Fig. 3: The SUVH-SDJ complex binding to the CHH hypermethylated regions of totipotency-regulating genes is required for LEC2-induced SE formation.

a Upper panel: schematic representation of the 400 bp region upstream of the ATG codon (+ 1) of the SUVH1 gene. PCR fragments labeled “a” to “c” were utilized for the ChIP-qPCR analysis, with “c” serving as a negative control. Lower panel: ChIP-qPCR of LEC2-GFP showing enrichment of the “a” region of the SUVH1 promoter at 96 HAI. P-values are shown, two-tailed Student’s t test. b EMSA of GST-LEC2 and the SUVH1 promoter region containing the LEC2 binding site. Four replicates showed similar results. The black arrowheads indicate the position of the shifted bands, non-specific bands, and the free probe, respectively. c Relative ratio of firefly LUC to REN activity in tobacco leaves. 35Spro::LEC2 was used as effector and SUVH1pro::LUC as reporter. d–i Phenotypes of seedlings of the indicated genotypes at 14 DAI. White arrowheads in d, e, and h point to individual SEs. Scale bars, 0.5 mm. j, k The statistical analysis of the frequencies of somatic embryogenesis calculated as the proportion of explants with at least one somatic embryo (SE) relative to all explants (j) and the average number of SEs per explant (k). n, number of transgenic lines analyzed. l ChIP-qPCR of SUVH1-6Myc showing enrichment of CHH methylation regions at the indicated gene promoters at 96 HAI. mCHH represents CHH hypermethylated regions. The data in a, c, j, l are presented as means ± SD, with (a, c, l) representing results from three biological replicates. For box plots in k, the horizontal line represents the median value, the boxes represent the interquartile range (25th to 75th percentiles), and the whiskers extend to the maximum and minimum values. Dots indicate individual values. Different letters in (c, j–l) indicate significant differences (P < 0.05), one-way ANOVA with Tukey’s multiple comparison test. Source data are provided as a Source Data file.