(A–B) Confocal images showing H1 localization under native promoter (A) and VC-specific promoter (pVC, B) during male gametogenesis. Msp, microspore; BCP, bicellular pollen; TCP, tricellular pollen. Bars, 5 μm. All pVC::H1.1-mRFP (short as pVC::H1) refers to line #2. (C) Ends analysis of all TEs or genes in VCs from pVC::H1 (line #2) and WT. (D–E) Kernel density plots illustrating frequency distribution of methylation differences in 50 bp windows between VCs from pVC::H1 and WT (D), and between WT sperm (Spm) and VC (E). (F) Snapshots showing CG methylation difference between the indicated cell types. Arrows point to DME targets that are hypermethylated by pVC::H1. (G) Snapshots demonstrating CG methylation in sperm and VCs at single-nucleotide resolution, with the cytosine most hypomethylated by DME marked in red. VC DME targets are underlined in black. (H) Scatter plot illustrating CG methylation differences between the indicated cell types at H1 hyperDMRs. 82.25% of H1 hyperDMRs show significant increase in sperm in comparison to VCs. (I) Box plot illustrating H3K9me2 level at VC DME targets that are significantly hypermethylated in pVC::H1 (H1-inhibited) or not (H1-independent), respectively. Difference between the two groups is significant (Kolmogorov-Smirnov test p<0.001). (J) VC DME targets were grouped according to H3K9me2 levels, aligned at the most demethylated cytosine (dashed lines), and plotted for average CG methylation difference as indicated in each 10 bp interval (left). Similarly, CG methylation in pVC::H1 and WT VCs was plotted for the group with the lowest and highest H3K9me2, respectively. Spm, sperm.