Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Paternal easiRNAs regulate parental genome dosage in Arabidopsis

Nature Genetics volume 50, pages 193–198 (2018)Cite this article

Subjects

Abstract

The regulation of parental genome dosage is of fundamental importance in animals and plants, as exemplified by X-chromosome inactivation and dosage compensation. The ‘triploid block’ is a classic example of dosage regulation in plants that establishes a reproductive barrier between species differing in chromosome number1,2. This barrier acts in the embryo-nourishing endosperm tissue and induces the abortion of hybrid seeds through a yet unknown mechanism3. Here we show that depletion of paternal epigenetically activated small interfering RNAs (easiRNAs) bypasses the triploid block in response to increased paternal ploidy in Arabidopsis thaliana. Paternal loss of the plant-specific RNA polymerase IV suppressed easiRNA formation and rescued triploid seeds by restoring small-RNA-directed DNA methylation at transposable elements (TEs), correlating with reduced expression of paternally expressed imprinted genes (PEGs). Our data suggest that easiRNAs form a quantitative signal for paternal chromosome number and that their balanced dosage is required for post-fertilization genome stability and seed viability.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Pol IV-mutant pollen is able to rescue seed abortion in triploid seeds.
Fig. 2: Pol IV is responsible for the biogenesis of pollen TE-derived sRNAs in the size range of 19–24 nt.
Fig. 3: Increase of pollen-derived 21/22-nt easiRNAs correlates with lower CHH methylation in the endosperm of triploid seeds.
Fig. 4: Pollen-derived 21/22-nt easiRNAs associate with gene expression changes in the endosperm of triploid seeds.

Similar content being viewed by others

References

  1. Johnston, S. A., den Nijs, T. P., Peloquin, S. J. & Hanneman, R. E. Jr. The significance of genic balance to endosperm development in interspecific crosses. Theor. Appl. Genet. 57, 5–9 (1980).

    Article  CAS  PubMed  Google Scholar 

  2. Schatlowski, N. & Köhler, C. Tearing down barriers: understanding the molecular mechanisms of interploidy hybridizations. J. Exp. Bot. 63, 6059–6067 (2012).

    Article  CAS  PubMed  Google Scholar 

  3. Scott, R. J., Spielman, M., Bailey, J. & Dickinson, H. G. Parent-of-origin effects on seed development in Arabidopsis thaliana. Development 125, 3329–3341 (1998).

    CAS  PubMed  Google Scholar 

  4. Rodrigues, J. A. & Zilberman, D. Evolution and function of genomic imprinting in plants. Genes Dev. 29, 2517–2531 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Kradolfer, D., Wolff, P., Jiang, H., Siretskiy, A. & Köhler, C. An imprinted gene underlies postzygotic reproductive isolation in Arabidopsis thaliana. Dev. Cell 26, 525–535 (2013).

    Article  CAS  PubMed  Google Scholar 

  6. Wolff, P., Jiang, H., Wang, G., Santos-González, J. & Köhler, C. Paternally expressed imprinted genes establish postzygotic hybridization barriers in Arabidopsis thaliana. eLife 4, https://doi.org/10.7554/eLife.1 (2015).

  7. Lu, J., Zhang, C., Baulcombe, D. C. & Chen, Z. J. Maternal siRNAs as regulators of parental genome imbalance and gene expression in endosperm of Arabidopsis seeds. Proc. Natl. Acad. Sci. USA 109, 5529–5534 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. d’Erfurth, I. et al. Turning meiosis into mitosis. PLoS Biol. 7, e1000124 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  9. Law, J. A. & Jacobsen, S. E. Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat. Rev. Genet. 11, 204–220 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Havecker, E. R. et al. The Arabidopsis RNA-directed DNA methylation argonautes functionally diverge based on their expression and interaction with target loci. Plant Cell 22, 321–334 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. McCue, A. D., Nuthikattu, S., Reeder, S. H. & Slotkin, R. K. Gene expression and stress response mediated by the epigenetic regulation of a transposable element small RNA. PLoS Genet. 8, e1002474 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Nuthikattu, S. et al. The initiation of epigenetic silencing of active transposable elements is triggered by RDR6 and 21-22 nucleotide small interfering RNAs. Plant Physiol. 162, 116–131 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Creasey, K. M. et al. miRNAs trigger widespread epigenetically activated siRNAs from transposons in Arabidopsis. Nature 508, 411–415 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. McCue, A. D. et al. ARGONAUTE 6 bridges transposable element mRNA-derived siRNAs to the establishment of DNA methylation. EMBO J. 34, 20–35 (2015).

    Article  CAS  PubMed  Google Scholar 

  15. Martínez, G., Panda, K., Köhler, C. & Slotkin, R. K. Silencing in sperm cells is directed by RNA movement from the surrounding nurse cell. Nat. Plants 2, 16030 (2016).

    Article  PubMed  Google Scholar 

  16. Slotkin, R. K. et al. Epigenetic reprogramming and small RNA silencing of transposable elements in pollen. Cell 136, 461–472 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Li, S. et al. Detection of Pol IV/RDR2-dependent transcripts at the genomic scale in Arabidopsis reveals features and regulation of siRNA biogenesis. Genome Res. 25, 235–245 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Zhai, J. et al. A one precursor one siRNA model for Pol IV-dependent siRNA biogenesis. Cell 163, 445–455 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Wei, W. et al. A role for small RNAs in DNA double-strand break repair. Cell 149, 101–112 (2012).

    Article  CAS  PubMed  Google Scholar 

  20. Gasciolli, V., Mallory, A. C., Bartel, D. P. & Vaucheret, H. Partially redundant functions of Arabidopsis DICER-like enzymes and a role for DCL4 in producing trans-acting siRNAs. Curr. Biol. 15, 1494–1500 (2005).

    Article  CAS  PubMed  Google Scholar 

  21. Vazquez, F. et al. Endogenous trans-acting siRNAs regulate the accumulation of Arabidopsis mRNAs. Mol. Cell 16, 69–79 (2004).

    Article  CAS  PubMed  Google Scholar 

  22. Peragine, A., Yoshikawa, M., Wu, G., Albrecht, H. L. & Poethig, R. S. SGS3 and SGS2/SDE1/RDR6 are required for juvenile development and the production of trans-acting siRNAs in Arabidopsis. Genes Dev. 18, 2368–2379 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Schatlowski, N. et al. Hypomethylated pollen bypasses the interploidy hybridization barrier in Arabidopsis. Plant Cell 26, 3556–3568 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Brennecke, J. et al. An epigenetic role for maternally inherited piRNAs in transposon silencing. Science 322, 1387–1392 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Martienssen, R. A. Heterochromatin, small RNA and post-fertilization dysgenesis in allopolyploid and interploid hybrids of Arabidopsis. New Phytol. 186, 46–53 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Kato, A. & Birchler, J. A. Induction of tetraploid derivatives of maize inbred lines by nitrous oxide gas treatment. J. Hered. 97, 39–44 (2006).

    Article  CAS  PubMed  Google Scholar 

  27. Baumberger, N. & Baulcombe, D. C. Arabidopsis ARGONAUTE1 is an RNA Slicer that selectively recruits microRNAs and short interfering RNAs. Proc. Natl. Acad. Sci. USA 102, 11928–11933 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Lobbes, D., Rallapalli, G., Schmidt, D. D., Martin, C. & Clarke, J. SERRATE: a new player on the plant microRNA scene. EMBO Rep. 7, 1052–1058 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Zheng, X., Zhu, J., Kapoor, A. & Zhu, J. K. Role of Arabidopsis AGO6 in siRNA accumulation, DNA methylation and transcriptional gene silencing. EMBO J. 26, 1691–1701 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Allen, E. et al. Evolution of microRNA genes by inverted duplication of target gene sequences in Arabidopsis thaliana. Nat. Genet. 36, 1282–1290 (2004).

    Article  CAS  PubMed  Google Scholar 

  31. Herr, A. J., Jensen, M. B., Dalmay, T. & Baulcombe, D. C. RNA polymerase IV directs silencing of endogenous DNA. Science 308, 118–120 (2005).

    Article  CAS  PubMed  Google Scholar 

  32. Pontier, D. et al. Reinforcement of silencing at transposons and highly repeated sequences requires the concerted action of two distinct RNA polymerases IV in Arabidopsis. Genes Dev. 19, 2030–2040 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Xie, Z. et al. Genetic and functional diversification of small RNA pathways in plants. PLoS Biol. 2, E104 (2004).

    Article  PubMed  PubMed Central  Google Scholar 

  34. Hehenberger, E., Kradolfer, D. & Köhler, C. Endosperm cellularization defines an important developmental transition for embryo development. Development 139, 2031–2039 (2012).

    Article  CAS  PubMed  Google Scholar 

  35. Moreno-Romero, J., Jiang, H., Santos-González, J. & Köhler, C. Parental epigenetic asymmetry of PRC2-mediated histone modifications in the Arabidopsis endosperm. EMBO J. 35, 1298–1311 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Schoft, V. K. et al. SYBR Green-activated sorting of Arabidopsis pollen nuclei based on different DNA/RNA content. Plant Reprod. 28, 61–72 (2015).

    Article  CAS  PubMed  Google Scholar 

  37. Goto, K. & Meyerowitz, E. M. Function and regulation of the Arabidopsis floral homeotic gene PISTILLATA. Genes Dev. 8, 1548–1560 (1994).

    Article  CAS  PubMed  Google Scholar 

  38. Harris, C. J., Molnar, A., Müller, S. Y. & Baulcombe, D. C. FDF-PAGE: a powerful technique revealing previously undetected small RNAs sequestered by complementary transcripts. Nucleic Acids Res. 43, 7590–7599 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Trapnell, C., Pachter, L. and Salzberg, S. L. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25, 1105–1111 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Feng, J. et al. GFOLD: a generalized fold change for ranking differentially expressed genes from RNA-seq data. Bioinformatics 28, 2782–2788 (2012).

    Article  CAS  PubMed  Google Scholar 

  41. Hong, F. et al. RankProd: a bioconductor package for detecting differentially expressed genes in meta-analysis. Bioinformatics 22, 2825–2827 (2006).

    Article  CAS  PubMed  Google Scholar 

  42. Johnson, N. R., Yeoh, J. M., Coruh, C. & Axtell, M. J. Improved placement of multi-mapping small RNAs. G3 (Bethesda) 6, 2103–2111 (2016).

    Article  CAS  Google Scholar 

  43. Krueger, F. & Andrews, S. R. Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications. Bioinformatics 27, 1571–1572 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank R. Martienssen and F. Borges for critical comments on the manuscript. We thank H. Jiang for contributing data to Fig. 1. This research was supported by a European Research Council Starting Independent Researcher grant 280496 (to C.K.), a grant from the Swedish Science Foundation 2014-3820 (to C.K.), and a grant from the Knut and Alice Wallenberg Foundation 2012.0087 (to C.K.). G.M. was supported by a Marie Curie IOF Postdoctoral Fellowship (PIOF-GA-2012-330069). Sequencing was performed by the SNP&SEQ Technology Platform, Science for Life Laboratory at Uppsala University, a national infrastructure supported by the Swedish Research Council (VRRFI) and the Knut and Alice Wallenberg Foundation.

Author information

Author notes

  1. Philip Wolff

    Present address: John Innes Centre, Norwich, UK

  2. Jordi Moreno-Romero

    Present address: Center for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, Barcelona, Spain

  3. Christopher DeFraia

    Present address: Department of Biological Sciences, Ferris State University, Big Rapids, MI, USA

  4. German Martinez and Philip Wolff contributed equally to this work.

Authors and Affiliations

  1. Department of Plant Biology, Swedish University of Agricultural Sciences, Uppsala, Sweden

    German Martinez, Philip Wolff, Zhenxing Wang, Jordi Moreno-Romero, Juan Santos-González, Lei Liu Conze & Claudia Köhler

  2. Linnean Center for Plant Biology, Uppsala, Sweden

    German Martinez, Philip Wolff, Zhenxing Wang, Jordi Moreno-Romero, Juan Santos-González, Lei Liu Conze & Claudia Köhler

  3. Department of Molecular Genetics and Center for RNA Biology, The Ohio State University, Columbus, OH, USA

    Christopher DeFraia

  4. Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden

    Lei Liu Conze & R. Keith Slotkin

Authors
  1. German Martinez
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Philip Wolff
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zhenxing Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jordi Moreno-Romero
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Juan Santos-González
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Lei Liu Conze
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Christopher DeFraia
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. R. Keith Slotkin
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Claudia Köhler
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

G.M., P.W., R.K.S., and C.K. designed the experiments. G.M., P.W., Z.W., J.M.-R., and C.D.F. performed the experiments and generated the data. G.M., J.S.-G., and L.L.C. carried out the bioinformatic analysis. G.M., P.W., J.S.-G., L.L.C., R.K.S., and C.K. analyzed the data. G.M., P.W., and C.K. wrote the manuscript.

Corresponding author

Correspondence to Claudia Köhler.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Integrated supplementary information

Supplementary Figure 1 Small RNA distribution in pollen.

Size distribution of 18-30 small RNA reads in 1n and 2n Col wild-type and nrpd1a pollen.

Supplementary Figure 2 sRNA strand origin for miRNAs and TE-derived siRNAs.

Percentage of plus and minus strand for miRNAs and TE-derived sRNAs in 1n and 2n pollen from Col wt and nrpd1a mutants.

Supplementary Figure 3 RDR6 and DCL4 are responsible for the biogenesis of pollen TE-derived sRNA in the size range of 19-24 nt.

sRNA profiles of TE-derived sRNAs of 1n pollen from Col wild-type, rdr6 and dcl4 rdr6 mutants.

Supplementary Figure 4 Diploid pollen is substantially larger than haploid pollen.

Representative pictures of 1nand 2n Col wild-type and nrpd1a pollen. A minimum of 20 pollen grains per genotype have been analyzed with similar results.

Supplementary Figure 5 Pol IV mutant pollen is able to rescue seed abortion in triploid seeds derived from maternal Ler parents.

Frequency of non-collapsed seeds derived from crosses of wild-type (Ler) maternal parents with osd1 and osd1 nrpd1a. Asterisks mark significant differences (Chi square test, P=2.04E-49 (experiment 1, n=479) and 2.12E-22 (experiment 2, n=303) to the cross Col x osd1.

Supplementary Figure 6 Distribution of TEs among different TE families.

Upper panel shows all TEs, lower panel shows TEs losing CHH methylation in 3x seeds and gaining CHH methylation in 3x nrpd1a seeds. TEs shown in the lower panel are not significantly enriched for a particular TE family (Chi square test). Analysis is based on two biological replicates.

Supplementary Figure 7 Pollen-derived 21/22-nt easiRNAs correlate with loss of CHH methylation in triploid seeds.

(a)Loss of CHH methylation in the endosperm of triploid seeds associates with increasing levels of 21/22-nt easiRNAs in 1n pollen. Plotted are increasing levels of 21/22-nt easiRNAs in 1n pollen sorted by quantiles of 50-bp genome bins against differences of CHH methylation in the endosperm of Ler x osd1 (3x) and Ler x Col (2x) seeds. Differences between categories are significant (P=0, Kolmogorov-Smirnov test). (b) Loss of CHH methylation in the endosperm of triploid seeds associates with gain of Pol IV-dependent 21/22-nt easiRNAs in 1n pollen. Plotted are differences in 21/22-nt easiRNAs in 1n wt and 1n nrpd1a pollen against differences of CHH methylation in the endosperm of 3x and 2x seeds. Differences between categories are significant (P=0, Kolmogorov-Smirnov test). (c) Gain of CHH methylation in the endosperm of triploid nrpd1a seeds associates with gain of Pol IV-dependent 21/22-nt easiRNAs in 1n pollen. Plotted are differences in 21/22-nt easiRNAs in 1n wt and 1n nrpd1a pollen against differences of CHH methylation in the endosperm of 3x nrpd1a and 3x seeds. Differences between categories are significant (P=0, Kolmogorov-Smirnov test). Each box encloses the middle 50% of the distribution, the horizontal line marks the median and the notches the confidence intervals of the median, vertical lines mark the minimum and maximum values that fall within 1.5 times the height of the box. Replicates are specified in the online methods.

Supplementary Figure 8 Expression of NRPD1a in the endosperm of diploid (2x) and triploid (3x) wild-type and nrpd1a seeds.

RPKM values of NRPD1a in the endosperm derived from Ler x Col (2x), Ler x nrpd1a (2x nrpd1a), Ler x osd1 (3x), and Ler x osd1 nrpd1a (3x nrpd1a) seeds. Lines represent mean values. Data are derived from two biological replicates.

Supplementary Figure 9 Small RNA distribution in diploid and triploid seeds.

Size distribution of 18-30 small RNA reads in seeds derived from Ler x Col (2x seeds), Ler x nrpd1a (2x nrpd1a seeds), Ler x osd1 (3x seeds), Ler x osd1 nrpd1a (3x nrpd1a seeds) crosses. R1, R2; replicate 1 and 2, respectively.

Supplementary Figure 10 Increasing gene expression levels in 3x compared to 2x seeds associate with decreasing gene expression levels in 3x nrpd1a seeds compared to 3x seeds.

Boxes show log2-fold expression changes in the endosperm of 3x versus 2x seeds sorted into deciles of log2-fold expression changes in the endosperm of 3x nrpd1a seeds versus 3x seeds. Differences between the top 10% and bottom 10% categories are significant (P=0, Kolmogorov-Smirnov test).

Supplementary Figure 11 Pol IV-dependent sRNAs flank PEG coding regions.

sRNAs mapping to 2kb flanking regions of PEGs (specified in Fig. 4c) and flanking regions of all genes (left and right panels, respectively) in 1n and 2n wild-type and nrpd1a pollen.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–11, Supplementary Tables 1–6

Life Sciences Reporting Summary

Supplementary Dataset 1

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Martinez, G., Wolff, P., Wang, Z. et al. Paternal easiRNAs regulate parental genome dosage in Arabidopsis. Nat Genet 50, 193–198 (2018). https://doi.org/10.1038/s41588-017-0033-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41588-017-0033-4

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing