structure, unlike vectors used in our previous study [11]. Consequently, 21 independent transformant lines were obtained, of which 12 lines represented single copy lines, as confirmed by Southern blot analysis using bar and sGFP gene probes (Figure 1B, Table S1). For comparison, six lines of single copy transgenic tobacco (Nicotiana tabacum cv. SR1) plants transformed with the same construct were also screened from 31 lines produced. Among the gentian lines, two lines (#5 and #8) exhibited low expression of sGFP mRNA, but all other lines exhibited no sGFP expression in the leaf tissues of in vitro cultured plants (Figure 1C). In the leaves of all six single copy transgenic tobacco plants, strong sGFP expression was observed. The mRNA expression of the bar gene, which was used as a selection marker, varied among the transformants (Figure 1C). GFP fluorescence was observed in the transformed calli in an early stage after transformation (Figure S1), thus silencing of the sGFP transgene did not occur at the callus stage. Methylation status of the 35S-sGFP region To investigate whether the 35Spro sequence underwent cytosine methylation in the transgenic plants, bisulfite genomic sequencing [20] was performed. To avoid incomplete bisulfite conversion in our genomic DNA samples, strict conditions (i.e. high-temperature for a long time, see Materials and Methods) were used, and methylation of the endogenous CHS promoter region [11] was evaluated for conversion efficiency (data not shown). Consequently, the bisulfite- treated products were found to be sufficiently converted (.99.8%). Accordingly, genomic DNAs derived from leaf tissues of all the single copy lines (12 gentians and six tobaccos) were subjected to bisulfite conversion (Table S1). The 35Spro with the sGFP coding region (from 2257 to +110) was PCR-amplified (see Figure 1A), and at least eight independent clones were sequenced. Figure 2 shows the frequencies of methylated cytosines of each 35Spro and sGFP 35S De Novo Methylation region analyzed from the transgenic gentian and tobacco lines. The cytosine methylation frequencies were divided into symmetric (CpG and CpWpG) and asymmetric (CpHpH) sequence contexts. All the gentian samples showed hypermethylation at CpG and CpWpG sites and moderate methylation frequencies at CpHpH sites in the 35Spro region, whereas no methylation was observed in the same region from the tobacco samples (Figure 2; see detail in Figures S2A and S2H). Compared with the 35S region, lower frequencies of CpG/CpWpG methylation of the sGFP region were observed in all gentian samples, and the methylation frequencies at CpG/CpWpG sites were correlated between the two regions in each gentian line (Figure 2). Within the promoter sequence, high methylation frequencies were commonly observed at a region 59 to the as-1 element (282 to 262) [13,21] (Figure S2A), which was consistent with our previous result using tandem 35S constructs [11]. Modified 35S promoters introduced transgenic gentian To confirm whether the as-1 element is involved in de novo methylation in gentian, modified 35S promoters, which were substituted for the 35Spro of the pSMABR35SsGFP vector, were constructed (Figure 1A). The six different modified 35S constructs were introduced through Agrobacterium-mediated transformation and 194 lines of independent transformants were obtained. After copy-number estimation by Southern blot analysis, 14, 10, 11, 14, 10, and 15 lines for 35S(Das-1), 35S(nos-1), 35S(PhCHS), 35S(GtCHS), 35S core, and 35S(Dcore), respectively, were identified as single copy lines (Table S1). Subsequently, the methylation states of the modified promoter regions of all the single copy transgenic plant lines were determined using bisulfite analysis (Figure S2). A point mutation of the as-1 element (see Figure 1A) conferred a substantial reduction of GFP fluorescence in the 35S(Das-1) transformed gentian calli (Figure S1), supporting the hypothesis Figure 2. Methylation frequencies of the 35S and sGFP regions in the transgenic gentian and tobacco plants. Average percent methylation of CpG/CpWpG (upper panels) and CpHpH (lower panels) sites in the 35Spro (2257 to 21; left panels) and sGFP 59 coding (+1 to +110; right panels) regions of the single copy unmodified 35S transgenic gentian and tobacco lines. doi:10.1371/journal.pone.0009670.g002 PLoS ONE | www.plosone.org 3 March 2010 | Volume 5 | Issue 3 | e9670