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Sexual-lineage-specific DNA methylation regulates meiosis in Arabidopsis



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.

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  1. 1.

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

  2. 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).

  3. 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).

  4. 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).

  5. 5.

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

  6. 6.

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

  7. 7.

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

  8. 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).

  9. 9.

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

  10. 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).

  11. 11.

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

  12. 12.

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

  13. 13.

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

  14. 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).

  15. 15.

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

  16. 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).

  17. 17.

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

  18. 18.

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

  19. 19.

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

  20. 20.

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

  21. 21.

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

  22. 22.

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

  23. 23.

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

  24. 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).

  25. 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).

  26. 26.

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

  27. 27.

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

  28. 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).

  29. 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).

  30. 30.

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

  31. 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).

  32. 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).

  33. 33.

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

  34. 34.

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

  35. 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).

  36. 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).

  37. 37.

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

  38. 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).

  39. 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).

  40. 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).

  41. 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).

  42. 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).

  43. 43.

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

  44. 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).

  45. 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).

  46. 46.

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

  47. 47.

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

  48. 48.

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

  49. 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).

  50. 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).

  51. 51.

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

  52. 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).

  53. 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).

  54. 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).

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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

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.

Competing interests

The authors declare no competing financial interests.

Correspondence to Xiaoqi Feng.

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.

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Further reading

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.
Supplementary Figure 1: DNA methylation profiles at genes and transposons in Arabidopsis male-sexual-lineage cells
Supplementary Figure 2: Examples of sexual-lineage-hypermethylated loci (SLHs)
Supplementary Figure 3: SLMs have little CHH/G methylation in the columella and embryo
Supplementary Figure 4: CG methylation in seedlings of wild type (WT) and RdDM mutants is strongly correlated
Supplementary Figure 5: Examples of genes suppressed by sexual-lineage-specific methylation in meiocytes
Supplementary Figure 6: Examples of sexual-lineage-specific methylation at pre-tRNA genes
Supplementary Figure 7: Pre-tRNA genes are hypermethylated in male sex cells
Supplementary Figure 8: Meiotic defects in RdDM mutants and MPS1-interference lines
Supplementary Figure 9: Separation of sperm- and vegetative-cell nuclei via fluorescence-activated cell sorting (FACS)