Genome-wide demethylation of Arabidopsis endosperm

Abstract

Parent-of-origin-specific (imprinted) gene expression is regulated in Arabidopsis thaliana endosperm by cytosine demethylation of the maternal genome mediated by the DNA glycosylase DEMETER, but the extent of the methylation changes is not known. Here, we show that virtually the entire endosperm genome is demethylated, coupled with extensive local non-CG hypermethylation of small interfering RNA-targeted sequences. Mutation of DEMETER partially restores endosperm CG methylation to levels found in other tissues, indicating that CG demethylation is specific to maternal sequences. Endosperm demethylation is accompanied by CHH hypermethylation of embryo transposable elements. Our findings demonstrate extensive reconfiguration of the endosperm methylation landscape that likely reinforces transposon silencing in the embryo.

Fig. 1

Profiles of DNA methylation in embryo, wild-type endosperm, and dme endosperm. (A to F) TAIR8-annotated genes [(A), (C), and (E)] or transposons [(B), (D), and (F)] were aligned at the 5′ end (left panel) or the 3′ end (right panel), and average methylation levels for each 100-bp interval are plotted from 2 kb away from the gene (negative numbers) to 4 kb into the gene (positive numbers). Embryo methylation is represented by the red trace, wild-type (WT) endosperm by the blue trace, dme endosperm by the green trace, and aerial tissues by the black trace. The dashed line at zero represents the point of alignment. CG methylation is shown in (A) and (B), CHG in (C) and (D), CHH in (E) and (F).

Fig. 2

Associations between endosperm methylation, siRNAs, and expression. (A) Box plots showing siRNA abundance within 50-bp windows in the entire Arabidopsis genome (All) and in sequences hypermethylated in WT endosperm compared with the embryo in the CHG and CHH contexts. (B) Box plots showing differences in gene expression between embryo and endosperm for all genes (n = 21,021), genes with 5′ hypomethylation in endosperm (n = 1097), and genes with 3′ hypomethylation in endosperm (n = 505). Each box encloses the middle 50% of the distribution, with the horizontal line marking the median and the dot marking the mean. The lines extending from each box mark the minimum and maximum values that fall within 1.5 times the height of the box.

Fig. 3

Genome-wide demethylation of endosperm. (A to C) Kernel density plots of the differences between embryo and WT endosperm methylation (blue trace) and the differences between embryo and dme endosperm methylation (red trace). The green trace in (B) and (C) represents methylation differences between embryo and dme endosperm for windows with absolute fractional methylation increase in WT endosperm compared with embryo of at least 0.4 in the CHG context (B) (n = 135) or at least 0.2 in the CHH context (C) (n = 6168). Methylation differences for the 3′ MEA repeats, FWA, FIS2, PHE1, and MPC are indicated; specifics are listed in table S2. (D and E) All TAIR8-annotated genes (28,244) were aligned at the 5′ end and stacked from the top of chromosome 1 to the bottom of chromosome 5. Embryo methylation is displayed as a heat map in the left panel, differences between embryo and WT endosperm in the right panel. CG methylation is shown in (D), CHG in (E).