Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Jul;140(14):2953-60.
doi: 10.1242/dev.092981. Epub 2013 Jun 12.

RNA-directed DNA methylation regulates parental genomic imprinting at several loci in Arabidopsis

Thiet Minh Vu  1 , Miyuki Nakamura, Joseph P Calarco, Daichi Susaki, Pei Qi Lim, Tetsu Kinoshita, Tetsuya Higashiyama, Robert A Martienssen, Frédéric Berger
Affiliations

RNA-directed DNA methylation regulates parental genomic imprinting at several loci in Arabidopsis

Thiet Minh Vu et al. Development. 2013 Jul.

Abstract

In mammals and plants, parental genomic imprinting restricts the expression of specific loci to one parental allele. Imprinting in mammals relies on sex-dependent de novo deposition of DNA methylation during gametogenesis but a comparable mechanism was not shown in plants. Rather, paternal silencing by the maintenance DNA methyltransferase 1 (MET1) and maternal activation by the DNA demethylase DEMETER (DME) cause maternal expression. However, genome-wide studies suggested other DNA methylation-dependent imprinting mechanisms. Here, we show that de novo RNA-directed DNA methylation (RdDM) regulates imprinting at specific loci expressed in endosperm. RdDM in somatic tissues is required to silence expression of the paternal allele. By contrast, the repression of RdDM in female gametes participates with or without DME requirement in the activation of the maternal allele. The contrasted activity of DNA methylation between male and female gametes appears sufficient to prime imprinted maternal expression. After fertilization, MET1 maintains differential expression between the parental alleles. RdDM depends on small interfering RNAs (siRNAs). The involvement of RdDM in imprinting supports the idea that sources of siRNAs such as transposons and de novo DNA methylation were recruited in a convergent manner in plants and mammals in the evolutionary process leading to selection of imprinted loci.

Keywords: Arabidopsis; DNA methylation; Endosperm; Imprinting; RdDM; Seed.

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.
Potential imprinted genes regulated by the RdDM pathway. (A) Allele-specific RT-PCR analysis showed the maternal expression of At1g61090, At2g34880 and At3g21830. (B,C) Maternal expression of (B) SDC and (C) MOP9.5 was confirmed by RT-PCR sequencing chromatographs at selected SNP present in different accessions. RNA was extracted 3 days after pollination (DAP) from seeds obtained from reciprocal crosses between two different accessions containing single nucleotide polymorphisms (SNPs), which allow maternal and paternal expression of the genes to be distinguished. SNPs were recognized by enzyme restriction digestion or sequencing. Primer sequences and restriction enzymes used are provided in supplementary material Table S1. The maternally expressed gene FIS2 was used as a positive control. (D-G) qRT-PCR analysis of expression of candidate imprinted genes in seedlings from mutants in RdDM and MET1 pathways. drms strands from drm1, drm2. The qPCR results were normalized by ACT11 using three technical replicates and two biological replicates. Error bars indicate s.d. The y-axis represents levels relative to the ACT11 control.
Fig. 2.
Fig. 2.
Expression pattern of SDC. (A-F) Expression of HISTONE 2B fused to the MONOMERIC RED FLUORESCENT PROTEIN under the control of the SDC promoter (pSDC::H2B-RFP) in (A) ovules before fertilization, (C) seeds at 2 DAP and (E) mature pollen. (B,D,F) Confocal sections corresponding to the sections shown in A, C and E, respectively, show GFP signal reporting the expression of DNA LIGASE1, which marks all nuclei in ovules and seeds. The red signal in the cell wall surrounding the embryo sac (A) and endosperm (C) originates from autofluorescence. cc, central cell nucleus; ec, egg cell nucleus; s, s sperm nuclei; v, vegetative nucleus; end, endosperm; emb and arrowheads, embryo nuclei (confocal sections; RFP channel for A and C; GFP channel for B and D). Scale bars: 30 μm. (G) RT-PCR analysis of SDC expression in different parts of the developing seed (7 DAP), FWA is the positive control for the endosperm-specific expression; UBQ10 is the loading control. (H) Allele-specific RT-PCR analysis of SDC in reciprocal crosses among different accessions. RT-PCR was performed on total RNA extracted from 4 DAP seeds produced by reciprocal crosses between Cvi and the other accessions, including Col, RLD and Ler. ACT2 was used as a loading control.
Fig. 3.
Fig. 3.
Maternal expression of MOP9.5 in endosperm. (A) RT-PCR analysis of MOP9.5 expression in endosperm, embryo and seed coat isolated from 7 DAP seeds. (B) Allele-specific RT-PCR analysis of MOP9.5 expression in seeds produced by reciprocal crosses between Col and C24. RT-PCR was performed on total RNA extracted from 4 DAP seeds. Primers used to amplify MOP9.5 span an intron, therefore PCR products from genomic DNA and cDNA are different sizes. ACT2 is the loading control. (C) qRT-PCR analysis of expression levels of MOP9.5 and FWA in unfertilized ovules and 4 DAP seeds. qPCR results showed that levels of transcripts of MOP9.5 increased nearly three times in 4 DAP seeds compared with unfertilized ovules. Error bars indicate s.d.
Fig. 4.
Fig. 4.
RdDM silences the paternal alleles of imprinted genes. (A) Allele-specific RT-PCR analysis of parental origin of SDC transcripts. FWA and MINI3 are positive controls for maternal expression and bi-parental expression, respectively; GAPDH is the loading control. Total RNA was extracted from 2 DAP seeds. (B) The RFP patterns report the expression of SDC in in seeds from crosses between ovules carrying pSDC::H2B-RFP and wild-type pollen; and a wild-type mother plant crossed with pollen carrying pSDC::H2B-RFP; a wild-type mother plant crossed with nrpd2a pollen carrying pSDC::H2B-RFP. (confocal sections, light blue reports background auto-fluorescence from plastids that outlines endosperm nuclei; the red signal in the cell wall surrounding the endosperm originates from auto-fluorescence). In the central panel, red auto-fluorescence from bleached chloroplasts around endosperm was observed. chz marks the chalazal pole of endosperm. Scale bars: 20 μm. (C) Allele-specific RT-PCR analysis of MOP9.5 expression in seeds produced by reciprocal crosses between C24 × Col and C24 × nrpd2a-1, GAPDH was used as the loading control. (D) Allele-specific RT-PCR analysis of SDC expression in seeds from reciprocal crosses between Col and Cvi, and a Cvi mother crossed with a single mutant in the Col background, with met1-3/+, with nrpd2a and with the double mutant nrpd2a/met1-3/+. ACT2 is the loading control.
Fig. 5.
Fig. 5.
Origin of the activation of maternal alleles. (A) RT-qPCR analysis of SDC and MOP9.5 expression in dme mutant and wild-type seeds. FWA is used as the positive control. The transcripts levels were normalized against ACTIN11 (ACT11) transcripts levels in wild-type seeds. Error bars indicate s.d. (B) Expression of the PolIV/PolV major subunits in isolated female gametophytes. DD65 and DD31 were used as markers for central cells (CC) and synergid cells (SY) isolated from ovules (OV). RPS5A is used as the loading control.
Fig. 6.
Fig. 6.
Maternal maintenance of imprinted genes in endosperm. (A) The 5′ upstream region and transcriptional start site (TSS) of SDC. Triangles represent the 32 bp tandem repeat sequences. Gray boxes and the black box represent the 5′-UTR exon and coding sequence of SDC, respectively. (B) Percentage methylation of the 32 bp repeat region (left) and of the -247 bp to +206 bp region relative to the predicted TSS (right) in the embryo and endosperm (7 DAP). DNA isolated from dissected embryos and endosperm that had been crossed between RLD and Cvi were subjected to bisulfite sequencing (n=12). Conserved cytosine residues both in RLD and Cvi shown in supplementary material Fig. S4 were used for the analysis. (C,D) RT-PCR analysis of SDC (C) and MOP9.5 (D) expression in 3 DAP seeds collected from wild-type and mutant plants for SDC and MOP9.5. GAPDH is the loading control. (E) Light microscope pictures of wild-type seeds, mop9.5 (FLAG_508H08) and sdc (SALK_017593) in the seed at the globular embryo stage. Seeds were cleared by chloral hydrate (DIC microscopy). Scale bars: 100 μm.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bartolomei M. S. (2009). Genomic imprinting: employing and avoiding epigenetic processes. Genes Dev. 23, 2124–2133 - PMC - PubMed
    1. Belmonte M. F., Kirkbride R. C., Stone S. L., Pelletier J. M., Bui A. Q., Yeung E. C., Hashimoto M., Fei J., Harada C. M., Munoz M. D., et al. (2013). Comprehensive developmental profiles of gene activity in regions and subregions of the Arabidopsis seed. Proc. Natl. Acad. Sci. USA 110, E435–E444 - PMC - PubMed
    1. Berger F., Vu M.T., Li J., Chen B. (2013). Hypothesis: Selection of imprinted genes is driven by silencing deleterious gene activity in somatic tissues. Cold Spring Harb. Symp. Quant. Biol. 77, doi 10.1101/sqb.2012.77.014514 - PubMed
    1. Berger F., Twell D. (2011). Germline specification and function in plants. Annu. Rev. Plant Biol. 62, 461–484 - PubMed
    1. Borges F., Gomes G., Gardner R., Moreno N., McCormick S., Feijó J. A., Becker J. D. (2008). Comparative transcriptomics of Arabidopsis sperm cells. Plant Physiol. 148, 1168–1181 - PMC - PubMed

Publication types