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
- University of Warwick, United Kingdom
- Max Planck Institute for Developmental Biology, Germany
- University of Perugia, Italy
- Instituto Gulbenkian de Ciencia, Portugal
Figures
Figure 1 with 1 supplement
Improved salt resistance of progeny from parents exposed to hyperosmotic stress.
(A) Diagram of the experimental design. Plants were grown on control or hyperosmotic medium (25 mM and 75 mM NaCl) for five consecutive generations. From each generation, progeny in P1 and P2 were …
Figure 1—figure supplement 1
High-salinity tolerance assays.
(A) Germination rates of P1 and P2 seeds on control medium or medium supplemented with 200 mM NaCl. For each sample and treatment 300 seeds were analysed in two triplicates. (B) Chlorophyll content …
Figure 2 with 5 supplements
Hyperosmotic stress-induced differentially methylated regions (DMRs) in the absence of stress stimulus.
(A) Annotation of cytosines in MRs and DMRs between P0 control and P0 hyperosmotic treated samples in different generations (see Figure 1A). (B) Methylation frequencies by sequence context in DMRs …
Figure 2—figure supplement 1
DNA methylation variation after multigenerational hyperosmotic stress.
(A) Principal component (PC) analysis of methylation frequencies at DMPs in each generation, with full information across all samples of that generation. Numbers in brackets indicate the percentage …
Figure 2—figure supplement 2
Effect of hyperosmotic-stress on global methylation.
Methylation frequencies in MRs of plants from different generations, divided by sequence context. 25, 25 mM NaCl; 75, 75 mM NaCl; C, control.
Figure 2—figure supplement 3
Hyperosmotic-stress induced methylation changes in DMRs.
Methylation frequencies in MRs of plants from different generations, divided by sequence context (unpaired Student’s t-test; * p<0.05, ** p<0.01, *** p<0.001, ns p>0.05). C, control; 25, 25 mM NaCl; …
Figure 2—figure supplement 4
Overlap of HS-DMRs with MA-DMRs.
(A-B) Clustering of DMRs between P0 control and salt-treated samples in generations 1 and 3, separated according to overlap with DMRs in MA lines (Hagmann et al., 2015).
Figure 2—figure supplement 5
Gene Ontology analysis of genes associated to HS-DMRs.
Heatmaps of significantly enriched GO categories among hypo-/hypermethylated salt-stress induced DMRs that overlap or do not overlap with DMRs identified in MA lines (Hagmann et al., 2015) (p-value<0…
Figure 3 with 2 supplements
Dynamics of methylation frequency changes in DMRs.
(A) DMRs that are hypo- (darker colours) or hyper-methylated (lighter colours) in stress-treated P0 and their P1 and P2 progeny compared to the average of all control samples. Methylation states …
Figure 3—figure supplement 1
Methylation dynamics of HS-DMRs across three generations.
One-directional clustering of DMRs in generations 1 and 5 by methylation frequency difference, divided by sequence context. Methylation frequency differences were calculated by subtracting the …
Figure 3—figure supplement 2
Methylation at hyperosmosis-induced DMRs in drm1drm2 double mutants.
Methylation frequency difference in the P1 progeny of salt-stressed Col-0 wild-type and drm1 drm2 plants, separated by sequence context. Methylation frequency differences were calculated by …
Figure 4 with 2 supplements
Parent-of-origin effects on stress-induced epimutations.
(A) Survival of F1 seedlings derived from reciprocal crosses between Col-0 wild type or dme-6 mutants that had been exposed to hyperosmotic stress for two generations and untreated wild-type (wt), …
Figure 4—figure supplement 1
Isolation of sperm cells and vegetative nuclei by fluorescent-activated-cell-sorting.
(A) Confocal microscopy image (25x) of pollen from the A. thaliana pMGH3::MGH3-eGFP/pACT11::H2B marker line. pMGH3::MGH3-eGFP expression marks the sperm cell nuclei (green); pACT11p::H2B-mRFP …
Figure 4—figure supplement 2
Methylation at hyperosmotic stress-induced DMRs in the dme-6 mutants.
Methylation frequency difference in sperm cells (SC) and vegetative nuclei (VN); original sequencing data was taken from Ibarra et al. (2012). Differences were calculated by subtracting the …
Figure 5 with 4 supplements
Expression of two genes adjacent to hyperosmotic stress-induced DMRs.
(A) Methylation near MYB20 and CNI1. Black boxes on top represent genes, red boxes TEs. Methylation on the top and bottom strands at individual cytosines is shown as vertical bars below. (B–C) MYB20 …
Figure 5—figure supplement 1
Hyperosmotic stress response of genes next to HS-DMRs.
Heatmap showing expression changes of genes adjacent to HS-DMRs after exposure to hyperosmotic stress (0.5 to 24 hr) in shoots and roots (Zeller et al., 2009). Blue indicates downregulated genes …
Figure 5—figure supplement 2
Expression of genes adjacent to HS-DMRs.
Expression of four genes next to HS-DMRs in leaves of P1 and P2 progeny of P0 control ('naïve') and P0 salt-treated ('primed') plants, was analysed in leaves of 2-week-old wild type and rdd, cmt3 …
Figure 5—figure supplement 3
Expression of RdDM and demethylation pathway genes in response to hyperosmotic stress.
Heatmap showing expression of genes from RdDM and demethylation pathways after exposure to hyperosmotic stress (0.5 to 24 hr) in shoots and roots (Zeller et al., 2009). Gene names are listed on the …
Figure 5—figure supplement 4
Methylation profiles of HS-DMRs in DNA methylation and demethylation Arabidopsis mutants.
Heatmap of methylation frequency differences in HS-DMRs in different methylation contexts in twelve epigenetic mutants; original data from Stroud et al. (2013).
Figure 6 with 3 supplements
lncRNA-mediated control of CNI1 expression by a salt-induced DMR.
(A) Diagram of the CNI1 locus and key for expression experiments. Positions of insertion alleles, cni1-2 (Salk_100221) and cni1-3 (Salk_030235), are indicated, as are the DMR (dark green) in the …
Figure 6—figure supplement 1
Generation of CRISPR/Cas9 deletions at the CNI1 HS-DMR region.
(A) Sequence of the flanking CNI1 HS-DMR selected for targeted genome editing (methylated cytosines in lower case) and sequence of the CNI1 HS-DMR deletion line generated by CRISPR/Cas9 genome …
Figure 6—figure supplement 2
Methylation analysis of hairpin lines directing RdDM hypermethylation at the CNI1 HS-DMR.
(A) CHOP-PCR assay demonstrating that the HS-DMR downstream of CNI1 remains methylated in IR hairpin lines after exposure to hyperosmotic stress. A flanking region not was used as PCR control. (B) …
Figure 6—figure supplement 3
Expression analysis of CNI1 antisense lncRNA transcripts in response to hyperosmotic stress.
(A) Genome browser view of the genomic region flanking CNI1 (At5g27420). Tracks represent gene annotations (blue), transposons (yellow) and HS-DMR (black), lncRNAs (purple) and normalised signal of …
Figure 7
Working model for stress-dependent epigenetic regulation of CNI1.
In wild type, demethylation of a DMR in response to hyperosmotic stress stimulates lncRNA expression (CNI1-AS1), which in turn causes downregulation of CNI1 expression by an unknown mechanism. In …
Tables
Table 1
Association between differential DNA methylation induced by hyperosmotic stress and histone modifications (Sani et al., 2013).
https://doi.org/10.7554/eLife.13546.014| Chromatin mark | Hypo HS-DMRs | Hyper HS-DMRs | MRs |
|---|---|---|---|
| All | 70 | 280 | 72,074 |
| H3K4me2 (650/98) | 0/0 | 3/0 | 508/155 |
| H3K4me3 (1454/46) | 4/0 | 8/0 | 1065/40 |
| H3K9me3 (276/254) | 0/0 | 0/0 | 484/920 |
| H3K27me3 (1213/6520) | 3/15 | 7/99 | 1318/7774 |
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Intersections between high salt-induced DMRs (HS-DMRs), MRs and different chromatin marks (Sani et al., 2013). The two numbers behind each mark indicate significantly increased or decreased regions after hyperosmotic stress (Sani et al., 2013).
Additional files
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Supplementary file 1
Phenotypic data.
- https://doi.org/10.7554/eLife.13546.028
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Supplementary file 2
Methylation sequencing statistics.
- https://doi.org/10.7554/eLife.13546.029
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Supplementary file 3
Differentially methylated positions (DMPs).
- https://doi.org/10.7554/eLife.13546.030
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Supplementary file 4
Differentially methylated regions (DMRs).
- https://doi.org/10.7554/eLife.13546.031
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Supplementary file 5
Genes in proximity of HS-DMRs.
- https://doi.org/10.7554/eLife.13546.032
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Supplementary file 6
Intersection between stress-induced lncRNAs and HS-DRM associated genes.
- https://doi.org/10.7554/eLife.13546.033
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Supplementary file 7
List of oligonucleotides employed in PCR analyses.
- https://doi.org/10.7554/eLife.13546.034
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Supplementary file 8
Scoring matrix for assessing cytosine site statistics.
- https://doi.org/10.7554/eLife.13546.035
