35S De Novo Methylation Figure 3. De novo methylation maps of the 35S-sGFP region in the single copy transgenic gentians. Percent methylation at CpHpH sites of 35S-sGFP region in the single copy unmodified 35S transgenic gentian lines (#3 to #30) is represented by bar charts. Positions of cytosines in CpHpH (vertical bars) motifs on the analyzed regions are represented below. Positions of cytosines from 2148 to 285 are indicated by red bars. doi:10.1371/journal.pone.0009670.g003 binding is sequence-specific (Figure 5B). In addition, a slightly retarded band was observed when the as-2 [25] probe (2107 to 283) was used with gentian nuclear extract, whereas tobacco nuclear extract strongly bound to the probe. Discussion Species-specific 35S promoter silencing The present study focused on single copy transgenic plant lines to avoid the copy-number effect [26,27] and analyzed different genomic positions. We clearly showed that introduction of the single copy unmodified 35Spro was subject to silencing by hypermethylation in the transgenic gentian plants without exceptions. Although cytosine methylation was also observed in the NOS promoter, the frequency of methylation in the 35Spro region was demonstrably higher (Figure S4B). Consistent with these results, bar mRNA expression in some transgenic gentian lines (#8, #24, #27, #28 and #30) was comparable with that of tobacco, whereas sGFP mRNA expression of the transgenic gentians was considerably suppressed (Figure 1C). Reduced bar PLoS ONE | www.plosone.org 5 March 2010 | Volume 5 | Issue 3 | e9670