Transient decreases in methylation at 5'-CCGG-3' sequences in potato (Solanum tuberosum L.) meristem DNA during progression of tubers through dormancy precede the resumption of sprout growth

The 5-methylcytosine (5mC) content in DNA of tuber meristems isolated from field-grown potatoes (Solanum tuberosum L.) was determined during a 7-month storage period at 3 degrees C for three growing/postharvest seasons. No significant changes in 5mC levels were noted genome-wide or within 5'-CG-3' dinucleotide sequences, 5'-CG-3' islands or 5'-CA(T)G-3' trinucleotide sequences during storage. However, a consistent but transient 50-70% decrease in methylation at both cytosines within 5'-CCGG-3' sequences was detected that peaked 112-194 days after harvest. This result was corroborated by methylation-sensitive amplified fragment length polymorphism analysis of meristem DNA. Similar to tuber meristems undergoing progression through natural dormancy, premature chemical termination of dormancy resulted in rapid, transient 5'-CCGG-3' demethylation in meristem DNA. Minimum methylation levels at this sequence preceded initiation of high levels of de novo DNA synthesis by two days. Cytosine methylation status was also followed in in vitro-generated potato microtubers during 7 months of post-harvest storage. As in DNA from tuber bud meristems, no changes in genome-wide 5mC content or methylation at 5'-CA(T)G-3' or 5'-CG-3' island sequences were noted in microtuber DNA. However, there was a transient 46% drop in methylation at 5'-CG-3' dinucleotides concomitant with minimum levels of 5'-CCGG-3' methylation (30-60% below those in dormant microtubers) 57-98 days after harvest. As microtubers exited dormancy, there were sustained three- and seven-fold increases in RNA and DNA synthesis rates, peaking on or after 98 days of storage, respectively. Together, these data demonstrate that demethylation of 5'-CCGG-3' sequences occurs independently of tuber age during dormancy progression and precedes transcriptional activation of genes leading to cell division and meristem growth in leading to cell division and meristem growth in potatoes.