(A) Sperm ATAC-seq profiles reflect the reprogrammed state of sporophytic H3K27me3 target genes. Plotted are averaged levels of leaf H3K27me3 and sperm H3K4me3 (log2 ChIP-seq enrichment relative to control) together with sperm ATAC-seq signals. ChIP-seq data and clustering for the presence and absence of H3K4me3 enrichment in sperm were described previously (Borg et al., 2020). (B) Heatmap summarising the overlap enrichment of SN-specific, VN-specific, and common accessible chromatin regions (ACRs) with somatic Polycomb target genes, DUO1 target genes, and sperm-enriched genes. Fold-enrichment was determined using hypergeometric tests compared with random Arabidopsis genomic regions (n = 10,000,000 permutations). (C) Levels of H3K27me3 marks in leaf tissue (log2 ChIP-seq enrichment relative to H3) at SN-specific, VN-specific, and common ACRs. Each boxplot indicates minimum and maximum values as well as 25th, 50th, and 75th quartiles. (D) Relative proportion of SN-specific ACRs that overlap with a somatic H3K27me3 domain. (E) Distribution of genomic features associated with SN-specific ACRs. (F) Expression status of genes in the vicinity (<900 bp) of an SN-specific ACR with or without H3K27me3 in the sporophyte. Genes were classified as being expressed in sperm (TPM >1, light blue), by having enriched expression in sperm (dark blue), or by expression in the early zygote or embryos but not sperm (pink). Non-expressed genes in sperm are shown in grey. TPM >1 or <1 was used as a threshold for expressed and non-expressed genes, respectively. Statistical enrichment was determined using pairwise two-sided Fisher’s exact tests. (G) Chromatin accessibility at three sperm-specific genes associated with sperm differentiation – DUO1-ACTIVATED ZINC FINGER 1 (DAZ1), SHORT SUSPENSOR 1 (SSP1) and PLANT CADMIUM RESISTANCE 11 (PCR11). Tracks represent the ATAC-seq signal normalised to 1× Arabidopsis genome coverage for nuclei of the microspore (MN), germ cell (GN), sperm (SN), and vegetative cell (VN). (H) Differential expression between htr10, elf6;ref6;jmj13, and elf6;ref6;jmj13;htr10 pollen relative to wild type (WT). Each boxplot indicates the minimum and maximum values as well as 25th, 50th, and 75th quartiles. Sample size (n) of genes associated with an H3K27me3-repressed ACR (blue) and all sperm-expressed genes (grey) is shown. Plot is restricted to genes with >10 counts in at least one RNA-seq sample and/or replicate. Statistical analysis was performed using two-sided Kolmogorov–Smirnov tests. (I) Chromatin accessibility at four major developmental regulators transcribed during early sporophyte development – BABY BOOM (BBM), FLOWERING LOCUS C (FLC), LEAFY COTYLEDON 1 (LEC1), and WUSCHEL-RELATED HOMEBOX 2 (WOX2). Tracks represent the ATAC-seq signal normalised to 1× Arabidopsis genome coverage for nuclei of the microspore (MN), germ cell (GN), sperm (SN), and vegetative cell (VN). (J) Averaged ATAC-seq enrichment over genes with enriched expression during early (pink), mid (yellow), and late (grey) embryogenesis, which represent the pre-cotyledon phase, the transition phase, and the mature green phase, respectively (Hofmann et al., 2019). Plotted is the sperm ATAC-seq signal normalised to 1× Arabidopsis genome coverage.