plants using a GenElute Plant Genomic DNA Miniprep kit (Sigma-Aldrich) following the supplier’s instructions. HindIII- digested genomic DNAs (5-mg aliquots) were separated by electrophoresis on 0.8% (w/v) agarose gels, blotted onto nylon membranes, and then fixed by UV irradiation. The blots were hybridized with bar and sGFP gene probes, which were prepared by the AlkPhos Direct Labeling System (GE Healthcare) following the supplier’s instructions. For northern blot analysis, total RNAs were isolated from young leaf tissues using the Concert Plant RNA reagent (Invitrogen) following the supplier’s instructions. The isolated total RNAs (10 mg) were separated by electrophoresis on 1.2% (w/v) agarose- formaldehyde gels, blotted onto nylon membranes, and then fixed by UV irradiation. The same probes as used for Southern analysis were also used for northern hybridization. Hybridization, membrane washing and detection procedures were performed following the supplier’s instructions (AlkPhos Direct Detection System; GE Healthcare). To prepare bar and sGFP gene probes, the following primer pairs were used. For the 389-bp of bar gene ORF fragment, 59- GGATCCATGAGCCCAGAACG-39 (forward; F) and 59-AGC- CCGATGACAGCGACCAC-39 (reverse; R) were used. For the 706-bp of sGFP gene ORF fragment, 59-TGGTGAGCAAGGGC- GAG-39 (F) and 59-TCGTCCATGCCGAGAGTGAT-39 (R) were used. Bisulfite genomic sequencing Bisulfite genomic sequencing [20] was performed as described in our previous study [11], or by using an EpiTect Bisulfite Kit (Qiagen) following the supplier’s instructions, except for the bisulfite reaction conditions. For the EpiTect Bisulfite Kit, the bisulfite conversion reaction was performed in a thermal cycler as follows: 99.9uC for 10 min, 60uC for 25 min, 99.9uC for 10 min, 60uC for 85 min, 5 cycles of 99.9uC for 10 min and 60uC for 180 min, then maintaining at 20uC until the next step. PCR reaction using bisulfite-treated genomic DNA as a template and subsequent cloning and sequencing was carried out as described in the previous study [11], except that the pSTBlue-1 vector (Novagen) was used for cloning. PCR was performed in 15 ml of AccuPrime Taq buffer II (Invitrogen) containing primers for top-strand amplification (concentrations of each primer are shown in Table S2), 0.5 ml AccuPrime Taq polymerase, and a 1–2 ml aliquot of bisulfite-treated DNA. The PCR reaction was performed as follows: 95uC for 2 min 30 sec, 5 cycles of 95uC for 30 sec, 55uC for 35 sec and 68uC for 1 min 30 sec, and 35 cycles of 95uC for 30 sec, 55uC for 35 sec and 68uC for 1 min. EMSA Nuclear isolation was performed based on the methods of CelLyticTM PN (Sigma-Aldrich) and [28] with some modifications. Five grams of young leaves of wild-type gentian or tobacco plants were frozen in liquid nitrogen and ground to a powder. The powdered tissues were incubated with 40 ml of cold extraction buffer A [50 mM HEPES (pH 7.5), 0.4 M Sucrose, 5 mM MgCl2, 5 mM dithiothreitol (DTT), 0.1 mM phenylmethylsulfonyl fluo- ride (PMSF) and 0.4 mM Pefabloc SC] for 15 min on ice. The suspension was filtrated through two layers of Miracloth (Calbiochem), and centrifuged for 10 min at 7006g. The pellet was gently re-suspended in 1000 ml of extraction buffer B [extraction buffer A with 0.3% (v/v) Triton X-100], and the suspension was slowly transferred onto a 1500 ml of sucrose cushion (extraction buffer B with 1.8 M sucrose) and centrifuged for 10 min at 120006g. The supernatant was removed, and the 35S De Novo Methylation pellet was re-suspended in 60 ml of lysis buffer [20 mM HEPES (pH 7.9), 0.5 M NaCl, 20% (v/v) glycerol, 0.1 mM EDTA, 1mM DTT, 0.1 mM PMSF and 0.4 mM Pefabloc SC] and incubated for 30 min on ice. The suspension was centrifuged for 10 min at 12,0006g, and the supernatant was desalted using Micro Bio-Spin 6 Chromatography columns (Bio-Rad). All steps were carried out at 4uC. Production of DIG 39 end-labeled probes and detection of shift bands were performed by the DIG Gel Shift Kit, 2nd Generation (Roche). The oligonucleotides used for EMSA are listed in Table S3. Five micrograms of nuclear extracts were used for each reaction. Binding reactions (20 ml) contained 10 fmol of DIG 39 end-labeled probe, 20 mM HEPES (pH 7.6), 1 mM EDTA, 10 mM (NH4)2SO4, 1 mM DTT, 0.2% (v/v) Tween 20, 50 mM KCl, 5 mM MgCl2, 2 mg poly(dI-dC) and competitor DNA. Five microliters of loading buffer [0.256TBE, 60% (v/v) glycerol] was added to each reaction after 20 min of incubation at 25uC. Reactions were separated by electrophoresis on a 4% polyacryl- amide gel in 0.56TBE buffer at 4uC, electric blotted onto a nylon membrane (Biodyne PLUS; PALL), and fixed by UV irradiation. Supporting Information Figure S1 GFP expression of the transgenic gentian calli. GFP fluorescence images of unmodified or modified 35Spro introduced transgenic gentian callus lines were obtained by FluorImager595 using 530DF30 filter with argon ion laser excitation (488nm). Untransformed gentian callus was used as a negative control (below center; bar = 10 mm). All the transgenic lines (line numbers are indicated at lower left) were single copy. Found at: doi:10.1371/journal.pone.0009670.s001 (0.46 MB PDF) Figure S2 Representation of CpG, CpWpG and CpHpH methylation of the (modified) 35S-sGFP regions in the single copy transgenic gentians. (A–H) Cytosine methylation was analyzed in the unmodified 35S (A), 35S(Das-1) (B), 35S(nos-1) (C), 35S(PhCHS) (D), 35S(GtCHS) (E), 35S(Dcore) (F) and 35S core (G) transgenic gentian plants. Unmodified 35S transgenic tobacco plants were also analyzed as a control (H). Cytosine methylation patterns of the 35S enhancer region (2244 to 241) on the complementary (lower) strands were also analyzed in the unmodified 35S gentian lines #3, #5, #8 and #15 (I). The percentage of methylated cytosine is represented by bar charts (red, CpG; green, CpWpG; black, CpHpH), and each position of cytosines are represented below (black, 35Spro; blue, sGFP). Positions of start codons are indicated in pink, and positions of the known elements within the promoter regions are indicated by different colors (TATA box, aqua; as-1, olive; as-2, orange). Found at: doi:10.1371/journal.pone.0009670.s002 (1.15 MB PDF) Figure S3 Expressions of the sGFP and bar transgenes in the modified 35S transgenic gentians. Northern blot analyses of sGFP and bar transgenes in leaf tissues of the single copy 35S(nos-1) (A), 35S(PhCHS) (B), 35S(GtCHS) (C), 35S core and 35S(Dcore) (D) transgenic gentian plants are shown. Wild-type (WT) gentian plant and unmodified 35S transgenic tobacco plant line #26 (designated as C) was used as a control. For comparison, unmodified 35S transgenic gentian plant #8 (35S-8) was also analyzed. Found at: doi:10.1371/journal.pone.0009670.s003 (3.13 MB PDF) Figure S4 Representation of CpG, CpWpG and CpHpH methylation of NOS-bar and rbcS-35S regions in the unmodified 35S transgenic gentian lines, #15 and #19. (A) A schematic PLoS ONE | www.plosone.org 9 March 2010 | Volume 5 | Issue 3 | e9670