Fig. 5

Chromatin regulates small RNA transcription. a Representative expansion microscopy images of DAPI-stained nuclei (cyan), fibrillarin (yellow), and tubulin (red) in zygote-containing seeds (top). Increased magnification of seed coat and zygote sections marked in white squares (bottom). zy, zygote; en, endosperm; sc, seed coat. Scale bars represent 20 μm. b Violin and dot plot representing the DAPI-stained area of seed coat (sc) and zygote (zy) nuclei. Horizontal and vertical white colored bars represent median and interquartile range, respectively. P values based on the Wilcoxon rank sum test of DAPI area differences between seed coat (sc) and zygote (zy) are shown. c Circos plot representing densities of euchromatic (red) and heterochromatic (purple) TEs, and 5S rRNA loci on chromosomes 3, 4, and 5. Centromeres are indicated by dots. d Metaplots of 24-nt siRNA levels in h1 mutants relative to wild-type leaves for heterochromatic (top) or euchromatic (bottom) TEs. e–h TEs were divided into percentiles, ordered based on their 24-nt siRNA levels in h1 mutants, and plotted according to proportion of euchromatic and heterochromatic TEs (e), CHH methylation levels in h1 relative to wild type [27] (f), and relative enrichments of H1.1 (g) and H3K9me2 (h) [58]. i, j Metaplots displaying nucleosome occupancy of long heterochromatic TE groups 1–3 according to MNase-seq datasets [59] (i) or 24-nt siRNA levels (j). Annotated TE 5′ and 3′ ends are labeled and indicated by vertical gray lines. k Boxplots of 25–30-nt precursor siRNA levels of euchromatic (red) and heterochromatic (purple) TEs in dcl2/3/4 floral buds (fb), globular embryos (gl), and leaves (lf). Thick horizontal bars indicate medians, and the top and bottom edges of the box indicate the 75th and 25th percentiles, respectively. P values < 0.0001 based on Mann-Whitney U test of differences between globular and floral bud or leaf 25-to-30-nt siRNAs derived from heterochromatic TEs (purple) tissues are represented by ****. l Metaplots of 25–30-nt siRNA levels overlapping long heterochromatic TEs in dcl2/3/4 floral buds (fb), globular embryos (gl), and leaves (lf) (key). Annotated TE 5′ and 3′ ends are labeled at the bottom. See also Additional file 2: Figure S5