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Paternal easiRNAs regulate parental genome dosage in Arabidopsis

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

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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.

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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.

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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.

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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

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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.

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Correspondence to Claudia Köhler.

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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.

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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

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