Fig. 7

SlALKBH2 is transcriptionally regulated by DNA methylation. a The 5mC levels in the differentially methylated region (DMR) of SlALKBH2 promoter in wild-type (WT) and Cnr mutant fruit based on the Tomato Epigenome Database (http://ted.bti.cornell.edu/epigenome/). b The 5mC levels in the DMR of SlALKBH2 promoter in WT and sldml2 mutant fruit based on the DNA methylome database [38]. a, b The numbers indicate the cytosine positions relative to the start codon. Black represents the methylation frequency of cytosines at the indicated positions. DPA, days post-anthesis. c Schematic of the dual-luciferase system used for promoter activity assay. The SlALKBH2 promoter was cloned into the dual-luciferase reporter vector to activate the expression of firefly luciferase (Fluc). The renilla luciferase (Rluc) driven by the CaMV 35S promoter served as an internal control. LB, left border; RB, right border; Ter, terminator. d–f Co-expression of SlDML2 (SlDML2-HA) with the dual-luciferase reporter vector in the Nicotiana benthamiana leaves increased the relative Fluc activity (d), facilitated the Fluc gene expression (e), and reduced the 5mC level in SlALKBH2 promoter (f) compared with the empty plasmid control (HA). d The representative image from a total of six images (left panel). The Fluc activity was normalized against the Rluc activity, followed by normalization against the control (right panel). Data are presented as means ± standard deviation (n = 6). Asterisks indicate significant differences (***P < 0.0001; Student’s t test). e Gene expression was determined by quantitative RT-PCR analysis. Error bars represent the standard deviation of three independent experiments. Asterisks indicate significant differences (*P < 0.05; Student’s t test). f The box plot showing 5mC levels of all cytosines (n = 41) in the DMR analyzed by Sanger bisulfite sequencing. The plus sign represents the average level in each box