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First published online 26 February 2008
doi: 10.1242/jcs.023077

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Mechanism of PHERES1 imprinting in Arabidopsis

Institute of Plant Sciences and Zurich-Basel Plant Science Center, Swiss Federal Institute of Technology, ETH Centre, CH-8092 Zurich, Switzerland

View larger version (32K): [in this window] [in a new window]   Fig. 1. The maternal PHE1 allele is not reactivated in DNA-methylation-defective mutants. Allelic expression analysis of PHE1 in wild-type and met1, cmt3 and drm1/drm2 mutant plants after crosses of wild-type and mutant plants with the C24 accession. Analysis was performed before fertilization (0 DAP) and at indicated days after pollination (DAP). g, genomic DNA.

View larger version (41K): [in this window] [in a new window]   Fig. 2. DNA methylation is important for paternal PHE1 expression. (A) Relative PHE1 mRNA levels in wild-type flowers before fertilization (0 DAP) and siliques after pollination with wild-type or met-mutant pollen harvested at different days after pollination (DAP). Because the maternal PHE1 allele is silent, the detected PHE1 expression represents mostly the activity of the paternal PHE1 allele. (B) Allelic expression analysis of PHE1 after pollination of C24 plants with wild-type or met1-mutant pollen. Analysis was performed at DAP1 to DAP4.

View larger version (33K): [in this window] [in a new window]   Fig. 3. The 3' region of PHE1 contains DNA-methylation marks. (A, left) Southern blot of DNA from wild-type and met1-mutant plants after digestion with the methylation-sensitive restriction enzyme ClaI. (Right) Overview of the PHE1 locus with large and small arrows corresponding to the PHE1 coding region and repeats, respectively. The expected fragment sizes and the region covered by the probe are indicated. The methylated ClaI site is indicated by a filled circle, the repeat regions by arrows. (B) Cytosine methylation profile of the PHE1 downstream region in different tissues analyzed by bisulfite sequencing. Cytosine positions relative to the translational stop codon and sequence contexts are indicated on the x-axis. The ClaI site corresponds to position 2538. The methylation status in the met1 mutant was analyzed in flowers.

View larger version (42K): [in this window] [in a new window]   Fig. 4. Establishment of imprinting at the PHE1 locus is compromised in mutants whose PHE1 downstream region is disrupted. (A) Schematic overview of the location of the phe1-3-, phe1-4- and phe1-5-mutant alleles. Probes used for northern blot analysis are indicated. (B) Allelic expression analysis of PHE1 after reciprocal crosses of wild-type and phe1-3 mutant plants with the C24 accession. (C) Expression analysis of PHE1 in leaves of wild-type and phe1-3-mutant plants. (D) Northern blot analysis of RNA from leaves of wild-type and phe1-3-mutant plants with probes indicated in panel (A). (E) Allelic expression analysis of PHE1 in wild-type and phe1-4- and phe1-4-mutant plants after crosses of wild-type and mutant plants with the C24 accession. Lb, left border; Rb, right border; g, genomic DNA; ACT, ACTIN; wt, wild-type.

View larger version (24K): [in this window] [in a new window]   Fig. 5. Cytosine methylation profile of the PHE13000::GUS_3' transgene in wild-type and drm1/drm2-mutant background compared with the methylation profile of the endogenous PHE1 locus analyzed by bisulfite sequencing. Cytosine positions relative to the translational stop codon and sequence contexts (CG and CNG, CNN not indicated) are indicated on the x-axis.

View larger version (64K): [in this window] [in a new window]   Fig. 6. The PHE13000::GUS_3' transgene is maternally imprinted. (A,B) Expression analysis of a maternally or paternally derived (A) PHE13000::GUS_3' or (B) PHE13000::GUS transgene in seeds at DAP3. (C) Expression analysis of a paternally derived PHE13000::GUS_3' transgene in a wild-type or drm1/drm2 mutant background in seeds at DAP3. Quantification of results are shown in the respective panels on the right. Scale bars, 50 µm.

View larger version (22K): [in this window] [in a new window]   Fig. 7. Model for the establishment of PHE1 imprinting. In the central cell of the female gametophyte the FIS-PcG complex binds to a polycomb response element (PRE) in the promoter region of PHE1. The differentially methylated region (DMR) is unmethylated in the central cell of the female gametophyte and blocks transcription together with the FIS complex, resulting in stable PHE1 repression in the endosperm. The FIS complex is absent in pollen und the methylated DMR prevents silencing activity, causing the paternal allele to be active in the endosperm. In met1-mutant pollen, the DMR is demethylated and can block PHE1 expression. Whether this involves PcG complexes is currently not known. Maternal alleles are shown in red, paternal alleles in blue.

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