Figure 1.
Scheme of Experimental Procedures Leading to the Identification of MEGs and PEGs in Arabidopsis.
(A) Seeds derived after reciprocal crosses of Col-0 and Bur-0 accessions are phenotypically indistinguishable. Embyro (EMB), chalazal (CZE), micropylar (MPE) and peripheral (PE) endosperm are indicated. Scale bars, 50 µM. (B) Outline of filtering procedures leading to the identification of MEGs and PEGs.
Figure 2.
Allele-Specific Expression Analysis of MEGs and PEGs.
Seeds of reciprocal crosses of Col-0 and Bur-0 accessions were harvested at 4 DAP and allele-specific expression was tested by restriction-based allele-specific PCR analysis or sequencing. MEGs and PEGs that are imprinted in both directions of the cross are shown in (A) and (C), MEGs and PEGs that are accession-dependently imprinted are shown in (B) and (D). Asterisks indicate unspecific PCR bands. Size differences between controls and cDNA samples are caused by the presence of introns in amplified regions.
Figure 3.
Expression Analysis of MEGs and PEGs in Vegetative and Seed Tissues.
(A) Cluster analysis of MEGs and PEGs (including accession-dependent MEGs and PEGs) based on their expression in vegetative tissues and seeds. Each row represents a gene, and each column represents a tissue type. Tissue types are: seedlings, cotyledons, hypocotyl, leaves, stems, roots, shoot apical meristem (SAM), flowers at stages 10, 12, 15, siliques containing seeds with embryos in the globular to heart stage, heart stage and torpedo stage. Red or green indicate tissues in which a particular gene is highly expressed or repressed, respectively. (B) Cluster analysis of MEGs and PEGs (including accession-dependent MEGs and PEGs) based on their expression in embryo, endosperm and seed coat during different stages of seed development. Each row represents a gene, and each column represents a tissue type. Tissue types are: embryos from the preglobular stage to the mature stage, micropylar (MPE), peripheral (PE) and chalazal (CZE) endosperm derived from seeds containing embryos of the preglobular stage to the mature stage, and seed coat derived from seeds containing embryos of the preglobular stage to the mature stage. Red or green indicate tissues in which a particular gene is highly expressed or repressed, respectively. (C) Box plots of expression levels of MEGs (including accession-dependent MEGs; red) and PEGs (including accession-dependent PEGs; blue) compared to all genes (gray) in the chalazal endosperm region of seeds containing preglobular, globular and heart stage embryos. SLRs, Signal Log Ratios based on ATH1 microarray signals after RMA normalization.
Figure 4.
Impact of DNA Methylation and FIS PcG Function on the Regulation of Confirmed MEGs without Prominent Genic CG DNA Methylation.
(A) Allele-specific expression analysis of indicated MEGs in seeds derived from crosses of Col-0×Bur-0, Bur-0×Col-0, fis2×Bur-0 and Col-0×met1. Seeds were harvested at 4 DAP and allele-specific expression was tested by restriction-based allele-specific PCR analysis or sequencing. Asterisks indicate unspecific PCR bands. (B) Fold-changes of MEG expression in fis2 mutant seeds at 3 and 6 days after pollination (DAP) and from seeds derived from pollination with tetraploid pollen donors at 6 DAP compared to wild-type seeds at the corresponding time points. Data are based on ATH1 microarray signals after RMA normalization. Significantly deregulated genes are marked by an asterisk. (C) CG DNA methylation profiles of indicated MEGs in vegetative tissues (black line) or endosperm (red line) based on data published by [7], [41]. The gray bar represents the annotated gene body from transcription start (left) to transcription end (right). Red boxes represent transposable elements. Profiles are shown for 5% length intervals along the gene body and for 100 bp sequence intervals for the 2-kb regions upstream and downstream of each gene. The vertical dotted lines mark the gene body. The horizontal dashed line marks the DNA methylation level in vegetative tissues of TAIR8-annotated genes at the transcriptional start site. (D) H3K27me3 profiles of indicated MEGs in vegetative tissues (black line) or endosperm (red line) based on data published by [52], [64]. The gray bar represents the annotated gene body from transcription start (left) to transcription end (right). Red boxes represent transposable elements. Profiles are shown for 5% length intervals along the gene body and for 100 bp sequence intervals for the 2-kb regions upstream and downstream of each gene. The vertical dotted lines mark the gene body. The horizontal dashed line marks the H3K27me3 level of TAIR8-annotated genes at the transcriptional start site.
Figure 5.
Impact of DNA Methylation and FIS PcG Function on the Regulation of Confirmed MEGs with Prominent Genic CG DNA Methylation.
(A) Allele-specific expression analysis of indicated MEGs in seeds derived from crosses of Col-0×Bur-0, Bur-0×Col-0, fis2×Bur-0 and Col-0×met1. Seeds were harvested at 4 DAP and allele-specific expression was tested by restriction-based allele-specific PCR analysis or sequencing. (B) Fold-changes of MEG expression in fis2 mutant seeds at 3 and 6 days after pollination (DAP) and from seeds derived from pollination with tetraploid pollen donors at 6 DAP compared to wild-type seeds at the corresponding time points. Data are based on ATH1 microarray signals after RMA normalization. Significantly deregulated genes are marked by an asterisk. (C) CG DNA methylation profiles of indicated MEGs in vegetative tissues (black line) or endosperm (red line) based on data published by [7], [41]. The gray bar represents the annotated gene body from transcription start (left) to transcription end (right). Red boxes represent transposable elements. Profiles are shown for 5% length intervals along the gene body and for 100 bp sequence intervals for the 2-kb regions upstream and downstream of each gene. The vertical dotted lines mark the gene body. The horizontal dashed line marks the DNA methylation level in vegetative tissues of TAIR8-annotated genes at the transcriptional start site. (D) H3K27me3 profiles of indicated MEGs in vegetative tissues (black line) or endosperm (red line) based on data published by [52], [64]. The gray bar represents the annotated gene body from transcription start (left) to transcription end (right). Red boxes represent transposable elements. Profiles are shown for 5% length intervals along the gene body and for 100 bp sequence intervals for the 2-kb regions upstream and downstream of each gene. The vertical dotted lines mark the gene body. The horizontal dashed line marks the H3K27me3 level of TAIR8-annotated genes at the transcriptional start site.
Figure 6.
Impact of FIS PcG Function on the Regulation of Confirmed PEGs.
(A) Allele-specific expression analysis of indicated PEGs in seeds derived from crosses of Col-0×Bur-0, Bur-0×Col-0, fis2×Bur-0, and fie×Bur-0. Seeds were harvested at 4 DAP and allele-specific expression was tested by restriction-based allele-specific PCR analysis or sequencing. Asterisks indicate unspecific PCR bands. (B) Fold-changes of PEG expression in fis2 mutant seeds at 3 and 6 days after pollination (DAP) and from seeds derived from pollination with tetraploid pollen donors at 6 DAP compared to wild-type seeds at the corresponding time points. Data are based on ATH1 microarray signals after RMA normalization. Significantly deregulated genes are marked by an asterisk. (C) CG DNA methylation profiles of indicated PEGs in vegetative tissues (black line) or endosperm (red line) based on data published by [7], [41]. The gray bar represents the annotated gene body from transcription start (left) to transcription end (right). Red boxes represent transposable elements. Profiles are shown for 5% length intervals along the gene body and for 100 bp sequence intervals for the 2-kb regions upstream and downstream of each gene. The vertical dotted lines mark the gene body. The horizontal dashed line marks the DNA methylation level in vegetative tissues of TAIR8-annotated genes at the transcriptional start site. (D) H3K27me3 profiles of indicated PEGs in vegetative tissues (black line) or endosperm (red line) based on data published by [52], [64]. The gray bar represents the annotated gene body from transcription start (left) to transcription end (right). Red boxes represent transposable elements. Profiles are shown for 5% length intervals along the gene body and for 100 bp sequence intervals for the 2-kb regions upstream and downstream of each gene. The vertical dotted lines mark the gene body. The horizontal dashed line marks the H3K27me3 level of TAIR8-annotated genes at the transcriptional start site.
Figure 7.
Types of Transposable Elements in the Vicinity of MEGs and PEGs.
(A) Type of transposable elements present in MEGs (left panel) and PEGs (right panel) in comparison to their representation among detectable genes in our dataset (gray bars). TE superfamilies are as defined by TAIR (www.arabidopsis.org). (B) Distance of transposable elements in relation to the transcriptional start (5′ location) or stop (3′ location) of MEGs and PEGs.
Figure 8.
Some MEGs and PEGs Are Located in Clusters.
Chromosomal distribution of MEGs and accession-dependent MEGs (A) and PEGs and accession-dependent PEGs (B) along the chromosomes. Accession-dependent MEGs and PEGs are italicized. Genes located in clusters are boxed. Clustered MEGs and PEGs having homologs within the cluster are highlighted in green and orange, respectively. Non-imprinted homologs of clustered MEGs and PEGs are indicated by cross signs.