The Arabidopsis DNA Methylome Is Stable under Transgenerational Drought Stress

Abstract

Improving the responsiveness, acclimation, and memory of plants to abiotic stress holds substantive potential for improving agriculture. An unresolved question is the involvement of chromatin marks in the memory of agriculturally relevant stresses. Such potential has spurred numerous investigations yielding both promising and conflicting results. Consequently, it remains unclear to what extent robust stress-induced DNA methylation variation can underpin stress memory. Using a slow-onset water deprivation treatment in Arabidopsis (Arabidopsis thaliana), we investigated the malleability of the DNA methylome to drought stress within a generation and under repeated drought stress over five successive generations. While drought-associated epi-alleles in the methylome were detected within a generation, they did not correlate with drought-responsive gene expression. Six traits were analyzed for transgenerational stress memory, and the descendants of drought-stressed lineages showed one case of memory in the form of increased seed dormancy, and that persisted one generation removed from stress. With respect to transgenerational drought stress, there were negligible conserved differentially methylated regions in drought-exposed lineages compared with unstressed lineages. Instead, the majority of observed variation was tied to stochastic or preexisting differences in the epigenome occurring at repetitive regions of the Arabidopsis genome. Furthermore, the experience of repeated drought stress was not observed to influence transgenerational epi-allele accumulation. Our findings demonstrate that, while transgenerational memory is observed in one of six traits examined, they are not associated with causative changes in the DNA methylome, which appears relatively impervious to drought stress.

Figure 1.

A mild drought stress is associated with variations in DNA methylation. A, Representative plants that were either unstressed (U) or underwent a drought stress (D) involving 9 d of withheld watering. B, Representative drought stress-associated DMR identified by DSS. Rows represent individual samples. DNA methylation is shown at single C resolution for mCG, mCHG, and mCHH as blue, orange, and green bars, respectively. Underlying genomic elements are presented at the bottom, and the exact region identified as a DMR is shown on top (red bar). C, Numbers of filtered stress-associated DMRs occurring near annotated protein-coding genes and TEs for each methylation context. D, Detailed mapping of filtered stress-associated DMRs within, or near, annotated protein-coding genes and TEs for each context of methylation. Body refers to DMRs occurring within the genomic feature, Upstream refers to DMRs within 1 kb near the 5′ end of the feature, Downstream refers to DMRs within 1 kb of the 3′ end of the feature, and Intergenic refers to DMRs that are farther than 1 kb away from the nearest genomic feature. E, Drought stress-associated DMR identified upstream of a gene encoding NAC089, identified previously as a locus exhibiting transcriptional memory to repeated dehydration stress. Rows represent individual samples. DNA methylation levels are shown at single C resolution for mCG, mCHG, and mCHH as blue, orange, and green bars, respectively. Underlying genomic elements are presented at the bottom, and the region identified as a DMR is shown on top (red bar).

Figure 2.

Promoter methylation levels at drought-responsive genes. A, Strip chart depicting log₂ fold change in mRNA expression of all drought-responsive genes grouped based on k-means clustering. Dots represent individual drought-responsive loci. Numbers of genes in each cluster are presented in parentheses. B, Summarized methylation levels in the 1-kb region directly upstream of drought-responsive loci averaged across all genes in each expression group as defined in A. Bars denote mean, error bars denote standard error of the mean (n = 3), and asterisk denotes significant differences (P < 0.05) between treatments as determined by a Kruskal-Wallis rank sum test. C, Summarized methylation levels in the 1-kb region directly upstream of 437 randomly selected non-drought-responsive loci. Bars denote mean, error bars denote standard error of the mean (n = 3), and asterisk denotes significant differences (P < 0.05) between treatments as determined by a Kruskal-Wallis rank sum test. D, Heat maps of mCHG and mCHH levels summarized 1 kb directly upstream of drought-responsive loci for individual transcripts ordered by expression group (as defined in A) and subsequently by log₂ fold change (top = highest; bottom = lowest).

Figure 3.

Transgenerational repeated drought stress experiment. A, Scheme of the repeated drought stress treatment performed every generation. Initial water deprivation (D1) began on 1-week-old seedlings and lasted for 14 d. After a recovery period of 5 d, a second treatment was performed (D2) that lasted for 12 d. B, Multiple independent lineages were propagated by single-seed descent for five generations, from a founding inbred progenitor, with half of the lines being exposed to the repeated drought stress treatment every generation. Testing for transgenerational stress memory was performed on the P₁ and P₂ progeny of G₄ and G₅ plants with plants from each independent lineage. WGBS was performed on P₁ progeny, each from an independent lineage, of G₀ and G₅ plants (red circles).

Figure 4.

Progeny from drought-exposed lineages show enhanced seed dormancy. A, Independent dormancy assays performed on seeds from P₀ (n = 6; more than 25 seeds per plate) and P₁ (n = 9; more than 25 seeds per plate) progeny of G₄ plants (P₀ and P₁ seeds, respectively). Points denote mean proportions of seeds germinated; error bars denote se. HR denotes the calculated hazard ratio from a fitted Cox proportional hazards model, representing the likelihood of germination between groups (HR_D = drought versus unstressed lineage). B, Dehydration assay performed on detached rosettes of P₁ progeny of G₄ (n = 12) and G₅ (n = 11) plants (independent experiments). A second-order polynomial regression, with a 95% confidence interval (shading), was performed to determine the coefficient for the lineage predictor term (α₂). R₂^C denotes the conditional R₂ calculated to assess model fit. C, Survival under the terminal drought experiment on transgenerational descendants (unstressed, n = 44; drought, n = 51). Bars denote means, and error bars denote se across two independent experiments.

Figure 5.

Limited methylome variation associated with transgenerational drought stress. A, Heat maps representing two-dimensional hierarchical clustering of correlations (Pearson’s r) in genome-wide DNA methylation levels, in all sequence contexts, averaged across 100-bp bins confirms similar broad DNA methylome patterns between all G₅ descendants. B, Integrative Genomics Viewer (IGV) visualization of lineage-associated DMRs identified by DSS (red bars). Vertical blue, yellow, and green bars denote mean mCG, mCHG, and mCHH, respectively, at single C resolution.

Figure 6.

DNA methylation levels at loci reported to exhibit transgenerational stress-induced methylation variation. IGV visualization is shown for DNA methylation, in G₀ P₁ control plants and G₅ P₁ plants of both watered and drought-stressed lineages, across loci documented to exhibit transgenerational memory of stress-induced changes in DNA methylation: A, FAMA (low humidity-induced hypermethylation); B, SPCH (low humidity-induced hypermethylation); C, MYB20 (upstream HS-induced hypermethylation); and D, CNI1 (downstream HS-induced hypomethylation). Blue, orange, and green bars denote mean mCG, mCHG, and mCHH, respectively, at single C resolution.

Figure 7.

Exploring stochastic and spontaneous methylome variation across transgenerational lineages. A, Heat maps of average methylation across 100-bp tile-based DMRs identified from pairwise comparisons, with one-dimensional hierarchical clustering of rows, between all samples. B, Heat maps representing two-dimensional hierarchical clustering of correlations (Pearson’s r) of genome-wide DNA methylation, in all sequence contexts, averaged across 100-bp tiles for all G₀ and G₅ progeny.