Fig. 1: The DRM2/RdDM pathway is essential for LEC2-induced somatic embryogenesis. | Nature Communications

Fig. 1: The DRM2/RdDM pathway is essential for LEC2-induced somatic embryogenesis.

From: LEC2 induces somatic cell reprogramming through epigenetic activation of plant cell totipotency regulators

Fig. 1

a Expression heatmap illustrating the upregulation of genes involved in the DNA methylation pathway during LEC2-induced somatic embryogenesis. b, c The expression pattern of gDRM2-GFP in pER8-LEC2 with DMSO (referred to as Mock, b) or β‐estradiol (ES) treatment (referred to as pER8-LEC2, c); hours after induction (HAI) are indicated. The magenta signal represents the cell wall stained with 4',6-diamidino-2-phenylindole (DAPI). Scale bars, 10 μm. d Upper panel: schematic representation of the 800 bp region upstream of the ATG codon (+ 1) of the DRM2 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 “b” region of the DRM2 promoter at 24 HAI. P-values are shown, two-tailed Student’s t test. e EMSA of GST-LEC2 and the DRM2 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. f Relative ratio of Firefly LUC to Renilla LUC (REN) activity in tobacco leaves. 35Spro::LEC2 was used as effector and DRM2pro::LUC as reporter. Control and LUC indicate the corresponding empty vectors. g–j Phenotypes of seedlings of indicated genotypes and treatments at 14 days after induction (DAI). White arrowheads in h and j point to somatic embryos (SEs). Scale bars, 0.5 mm. k, l The statistical analysis of the frequencies of somatic embryogenesis k and the average number of SEs per explant l. n, number of transgenic lines analyzed. The data in d, f, k are presented as means ± SD, with d, f representing results from three biological replicates. For box plots in l, 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 f, k, 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.

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