failed to confer high expression [10,35,36]; further study would be needed to determine whether these species share a similar silencing machinery with gentian. To the best of our knowledge, the present study is the first attempt to determine the different epigenetic response to single copy transgenes in different plant species. Survey of asymmetric methylation to elucidate de novo methylation The CpHpH methylation analysis allowed us to infer that the consensus target sequence for de novo methylation was the 2148 to 285 region of the 35Spro. In the representation of methylation with all the cytosine sequence contexts (Figure S2A), high levels of methylation at the CpG/CpWpG sites were scattered, not only in the 2148 to 285 region, but also in other regions including the sGFP coding region in some lines. Importantly, the presence of the symmetric methylation patterns was not stable among the transgenic lines, unlike the uniform pattern of the CpHpH methylation (Figure 3). Moderate and high levels of methylation at CpG/CpWpG sites were also observed in the bar coding and rbcS terminator to 35S 59 regions, respectively, where distinct levels of CpHpH methylation were not observed (Figure S4). Thus, we hypothesized that the symmetric methylation of these regions was a consequence of methylation spreading from the de novo methylated 2148 to 285 region. Highly uniform de novo methylation of the 11 asymmetric cytosines spanning 2148 to 285 were detected in all 26 unmodified 35S and 35S(Das-1) lines (Figures 3 and 4), suggesting that a strict sequence-specific de novo methylation occurred, regardless of the presence of the as-1 element, the orientation of the DNA strands (Figure S2I), and genomic location. The frequencies of CpHpH methylation increased during culture (Figure S5); therefore, the establishment of stable de novo methylation status might require continuous growth and/or some developmental process. Although RdDM is known to be a major coordinator for sequence-specific de novo methylation in plants [37], alternative mechanisms probably exist because a certain number of methylated regions are not associated with siRNA expression in the Arabidopsis genome [9]. Small RNAs with homology to the 35S enhancer sequence could not be detected in either transgenic or wild-type gentians (Figure S6A) [11], therefore the de novo methylation of the 35S sequence might involve mechanisms other than the RdDM pathway. While no studies have identified an alternative coordinator in plants, possible mechanisms were proposed in mammals: that de novo methyltrans- ferases themselves might recognize particular DNA or chromatin; and that de novo methyltransferases might be recruited through protein-protein interactions with transcriptional repressors or other factors [38]. More recently, research on the Arabidopsis ibm1 mutation suggested that novel transcription-coupled mech- anisms direct gene body methylation [39], hence such RdDM- independent target methylation mechanisms might exist in plants. Accordingly, we found nuclear factors that bind to the highly de novo methylated region (2149 to 2124) of the 35S enhancer sequence in gentian. The form of the binding complex in gentian was distinct from that in tobacco (Figure 5B); therefore, some of the gentian nuclear factors might contribute to de novo methylation. With regard to host genome defense [3], 35S methylation is thought to be a specially diversified system in gentian, contributing to genomic homeostasis against parasitic sequences. In the previous study, we showed that hypomethylation was found in the wild-type gentian genomic regions corresponding to those adjoining the T-DNA boundary of the transgenic plants [11], suggesting that the T-DNA was not entirely integrated into 35S De Novo Methylation heterochromatin regions. In this regard, the 2C nuclear DNA content of gentian used in our experiment (10.57 pg) [40] is similar to that of tobacco (11.71 pg) [41]. No 35S-like sequences were detected in the wild-type gentian genome by Southern analysis (Figure S6B); therefore, it is much less likely that a virus sequence had been acquired in the ancestral gentian genome, comparable to the endogenous pararetroviruses in tobacco [42]. We thereby assume that cis-element(s) located within the de novo methylated region might be recognized as parasitic sequences. To confirm these possibilities, determination of the whole genome sequence of gentian would be required in the future. De novo methylation in the modified 35S promoters Distinct distribution patterns of CpHpH methylation were observed in both petunia and gentian CHS core promoter regions, respectively. In the 35S(GtCHS) lines, slight CpHpH methylation was shown in the converted gentian CHS core promoter regions, which is consistent with the CpHpH hypomethylation of the intrinsic CHS promoter in wild-type gentian (Figure 4). Paradox- ically, this result supports the existence of possible methylation machinery that acts sequence-specifically. On the other hand, the CpHpH methylation states of the petunia CHS core promoter regions varied among the 35S(PhCHS) lines (Figure 4). The variation of the CpHpH methylation patterns resembled those of the 35S(nos-1) lines, suggesting that their supplementary sequences involving cis-elements (i.e. nos-1 and TACPyAT boxes) might affect de novo methylation directly or indirectly. Another possibility is that these additional sequences might occasionally permit spreading of de novo methylation activity by modification of histone tails. In any case, the variations of the distributions of CpHpH methylation in the modified 35S promoters make it difficult to reconcile the assumption that endogenous siRNAs with homology to the 35S enhancer region cause the de novo methylation. Even though further experiments are required to uncover the de novo methylation machinery, the present identification of the target region of de novo methylation impels us to search for the causal factor, which binds to the sequence and probably attracts a de novo methyltransferase. Materials and Methods Production of transgenic gentians A binary vector, pSMABR35SsGFP, was constructed from pSMAB704 [43], by replacing the uidA coding sequence with the sGFP coding sequence [44] (Figure 1A). The T-DNA construct contains a CaMV-35S promoter-driven sGFP ORF with the NOS (nopaline synthase) terminator, and NOS promoter-driven bar (bialaphos resistance gene) [45] ORF with the Arabidopsis rbcS (rubisco small subunit) terminator. Modified 35S promoters were made from the pSMABR35SsGFP binary vector by replacing its XbaI/BamHI/EcoRV sites within the 35Spro with each modified fragment (Figure 1A). Binary vectors were introduced into Agrobacterium strain EHA101 [46]. Transgenic gentian (Gentiana triflora 6G. scabra cv. Polano-White) and tobacco (Nicotiana tabacum cv. SR1) plants were obtained by Agrobacterium-mediated transformation as described in our previous study [11]. Transgenic gentian and tobacco plants were maintained in vitro by subculturing the shoots (approx. 20 mm lengths) on 0.25% (w/v) gellan gum-solidified MS medium containing 3% (w/v) sucrose every three months and two months, respectively. Southern and northern blot analyses For Southern blot analysis and bisulfite genomic sequencing, genomic DNAs were isolated from young leaves of in vitro growing PLoS ONE | www.plosone.org 8 March 2010 | Volume 5 | Issue 3 | e9670