that as-1 binding factor(s) might principally act for 35S transcription in gentian callus tissues at an early stage after transformation. On the other hand, replacement of the nos-1 element [22] for the as-1 element allowed high GFP expression in transformed calli (Figure S1), followed by suppression in the regenerated plant tissues (Figure S3A), which is comparable to the unmodified 35S lines. In either case, hypermethylation of the modified 35Spro regions was observed in all the 35S(Das-1) and 35S(nos-1) transgenic plant lines, as well as the unmodified 35S lines (Figures S2A–C). As for the 35S(PhCHS) and 35S(GtCHS) lines, in which the 35S core region (290) was replaced by the core regions of the CHS promoters from petunia [23] and gentian [24], respectively, strong GFP expression at the callus stage (Figure S1) and subsequent suppression in transgenic plant lines (Figures S3B and S3C) were also observed. Hypermethylation was also observed in the modified 35Spro regions in all transgenic plant lines analyzed (Figures S2D and S2E). Surprisingly, all the 35S(Dcore) lines also exhibited hypermethy- lation of the introduced 35S enhancer region (2257 to 291), despite the fact that this construct lacks the 35S core promoter (290) region (Figure S2F). On the contrary, hypomethylation of the 35S core region was observed in some 35S core lines (Figure S2G; see also Figure S3D for examples of sGFP and bar expressions in both lines). As a result, the 35S core region, including the as-1 element, was considered to be dispensable for 35Spro-specific methylation in gentian. Analysis of de novo methylation using non-CpG/CpWpG cytosine sequences To identify the possible target region of de novo methylation of the 35Spro, we focused on the asymmetric cytosine sequences from the bisulfite data obtained. Figure 3 shows a schematic representation of CpHpH methylation of the 35S-sGFP region in the unmodified 35S introduced gentian lines. Interestingly, distinct peaks, spanning 11 cytosines from positions 2148 to 285 of the CpHpH methylation region, were shown in all the lines analyzed (Figure 3; indicated by red bars). High levels of CpHpH methylation were also observed within the region of the complementary strand (Figure S2I). In the remaining region, traces of methylated cytosines were scattered out the 59 region of the analyzed sequence, with a slight peak around the cytosine at position 2161. On the other hand, the 35S core region (comprising 17 cytosines from 266 to 212) and the adjoining sGFP 59 region (15 cytosines from +1 to +101) rarely contained methylated cytosines except in #29 line that had a moderate peak in the region (Figure 3). From the NOS promoter to the bar 39 region and the rbcS terminator to the 35S 59 region, no or few CpHpH methylations (indicated by black bars) were observed in the unmodified 35S lines (#15 and #19), despite a certain level of CpG/CpWpG methylation (red and green bars, respectively) being detected in both lines (Figure S4). A distinct distribution of CpHpH methylation was also observed in the 35S(Das-1) transgenic lines. All 14 lines showed prominent CpHpH methylation peaks at the same regions (2148 to 285) as those in the unmodified 35S lines (Figure 4; indicated by red bars). In addition, most of the lines had moderate CpHpH methylation in the 35S 59 region (2257 to 2161) and slight methylation in the 35S core and sGFP regions, similar to the unmodified 35S lines. Altered de novo methylation patterns in the modified 35Spro Despite the CpHpH methylation peak spanning 2148 to 285 region being observed in all of 35S(nos-1) lines, their distributions 35S De Novo Methylation of CpHpH methylation tended to extend towards the 35S core region, where the methylation levels were generally low in the unmodified 35S and 35S(Das-1) lines (Figure 4). The variability of the CpHpH methylation pattern was further characterized in the 11 35S(PhCHS) lines, in which two lines (#22 and #39) with strong CpHpH methylation patterns and six lines (#19, #31, #42, #43, #44, and #46) with moderate CpHpH methylation patterns were observed on the replaced petunia CHS core promoter region (Figure 4). On the other hand, no or very little CpHpH methylation was observed in the replaced gentian CHS core promoter region of all the 35S(GtCHS) lines. Among these CHS-core substituted lines, CpHpH methylation in the sequence corresponding to the 2148 to 285 region [note three cytosines at the 39 end of the region in the unmodified 35Spro were not contained in the 35S(PhCHS), 35S(GtCHS) and 35S(Dcore) promoters] of the unmodified 35Spro was also shown (Figure 4; indicated by red bars). However, the extent of methylation tended to be rather lower than those in the unmodified 35S, 35S(Das-1), and 35S(nos-1) lines. Similar CpHpH methylation patterns to the CHS-core substi- tuted lines were also observed in the 35S(Dcore) lines. All the lines had a moderate extent of CpHpH methylation in the 35S enhancer region. In the case of 10 lines of the 35S core lines, no (#13, #26, #32, #33) or very low amounts (#1, #2, #6, #19) of CpHpH methylation were predominantly observed, whereas moderate (#44) and strong (#38) CpHpH methylation was also detected (Figure 4). Increasing frequency of de novo methylation by culture of transgenic gentian plants CpHpH methylation states of the 35S(Dcore) lines, which had been obtained after Agrobacterium inoculation on Jun. 7, 2005, were compared over a time-series of culture. Genomic DNAs from the in vitro cultured plants were obtained on Oct. 16, 2006 and Mar. 22, 2007 (11 and 16 months of culture after regeneration, respectively). An obvious increase in CpHpH methylation frequencies in the samples collected later (meanwhile, there was one time of subculturing the apical shoots) was found as compared with the corresponding lines collected earlier (Figure S5). The methylation frequencies of CpG/CpWpG sequences also in- creased during the five months of culture, synchronously with CpHpH methylation (data not shown). Identification of possible causal agents of the 35S de novo methylation To identify possible causal agents for the de novo methylation of the 35S enhancer, we analyzed small RNA and genomic DNA in the wild-type and unmodified 35S transgenic gentian and tobacco plants. However, neither small RNA molecules (Figure S6A) nor genomic DNA sequences (Figure S6B) corresponding to the 35S enhancer could be detected in the gentian and tobacco plants. Gentian nuclear factors bind to the 2149 to 2124 region and the as-2 element To explore other possible candidates, we tried to detect DNA binding factor(s) on the 35S enhancer region from the wild-type gentian nuclei extracts by electrophoretic mobility shift assay (EMSA). Using eight different 26-bp probes covering the 2254 to 283 of the 35S enhancer region, distinctly different forms of complexes between gentian and tobacco nuclear extracts were identified using the 2149 to 2124 probe (Figure 5A). The probe was competed with unlabeled wild-type probe but not with the 2191 to 2166 or mutant (see Table S3) probes, indicating that the PLoS ONE | www.plosone.org 4 March 2010 | Volume 5 | Issue 3 | e9670