35S De Novo Methylation Figure 4. De novo methylation maps of the modified 35S promoter regions in the transgenic gentians. Percent methylation at CpHpH sites of the six different modified 35Spro regions in all of the single copy transgenic gentian lines tested are represented by bar charts. Analyzed regions of the modified 35S promoters (unmodified 35Spro sequences are indicated by white bars) with sGFP (black bar; indicated by arrows) are represented below each chart. Positions of cis-elements are indicated by marks. Regions of CHS core promoters and CHS 59 UTRs are shown by gray and dotted bars, respectively. In the 35S(GtCHS) region, percent methylation of the corresponding endogenous CHS region from wild-type plants is also shown at the bottom. Positions of cytosines corresponding to the sequence from 2148 to 285 of the unmodified 35Spro are indicated by red bars. doi:10.1371/journal.pone.0009670.g004 mRNA expression in other gentian lines was probably caused by symmetric methylation in the NOS promoter and/or bar gene coding region (Figure S4B), in which the methylation might occur by spreading from the 35S region [28]. In addition, transgenic gentian plants using promoters other than 35Spro showed stable transgene expression [11,14], implying that the 35Spro sequence acts as a possible inducer of cytosine methylation in gentian. Suppression of sGFP mRNA transcription is thought to be mainly caused by the methylation of two cytosine residues in the as-1 element, because a previous report also showed that the motif is sensitive to cytosine methylation in petunia [29]. Accordingly, the slight sGFP mRNA expression in the #8 and #5 lines might correlate with lower methylation frequencies (43.4 and 68.2%, respectively) at the two cytosines of the lines compared with those of the other lines (ranging from 68.8 to 100%). Contrastingly, all the single copy transgenic tobacco lines showed strong sGFP expression with no methylation of the 35Spro sequence, which is consistent with the previous studies showing that 79 [30] and 10 [31] independent single copy Arabidopsis transgenic lines using 35Spro revealed high and stable expression, without exception. 35Spro is the most widely used promoter for driving transgenes, not only in dicots [32], but also in some monocots [33,34], suggesting that the strict silencing phenomenon is specific to gentian. In some plant species, nevertheless, 35Spro PLoS ONE | www.plosone.org 6 March 2010 | Volume 5 | Issue 3 | e9670