Chromosome dosage has substantial effects on reproductive isolation and speciation in both plants and animals, but the underlying mechanisms are largely obscure1. Transposable elements in animals can regulate hybridity through maternal small RNA2, whereas small RNAs in plants have been postulated to regulate dosage response via neighboring imprinted genes3,4. Here we show that a highly conserved microRNA in plants, miR845, targets the tRNAMet primer-binding site (PBS) of long terminal repeat (LTR) retrotransposons in Arabidopsis pollen, and triggers the accumulation of 21–22-nucleotide (nt) small RNAs in a dose-dependent fashion via RNA polymerase IV. We show that these epigenetically activated small interfering RNAs (easiRNAs) mediate hybridization barriers between diploid seed parents and tetraploid pollen parents (the ‘triploid block’), and that natural variation for miR845 may account for ‘endosperm balance’ allowing the formation of triploid seeds. Targeting of the PBS with small RNA is a common mechanism for transposon control in mammals and plants, and provides a uniquely sensitive means to monitor chromosome dosage and imprinting in the developing seed.
Subscribe to Journal
Get full journal access for 1 year
only $18.75 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Birchler, J. A. & Veitia, R. A. Gene balance hypothesis: connecting issues of dosage sensitivity across biological disciplines. Proc. Natl. Acad. Sci. USA 109, 14746–14753 (2012).
Senti, K.-A. & Brennecke, J. The piRNA pathway: a fly’s perspective on the guardian of the genome. Trends Genet. 26, 499–509 (2010).
Martienssen, R. A. Heterochromatin, small RNA and post-fertilization dysgenesis in allopolyploid and interploid hybrids of Arabidopsis. New Phytol. 186, 46–53 (2010).
Köhler, C., Mittelsten Scheid, O. & Erilova, A. The impact of the triploid block on the origin and evolution of polyploid plants. Trends Genet. 26, 142–148 (2010).
Law, J. A. & Jacobsen, S. E. Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat. Rev. Genet. 11, 204–220 (2010).
Heard, E. & Martienssen, R. A. Transgenerational epigenetic inheritance: myths and mechanisms. Cell 157, 95–109 (2014).
Dresselhaus, T., Sprunck, S. & Wessel, G. M. Fertilization mechanisms in flowering plants. Curr. Biol. 26, R125–R139 (2016).
Calarco, J. P. et al. Reprogramming of DNA methylation in pollen guides epigenetic inheritance via small RNA. Cell 151, 194–205 (2012).
Slotkin, R. K. et al. Epigenetic reprogramming and small RNA silencing of transposable elements in pollen. Cell 136, 461–472 (2009).
Ibarra, C. A. et al. Active DNA demethylation in plant companion cells reinforces transposon methylation in gametes. Science 337, |1360–1364 (2012).
Gehring, M., Bubb, K. L. & Henikoff, S. Extensive demethylation of repetitive elements during seed development underlies gene imprinting. Science 324, 1447–1451 (2009).
Hsieh, T. F. et al. Genome-wide demethylation of Arabidopsis endosperm. Science 324, 1451–1454 (2009).
Schoft, V. K. et al. Function of the DEMETER DNA glycosylase in the Arabidopsis thaliana male gametophyte. Proc. Natl. Acad. Sci. USA 108, 8042–8047 (2011).
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).
Creasey, K. M. et al. miRNAs trigger widespread epigenetically activated siRNAs from transposons in Arabidopsis. Nature 508, 411–415 (2014).
Rathore, P., Geeta, R. & Das, S. Microsynteny and phylogenetic analysis of tandemly organised miRNA families across five members of Brassicaceae reveals complex retention and loss history. Plant Sci. 247, 35–48 (2016).
Willing, E.-M. et al. Genome expansion of Arabis alpina linked with retrotransposition and reduced symmetric DNA methylation. Nat. Plants 1, 14023 (2015).
Zhou, L. et al. Genome-wide identification and analysis of drought-responsive microRNAs in Oryza sativa. J. Exp. Bot. 61, 4157–4168 (2010).
Šurbanovski, N., Brilli, M., Moser, M. & Si-Ammour, A. A highly specific microRNA-mediated mechanism silences LTR retrotransposons of strawberry. Plant J. 85, 70–82 (2016).
Schorn, A. J., Gutbrod, M. J., LeBlanc, C. & Martienssen, R. LTR-retrotransposon control by tRNA-derived small RNAs. Cell 170, 61–71 (2017).
Quadrana, L. et al. The Arabidopsis thaliana mobilome and its impact at the species level. eLife 5, e15716 (2016).
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).
Pignatta, D. et al. Natural epigenetic polymorphisms lead to intraspecific variation in Arabidopsis gene imprinting. eLife 3, e03198 (2014).
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).
Borges, F. & Martienssen, R. A. The expanding world of small RNAs in plants. Nat. Rev. Mol. Cell Biol. 16, 727–741 (2015).
Wei, W. et al. A role for small RNAs in DNA double-strand break repair. Cell 149, 101–112 (2012).
Schalk, C. et al. Small RNA-mediated repair of UV-induced DNA lesions by the DNA DAMAGE-BINDING PROTEIN 2 and ARGONAUTE 1. Proc. Natl. Acad. Sci. USA 114, E2965–E2974 (2017).
Pumplin, N. et al. DNA methylation influences the expression of DICER-LIKE4 isoforms, which encode proteins of alternative localization and function. Plant Cell 28, 2786–2804 (2016).
Ronemus, M., Vaughn, M. W. & Martienssen, R. A. MicroRNA-targeted and small interfering RNA-mediated mRNA degradation is regulated by argonaute, dicer, and RNA-dependent RNA polymerase in Arabidopsis. Plant Cell 18, 1559–1574 (2006).
Schmickl, R. & Koch, M. A. Arabidopsis hybrid speciation processes. Proc. Natl. Acad. Sci. USA 108, 14192–14197 (2011).
d’Erfurth, I. et al. Turning meiosis into mitosis. PLoS Biol. 7, e1000124 (2009).
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).
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).
Dilkes, B. P. et al. The maternally expressed WRKY transcription factor TTG2 controls lethality in interploidy crosses of Arabidopsis. PLoS Biol. 6, 2707–2720 (2008).
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).
Wolff, P., Jiang, H., Wang, G., Santos-González, J. & Köhler, C. Paternally expressed imprinted genes establish postzygotic hybridization barriers in Arabidopsis thaliana. eLife 4, e10074 (2015).
Josefsson, C., Dilkes, B. & Comai, L. Parent-dependent loss of gene silencing during interspecies hybridization. Curr. Biol. 16, 1322–1328 (2006).
Kirkbride, R. C. et al. An epigenetic role for disrupted paternal gene expression in postzygotic seed abortion in Arabidopsis interspecific hybrids. Mol. Plant 8, 1766–1775 (2015).
Mozgová, I., Köhler, C. & Hennig, L. Keeping the gate closed: functions of the Polycomb repressive complex PRC2 in development. Plant J. 83, 121–132 (2015).
Jullien, P. E. & Berger, F. Parental genome dosage imbalance deregulates imprinting in Arabidopsis. PLoS Genet. 6, e1000885 (2010).
Martinez, G. et al. Paternal easiRNAs establish the triploid block in. Arabidopsis. Nat. Genet. https://doi.org/10.1038/s41588-017-0033-4 (2018).
Kato, A. & Birchler, J. A. Induction of tetraploid derivatives of maize inbred lines by nitrous oxide gas treatment. J. Hered. 97, 39–44 (2006).
Birchler, J. A. Interploidy hybridization barrier of endosperm as a dosage interaction. Front. Plant Sci. 5, 281 (2014).
Duharcourt, S., Lepère, G. & Meyer, E. Developmental genome rearrangements in ciliates: a natural genomic subtraction mediated by non-coding transcripts. Trends Genet. 25, 344–350 (2009).
Kidner, C. A. & Martienssen, R. A. The role of ARGONAUTE1 (AGO1) in meristem formation and identity. Dev. Biol. 280, 504–517 (2005).
Schatlowski, N. et al. Hypomethylated pollen bypasses the interploidy hybridization barrier in Arabidopsis. Plant Cell 26, 3556–3568 (2014).
Borges, F. et al. FACS-based purification of Arabidopsis microspores, sperm cells and vegetative nuclei. Plant Methods 8, 44 (2012).
Liao, Y., Smyth, G. K. & Shi, W. The Subread aligner: fast, accurate and scalable read mapping by seed-and-vote. Nucleic Acids Res. 41, e108 (2013).
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).
Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014).
We thank all members of the Martienssen laboratory for discussions. Research in the Martienssen laboratory is supported by the US National Institutes of Health (NIH; grant R01 GM067014), the National Science Foundation Plant Genome Research Program, and the Howard Hughes Medical Institute and Gordon and Betty Moore Foundation. The authors acknowledge assistance from the Cold Spring Harbor Laboratory Shared Resources, which are funded in part by the Cancer Center (Support Grant 5PP30CA045508). The osd1-1 mutant was kindly provided by R. Mercier (Institut Jean-Pierre Bourgin, INRA Versailles-Grignon, Versailles, France). Research in the Köhler laboratory was supported by a European Research Council Starting Independent Researcher grant, the Swedish Science Foundation and the Knut and Alice Wallenberg Foundation (to C.K.). G.M. was supported by a Marie Curie IOF Postdoctoral Fellowship (PIOF-GA-2012-330069). Sequencing for the Köhler laboratory 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.
The authors declare no competing financial interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Integrated supplementary information
(a) Expression of a GFP reporter with a miR845b target site in the 3′UTR was driven by the UBIQUITIN10 (UBQ10) promoter and visualized in wild-type somatic tissues. GFP silencing was observed only in pollen in eight independent transgenic lines. Scale bars represent 30 μm. (b) miR845b was active in Col-0 sperm cells, as expression of the GFP-miR845b construct driven by the sperm-specific MGH3 promoter was silenced. GFP expression in sperm cells was observed in Ler-0 pollen in four independent transgenic lines, since miR845b is not expressed in this ecotype. Scale bars represent 10 μm. (c) GFP sensor was silenced in wild-type pollen, but not in ago1-9 and dcl1-5 pollen. Scale bars represent 30 μm. (d) The GFP sensor in dcl1-5/+ heterozygotes was used to sort wild-type (GFP negative) and dcl1-5 (GFP positive) pollen. (e) Small RNA sequencing of FACS-sorted pollen populations revealed that most miRNAs, including miR845a and miR845b, were depleted in dcl1-5 pollen.
(a) A Single Nucleotide Polymorphism (SNP) in the miR845b* region of MIR845b between Col-0 and Ler-0. (b) SNPs in MIR845b change the predicted structure of the MIR845b stem-loop, which could impair processing by DCL1. (c) Ectopic expression of Col-MIR845b and Ler-MIR845b in leaves driven by the 35S promoter was quantified by RT-PCR (qRT-PCR), and confirms that the Ler-MIR845b is not efficiently processed in six independent transgenic lines. (d) MIR845a gene was identified in Col-0 (TAIR10 annotation). In Ler-0, there is a 1kb deletion at the MIR845a locus1. (e) This indel was confirmed by PCR in Col-0, Ler-0 and Col/Ler-0 F1 hybrid. (f) Venn diagram shows that in addition to MIR845a, there are 7 additional MIRNA genes annotated in Col-0 that are not present in Ler-0.
Supplementary Figure 3 Natural variation in transposon expression and easiRNA biogenesis in Col-0 and Ler-0 pollen
(a) Col-0 and Ler-0 pollen transcriptomes revealed that TE transcripts were overall more abundant in Ler-0 pollen. (b) These differences are reflected in the accumulation of easiRNAs in pollen isolated from each ecotype. For example, miR845-targeted TEs ATGP2 and ATCOPIA41 accumulate easiRNA in Col pollen, while ATCOPIA63 is only expressed in Ler-0 where it accumulates easiRNA, presumably via alternative miRNA. (c) LTR retrotransposon (Gypsy and Copia) that are predicted miR845 targets show enriched expression in Ler-0 pollen. RPM, reads per million. FPKM, fragments per kilobase per million.
(a) Normalized small RNA reads were mapped to aligned 5′ regions (metaplots) of all annotated Gypsy elements, including 2kb upstream and 4kb downstream of the LTR. This includes the 5′ LTR (~2kb), PBS (miR845b target site) and part of the gag gene. 21/22-nt siRNAs were more abundant in wild-type pollen but depleted in the dcl2/4 mutant pollen. Both 21/22- and 24-nt siRNAs were depleted nrpd1a (Pol IV) mutant pollen. (b) Most small RNAs matching to TEs were lost in the nrpd1a mutant pollen. Many miRNA were also reduced by 2-fold, likely due to destabilization of ARGONAUTE proteins in nrpd1a mutant pollen. RPM, reads per million.
(a) Average DNA methylation levels in Col-0 and Ler-0 sperm (SC) and vegetative nuclei (VN) were plotted in 100kb windows on all 5 chromosomes in Arabidopsis. (b) DNA methylation in the CHH context in SC and VN was not significantly changed in Ler-0 transgenics expressing Col-MIR845b (Ler:MIR845b) in pollen. (c) Histogram representation of average DNA methylation percentages in Col, Ler-0 and Ler:MIR845b SC and VN nuclei.
(a) Small RNA reads were mapped to aligned metaplots of maternally (MEGs, n=285) and paternally expressed genes (PEGs, n = 103)2, including 2kb upstream and downstream of annotated coding regions, and normalized by total mapped reads. MEGs and PEGs accumulate 21/22nt easiRNA in pollen. (b) Boxplots of normalized siRNA abundances matching 2kb regions flanking MEGs and PEGs in haploid (1n) and diploid (2n) pollen isolated from wild type Col-0, mir845b-1, nrpd1a-3, wild type Ler-0 and Ler-0 transgenics expressing Col-miR845b (Ler:MIR845b). 21/22nt easiRNA matching MEGs and PEGs depend on miR845b and polIV. (c) Boxplots of published small RNA datasets from diploid (2x2), tetraploid (4x4) and interploid (2x4) seeds 3 and from Col-0 and Ler-0 seeds 2. MEGs accumulated higher levels of 21/22-nt easiRNA in interploid (2x4) seed. All boxes represent lower and upper quartiles surrounding the median (center line). RPM, reads per million.
In pollen, biogenesis of 21-22-nt easiRNA is triggered by miR845, resulting from the activation of retrotransposons during meiosis and in the vegetative nucleus (VN). easiRNAs accumulate in the sperm cells (SC) that are passively transported to the embryo sac to perform double fertilization with the haploid egg (EC) and diploid central cell (CC), giving rise to the embryo (Eb) and endosperm (EN), respectively. a, In the wild-type endosperm, Maternally Expressed Genes (MEGs), such as MEA and FIS2, encode important components of the Polycomb repressor complex 2 (PRC2) that regulates expression of PEGs via deposition of H3K27me3. b, In interploid hybrids with paternal excess, the endosperm over-proliferates and fails to cellularize, leading to seed abortion (“the triploid block”)4. This interploidy hybridization barrier resembles maternal PRC2 mutants, where PEGs are also over-expressed and the endosperm over-proliferates leading to seed abortion phenotypes4. Similarly to mutations in PRC2 components, the triploid block can be suppressed by mutations in particular PEGs5,6. Our study suggests that miR845b-dependent easiRNAs are an important component of this pathway, perhaps by targeting TEs that flank imprinted genes. For example, increased paternal dosage and easiRNA activity, RNA-directed DNA methylation (RdDM) and H3K9me27, may contribute to down-regulation of MEGs and PRC2 activity, which in turn leads to up-regulation of PEGs and seed collapse.
Supplementary Figures 1–7 and Supplementary Tables 3 and 4
miR845 target sites in retrotransposons. Target sites were predicted with the psRNATarget software (2011 release), using default settings for all parameters, except the maximum expectation value (Exp < 5.0)
Predicted miR845 targets overlapping with mCHH hypomethylated DMRs in Ler-0 pollen
About this article
Cite this article
Borges, F., Parent, J., van Ex, F. et al. Transposon-derived small RNAs triggered by miR845 mediate genome dosage response in Arabidopsis. Nat Genet 50, 186–192 (2018). https://doi.org/10.1038/s41588-017-0032-5
Arabidopsis RNA Polymerase IV generates 21–22 nucleotide small RNAs that can participate in RNA-directed DNA methylation and may regulate genes
Philosophical Transactions of the Royal Society B: Biological Sciences (2020)
Nature Reviews Genetics (2020)
Genome Research (2020)
Genetic variation in herbivore resistance within a strawberry crop wild relative (Fragaria vesca L.)
Arthropod-Plant Interactions (2020)
High expression in maize pollen correlates with genetic contributions to pollen fitness as well as with coordinated transcription from neighboring transposable elements
PLOS Genetics (2020)