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.

  • Article
  • Published:

Sexual-lineage-specific DNA methylation regulates meiosis in Arabidopsis

Nature Genetics volume 50, pages 130–137 (2018)Cite this article

Subjects

Abstract

DNA methylation regulates eukaryotic gene expression and is extensively reprogrammed during animal development. However, whether developmental methylation reprogramming during the sporophytic life cycle of flowering plants regulates genes is presently unknown. Here we report a distinctive gene-targeted RNA-directed DNA methylation (RdDM) activity in the Arabidopsis thaliana male sexual lineage that regulates gene expression in meiocytes. Loss of sexual-lineage-specific RdDM causes mis-splicing of the MPS1 gene (also known as PRD2), thereby disrupting meiosis. Our results establish a regulatory paradigm in which de novo methylation creates a cell-lineage-specific epigenetic signature that controls gene expression and contributes to cellular function in flowering plants.

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

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

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

Fig. 1: Male meiocytes exhibit high CG/CHG and low CHH methylation.
Fig. 2: SLHs in Arabidopsis.
Fig. 3: SLHs are produced by RdDM.
Fig. 4: SLMs are novel RdDM targets specific to the sexual lineage.
Fig. 5: SLMs target genes and regulate gene expression in meiocytes.
Fig. 6: Pre-tRNA genes are hypermethylated in the male sexual lineage.
Fig. 7: RdDM is important for the splicing of MPS1 and normal meiosis.

Similar content being viewed by others

References

  1. Zemach, A. & Zilberman, D. Evolution of eukaryotic DNA methylation and the pursuit of safer sex. Curr. Biol. 20, R780–R785 (2010).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  3. He, X. J., Chen, T. & Zhu, J. K. Regulation and function of DNA methylation in plants and animals. Cell Res. 21, 442–465 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Du, J., Johnson, L. M., Jacobsen, S. E. & Patel, D. J. DNA methylation pathways and their crosstalk with histone methylation. Nat. Rev. Mol. Cell Biol. 16, 519–532 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Pikaard, C. S. & Mittelsten Scheid, O. Epigenetic regulation in plants. Cold Spring Harb. Perspect. Biol. 6, a019315 (2014).

    PubMed  PubMed Central  Google Scholar 

  6. Matzke, M. A. & Mosher, R. A. RNA-directed DNA methylation: an epigenetic pathway of increasing complexity. Nat. Rev. Genet. 15, 394–408 (2014).

    CAS  PubMed  Google Scholar 

  7. Kim, M. Y. & Zilberman, D. DNA methylation as a system of plant genomic immunity. Trends Plant Sci. 19, 320–326 (2014).

    CAS  PubMed  Google Scholar 

  8. Lipka, D. B. et al. Identification of DNA methylation changes at cis-regulatory elements during early steps of HSC differentiation using tagmentation-based whole genome bisulfite sequencing. Cell Cycle 13, 3476–3487 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Dawlaty, M. M. et al. Loss of Tet enzymes compromises proper differentiation of embryonic stem cells. Dev. Cell 29, 102–111 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Kubo, N. et al. DNA methylation and gene expression dynamics during spermatogonial stem cell differentiation in the early postnatal mouse testis. BMC Genomics 16, 624 (2015).

    PubMed  PubMed Central  Google Scholar 

  11. Slieker, R. C. et al. DNA methylation landscapes of human fetal development. PLoS Genet. 11, e1005583 (2015).

    PubMed  PubMed Central  Google Scholar 

  12. Xie, W. et al. Epigenomic analysis of multilineage differentiation of human embryonic stem cells. Cell 153, 1134–1148 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Cacchiarelli, D. et al. Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency. Cell 162, 412–424 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Benner, C., Isoda, T. & Murre, C. New roles for DNA cytosine modification, eRNA, anchors, and superanchors in developing B cell progenitors. Proc. Natl. Acad. Sci. USA 112, 12776–12781 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 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 

  16. Park, K. et al. DNA demethylation is initiated in the central cells of Arabidopsis and rice. Proc. Natl. Acad. Sci. USA 113, 15138–15143 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Calarco, J. P. et al. Reprogramming of DNA methylation in pollen guides epigenetic inheritance via small RNA. Cell 151, 194–205 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Ibarra, C. A. et al. Active DNA demethylation in plant companion cells reinforces transposon methylation in gametes. Science 337, 1360–1364 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Kawakatsu, T. et al. Unique cell-type-specific patterns of DNA methylation in the root meristem. Nat. Plants 2, 16058 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Hsieh, T. F. et al. Genome-wide demethylation of Arabidopsis endosperm. Science 324, 1451–1454 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Secco, D. et al. Stress induced gene expression drives transient DNA methylation changes at adjacent repetitive elements. eLife 4, (2015).

  22. Jiang, C. et al. Environmentally responsive genome-wide accumulation of de novo Arabidopsis thaliana mutations and epimutations. Genome Res. 24, 1821–1829 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Dowen, R. H. et al. Widespread dynamic DNA methylation in response to biotic stress. Proc. Natl. Acad. Sci. USA 109, E2183–E2191 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Bilichak, A., Ilnystkyy, Y., Hollunder, J. & Kovalchuk, I. The progeny of Arabidopsis thaliana plants exposed to salt exhibit changes in DNA methylation, histone modifications and gene expression. PLoS One 7, e30515 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Wibowo, A. et al. Hyperosmotic stress memory in Arabidopsis is mediated by distinct epigenetically labile sites in the genome and is restricted in the male germline by DNA glycosylase activity. eLife 5, e13546 (2016).

    PubMed  PubMed Central  Google Scholar 

  26. Feng, X., Zilberman, D. & Dickinson, H. A conversation across generations: soma-germ cell crosstalk in plants. Dev. Cell 24, 215–225 (2013).

    CAS  PubMed  Google Scholar 

  27. Kawashima, T. & Berger, F. Epigenetic reprogramming in plant sexual reproduction. Nat. Rev. Genet. 15, 613–624 (2014).

    CAS  PubMed  Google Scholar 

  28. Hsieh, P. H. et al. Arabidopsis male sexual lineage exhibits more robust maintenance of CG methylation than somatic tissues. Proc. Natl. Acad. Sci. USA 113, 15132–15137 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Stroud, H., Greenberg, M. V., Feng, S., Bernatavichute, Y. V. & Jacobsen, S. E. Comprehensive analysis of silencing mutants reveals complex regulation of the Arabidopsis methylome. Cell 152, 352–364 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Catoni, M. et al. DNA sequence properties that predict susceptibility to epiallelic switching. EMBO J. 36, 617–628 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Chen, C. et al. Meiosis-specific gene discovery in plants: RNA-Seq applied to isolated Arabidopsis male meiocytes. BMC Plant Biol. 10, 280 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Yang, H., Lu, P., Wang, Y. & Ma, H. The transcriptome landscape of Arabidopsis male meiocytes from high-throughput sequencing: the complexity and evolution of the meiotic process. Plant J. 65, 503–516 (2011).

    CAS  PubMed  Google Scholar 

  33. Martinez, G., Choudury, S. G. & Slotkin, R. K. tRNA-derived small RNAs target transposable element transcripts. Nucleic Acids Res. 45, 5142–5152 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Wang, X. et al. DNA methylation affects gene alternative splicing in plants: an example from rice. Mol. Plant 9, 305–307 (2016).

    CAS  PubMed  Google Scholar 

  35. Regulski, M. et al. The maize methylome influences mRNA splice sites and reveals widespread paramutation-like switches guided by small RNA. Genome Res. 23, 1651–1662 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Naftelberg, S., Schor, I. E., Ast, G. & Kornblihtt, A. R. Regulation of alternative splicing through coupling with transcription and chromatin structure. Annu. Rev. Biochem. 84, 165–198 (2015).

    CAS  PubMed  Google Scholar 

  37. Lev Maor, G., Yearim, A. & Ast, G. The alternative role of DNA methylation in splicing regulation. Trends Genet. 31, 274–280 (2015).

    CAS  PubMed  Google Scholar 

  38. Jiang, H. et al. MULTIPOLAR SPINDLE 1 (MPS1), a novel coiled-coil protein of Arabidopsis thaliana, is required for meiotic spindle organization. Plant J. 59, 1001–1010 (2009).

    CAS  PubMed  Google Scholar 

  39. De Muyt, A. et al. A high throughput genetic screen identifies new early meiotic recombination functions in Arabidopsis thaliana. PLoS Genet. 5, e1000654 (2009).

    PubMed  PubMed Central  Google Scholar 

  40. Oliver, C., Santos, J. L. & Pradillo, M. Accurate chromosome segregation at first meiotic division requires AGO4, a protein involved in RNA-dependent DNA methylation in Arabidopsis thaliana. Genetics 204, 543–553 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Hollister, J. D. & Gaut, B. S. Epigenetic silencing of transposable elements: a trade-off between reduced transposition and deleterious effects on neighboring gene expression. Genome Res. 19, 1419–1428 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Baubec, T., Finke, A., Mittelsten Scheid, O. & Pecinka, A. Meristem-specific expression of epigenetic regulators safeguards transposon silencing in Arabidopsis. EMBO Rep. 15, 446–452 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Tran, R. K. et al. DNA methylation profiling identifies CG methylation clusters in Arabidopsis genes. Curr. Biol. 15, 154–159 (2005).

    CAS  PubMed  Google Scholar 

  44. Takuno, S. & Gaut, B. S. Body-methylated genes in Arabidopsis thaliana are functionally important and evolve slowly. Mol. Biol. Evol. 29, 219–227 (2012).

    CAS  PubMed  Google Scholar 

  45. Zilberman, D., Gehring, M., Tran, R. K., Ballinger, T. & Henikoff, S. Genome-wide analysis of Arabidopsis thaliana DNA methylation uncovers an interdependence between methylation and transcription. Nat. Genet. 39, 61–69 (2007).

    CAS  PubMed  Google Scholar 

  46. Coleman-Derr, D. & Zilberman, D. Deposition of histone variant H2A.Z within gene bodies regulates responsive genes. PLoS Genet. 8, e1002988 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Stroud, H. et al. Non-CG methylation patterns shape the epigenetic landscape in Arabidopsis. Nat. Struct. Mol. Biol. 21, 64–72 (2014).

    CAS  PubMed  Google Scholar 

  48. Zemach, A. et al. The Arabidopsis nucleosome remodeler DDM1 allows DNA methyltransferases to access H1-containing heterochromatin. Cell 153, 193–205 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Liang, C., Liu, X., Sun, Y., Yiu, S. M. & Lim, B. L. Global small RNA analysis in fast-growing Arabidopsis thaliana with elevated concentrations of ATP and sugars. BMC Genomics 15, 116 (2014).

    PubMed  PubMed Central  Google Scholar 

  50. 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).

    PubMed  Google Scholar 

  51. Armstrong, S. J., Sanchez-Moran, E. & Franklin, F. C. Cytological analysis of Arabidopsis thaliana meiotic chromosomes. Methods Mol. Biol. 558, 131–145 (2009).

    PubMed  Google Scholar 

  52. Sonobe, S. & Shibaoka, H. Cortical fine actin-filaments in higher-plant cells visualized by rhodamine-phalloidin after pretreatment with m-maleimidobenzoyl N-hydroxysuccinimide ester. Protoplasma 148, 80–86 (1989).

    Google Scholar 

  53. Nakagawa, T. et al. Development of series of gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation. J. Biosci. Bioeng. 104, 34–41 (2007).

    CAS  PubMed  Google Scholar 

  54. Deal, R. B. & Henikoff, S. The INTACT method for cell type-specific gene expression and chromatin profiling in Arabidopsis thaliana. Nat. Protoc. 6, 56–68 (2011).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank D. Zilberman for intellectual contributions to this work; D. Zilberman, C. Dean, K. Bomblies, V. Kumar, S. Brady and S. Kamoun for comments on the manuscript; H. Dickinson and J. Hellberg for developing the meiocyte isolation method; G. Oldroyd (Sainsbury Laboratory, Cambridge University) for the pGWB13-Bar vector; E. Fiume (Institut Jean-Pierre Bourgin, Paris) for the pMDC107-NTF vector; M. Hartley, M. Couchman and T. S. G. Olsson at the John Innes Centre Computing Infrastructure for Science Facility for bioinformatics support; and the Norwich Bioscience Institute Partnership Computing Infrastructure for Science group for High Performance Computing resources and the John Innes Centre Bioimaging Facility for assistance with microscopy. This work was funded by a Biotechnology and Biological Sciences Research Council (BBSRC) David Phillips Fellowship (BB/L025043/1) to X.F., a BBSRC grant (BB/M01973X/1) to J.D.H. and a Sainsbury PhD Studentship to J.W.

Author information

Author notes

  1. James Walker and Hongbo Gao contributed equally to this work.

Authors and Affiliations

  1. Department of Cell and Developmental Biology, John Innes Centre, Norwich, UK

    James Walker, Hongbo Gao, Jingyi Zhang, Billy Aldridge, Martin Vickers & Xiaoqi Feng

  2. Department of Genetics, University of Leicester, Leicester, UK

    James D. Higgins

Authors
  1. James Walker
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hongbo Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jingyi Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Billy Aldridge
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Martin Vickers
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. James D. Higgins
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Xiaoqi Feng
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

X.F. designed the study; J.W., H.G., J.Z., B.A., J.D.H. and X.F. performed the experiments; J.W., H.G., M.V. and X.F. analyzed the data; and X.F. wrote the manuscript.

Corresponding author

Correspondence to Xiaoqi Feng.

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 DNA methylation profiles at genes and transposons in Arabidopsis male-sexual-lineage cells

A. thaliana genes (a, c, e) or transposable elements (TEs; b, d, f) were aligned at the 5’ end (left panel) or the 3’ end (right panel), and average methylation levels in the CG (a-b), CHG (c-d) or CHH (e-f) context for each 100-bp interval are plotted. drm, drm1drm2. The dashed line at zero represents the point of alignment.

Supplementary Figure 2 Examples of sexual-lineage-hypermethylated loci (SLHs)

Examples of typical SLHs located at the transcriptional start and termination sites and body of genes (a-c), and SLHs (that are also SLMs) with remnant CG methylation in drm1drm2 (drm) mutant sex cells and wild-type somatic tissues (d, e). SLHs are underlined in red (refer to Supplementary Table 2 for a full list).

Supplementary Figure 3 SLMs have little CHH/G methylation in the columella and embryo

Box plots showing the absolute methylation at SLMs (533 loci before filtering out columella overlaps; see Methods) in somatic tissues (Sd, seedling; Rs, rosette leaf; Ca, cauline leaf; Ro, root), sex cells (Me, meiocyte; Mi, microspore; Sp, sperm), columella root cap (Co) and embryo (Em).

Supplementary Figure 4 CG methylation in seedlings of wild type (WT) and RdDM mutants is strongly correlated

Scatter plots showing linear correlation between CG methylation at SLMs in seedlings of WT and drm2 (Pearson’s R = 0.58), and WT and rdr2 (Pearson’s R = 0.70).

Supplementary Figure 5 Examples of genes suppressed by sexual-lineage-specific methylation in meiocytes

Similar to Fig. 5b, snapshots of cytosine methylation in wild-type male sex cells, drm1drm2 (drm) meiocyte, and wild-type rosette leaves, and transcriptional expression (in log2RPKM) in wild-type and drm meiocyte are shown. SLMs are underlined in red.

Supplementary Figure 6 Examples of sexual-lineage-specific methylation at pre-tRNA genes

Snapshots of cytosine methylation, similar to Supplementary Fig. 2, in wild-type (WT) male sex cells (black), drm1drm2 (drm) mutant sex cells (red), and four somatic tissues (green). SLMs are underlined in red. a, Examples of SLMs at pre-tRNA genes encoding phenylalanine, methionine, glycine or valine anticodons. b, SLM at the methionine pre-tRNA gene (magenta box) located in the last intron of MPS1.

Supplementary Figure 7 Pre-tRNA genes are hypermethylated in male sex cells

Similar to Fig. 6b, these box plots show the absolute CHG (a) and CG (b) methylation at 3 groups of pre-tRNA genes in sex cells (Me, meiocyte; Mi, microspore; Sp, sperm; Ve, vegetative cell), somatic tissues (Sd, seedling; Rs, rosette leaf; Ca, cauline leaf; Ro, root), and drm (drm1drm2) mutant sex cells (dM, drm meiocyte; dS, drm sperm; dV, drm vegetative cell). Refer to Fig. 6 legend for the 3 groups of pre-tRNA genes.

Supplementary Figure 8 Meiotic defects in RdDM mutants and MPS1-interference lines

a-e, Male meiosis II in wild type (WT; a), drm1drm2 (drm; b, d) and rdr2 (c, e) mutants, and the MPS1 interference lines (g). All instances of WT male meiosis we observed (301 observations) were normal and lead to tetrads at the end of meiosis II (a). However, in 7.1% (380 total observations) and 7.8% (502 total observations) instances of drm (b) and rdr2 (c) male meiosis, respectively, chromosomes fail to separate, so that triads are observed at telophase II. Occasionally we also observed pentads in drm (d) and rdr2 (e) mutants. f, RT-PCR showing the expression of MPS1 transcript retaining last intron (149 bp) in drm mutant and 9 T1 plants of the interference lines, but not in WT. ACT8 as control shows 156 bp bands. g, Interference lines exhibit even higher percentages of triads (I1, 13.4%, 463 total observations; I2, 15.1%, 292 total observations). n, the number of chromosomes in the haploid genome. Scale bars, 10 μm.

Supplementary Figure 9 Separation of sperm- and vegetative-cell nuclei via fluorescence-activated cell sorting (FACS)

A representative flow cytometry plot showing two clear populations which indicate SYBR Green-stained sperm nuclei (SN) and vegetative cell nuclei (VN), respectively. AM and AN indicate the percentages of sorted SN and VN in total events, respectively.

Supplementary information

Supplementary Figures and Tables

Supplementary Figures 1–9 and Supplementary Tables 1, 3, 6 and 7

Life Sciences Reporting Summary.

Supplementary Table 2

List of loci that are significantly differentially methylated between the sexual lineage and somatic tissues.

Supplementary Table 4

Expression of 83 meiotic genes in our meiocyte RNA-seq data in comparison to those from published meiocyte transcriptomes.

Supplementary Table 5

List of differentially expressed genes and their association with SLMs.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Walker, J., Gao, H., Zhang, J. et al. Sexual-lineage-specific DNA methylation regulates meiosis in Arabidopsis . Nat Genet 50, 130–137 (2018). https://doi.org/10.1038/s41588-017-0008-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41588-017-0008-5

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research