm at 8 days after pollination (dap), endosperm at 22 dap (w.t. and homozygous for opaque-2), three day etiolated shoot, three day root, immature ear (4 centimeters long) and mature pollen. We used as probes fragments of a cluster of zein genes coding for 22 kd polypeptides, an opaque-2 dependent cDNA clone coding for a 27 kd zein, and a novel cDNA clone related in sequence to a RSP gene. The transcripts of these genes were detected by Slot Dot analysis of total or polyadenylated RNAs (detection threshold: approx. 0.0004% of total RNA) only in the endosperm cells, but not yet at 8 dap, an early and for most aspects undifferentiated stage of development. The DNA of zein and RSP-related genes (coding regions and surrounding sequences) was found to be extensively undermethylated in the endosperm, while we observed a single, more methylated pattern in the other tissues. Data on pollen DNA obtained with the 27 kd zein clone revealed a similarly methylated pattern. At 8 dap, the demethylated pattern is already and completely established, indicating that DNA demethylation precedes and is not necessarily coupled with the active transcription of these genes. In agreement with these data, we found the demethylated pattern of the 27 kd zein sequences in the 22 dap opaque-2 endosperm as well, where the level of the corresponding transcripts is significantly reduced. The 5-methyl cytosine content of endosperm DNA is not significantly different from that of DNA from the immature ear, as assessed by HPLC analysis, or from that of DNA from the other tissues, as assessed by comparison between ethidium bromide stained or end-labelled digests. Moreover, hybridization experiments with a sequence abundantly represented in root and endosperm RNA, revealed very similar methylation patterns in the two tissues.
Michele W. Bianchi and Angelo Viotti
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