Partial maintenance of organ-specific epigenetic marks during plant asexual reproduction leads to heritable phenotypic variation

Abstract

Plants differ from animals in their capability to easily regenerate fertile adult individuals from terminally differentiated cells. This unique developmental plasticity is commonly observed in nature, where many species can reproduce asexually through the ectopic initiation of organogenic or embryogenic developmental programs. While organ-specific epigenetic marks are not passed on during sexual reproduction, the fate of epigenetic marks during asexual reproduction and the implications for clonal progeny remain unclear. Here we report that organ-specific epigenetic imprints in Arabidopsis thaliana can be partially maintained during asexual propagation from somatic cells in which a zygotic program is artificially induced. The altered marks are inherited even over multiple rounds of sexual reproduction, becoming fixed in hybrids and resulting in heritable molecular and physiological phenotypes that depend on the identity of the founder tissue. Consequently, clonal plants display distinct interactions with beneficial and pathogenic microorganisms. Our results demonstrate how novel phenotypic variation in plants can be unlocked through altered inheritance of epigenetic marks upon asexual propagation.

Keywords: Arabidopsis thaliana; DNA methylation; asexual reproduction; epigenetics; transgenerational inheritance.

Fig. 1.

Differential expression of defense-related genes in RO and LO plants. (A) Experimental design. RO and LO regenerated plants (n = 5 independent lines each) were propagated through self-fertilization over three generations. (B) GO analysis of DEGs (FDR <0.01; absolute log-twofold change >1.5) between leaves of RO and LO G₂ plants revealing enrichment for defense-related functions. (C) Heatmap of scaled, normalized log-transformed read counts for genes underlying the significant enrichment of GO terms in B. FPKM, fragments per kilobase per million. (D) Interaction gene network of DEGs. To facilitate network analysis, we lowered the FDR threshold to <0.05. Of the resulting 4,585 DEGs, 2,752 were represented in the ANAP protein interaction database. Nodes represent genes; triangles highlight defense-related genes from B; edges indicate evidence for gene interaction. Blue filled circles, high expression in LO plants; orange filled circles, high expression in RO plants; red outlined circles, genes with stress-related GO terms.

Fig. 2.

Plants regenerated from roots or leaves interact differently with beneficial and pathogenic microbes. (A) PCA of Bray–Curtis distances of bacterial communities present in roots of nonregenerated and regenerated plants grown in natural soils (n = 10). (B) Canonical analysis of principal coordinates (based on Bray–Curtis distances) showing different root-associated communities of SynComs colonized on nonregenerated and regenerated plants (n = 12). (C) Susceptibility of nonregenerated and regenerated plants to P. syringae pv. tomato strain DC3000 infection. Bacterial growth was determined at 3 d after inoculation (100 cfu mL⁻¹). Data are mean ± SD values from three independent experiments. Statistical significance according to Fisher’s exact test: **P < 0.01, ***P < 0.001; NS, not significant. (D) Susceptibility of nonregenerated and regenerated plants to H. arabidopsidis (Hpa) Noks1 infection, as indicated by the number of conidiospores on leaves at 3 d after inoculation with a suspension of 300,000 spores/mL. Data are mean ± SD values from two independent experiments. P values were determined using Student’s t test.

Fig. 3.

DNA methylation variation in regenerated plants is stably inherited after selfing and back-crossing. (A) PCA of DNA methylation levels at DMPs identified from pairwise sample comparisons. Numbers in brackets indicate the fraction of overall variance explained by the respective PC. (B) Clustering of LO and RO leaf samples in generation G₂, based on 765 DMRs identified in all-against-all pairwise comparisons. (C) PCA of methylation at 255 DMRs identified in G₂ RO vs. LO leaf comparison, divided by cytosine sequence context. (Right) PCA on methylation in all contexts within randomly chosen non-DMRs. Numbers in brackets indicate the amount of variance explained by the respective PC. (D) Gains and losses of DNA methylation in DMRs identified between RO and LO leaves in the G₂ generation. Color keys indicate methylation rate differences in relation to leaves of nonregenerated plants. (Right) Differences in a random subset of non-DMRs. (E) Methylation frequencies at DMRs in leaves of nonregenerated Col-0, RO, and reciprocal crosses (F₁) between nonregenerated Col-0 and RO plants. (F) Methylation analysis of progeny from F₁ reciprocal hybrids. The heatmap shows DMR methylation levels in individual F₁ hybrid plants (#7 and #16) and each of four independent descendants. The bar plot shows the frequency of hypomethylation in F₂ plants of DMRs that were hypomethylated in the F₁ hybrid.

Fig. 4.

Activity of an RSM1 regulatory element is affected by DNA methylation. (A) Snapshot of DNA methylation in different sequence contexts at the RSM1 locus in wild-type (WT) Col-0 plants and plants carrying an inverted repeat transgene (RSM1-IR). The black horizontal bar indicates the region targeted by RSM1-IR–induced RdDM for DNA hypermethylation. Green ticks, CG methylation; blue, CHG methylation; orange, CHH methylation. (B) RSM1 expression in leaves and roots of WT plants, RSM1-IR plants, and RSM1-IR plants complemented with synRSM1. White bar, leaf; black bar, root. (C) Induced DNA hypermethylation of RSM1-DMR affects morphology. (D) Genetic complementation of RSM1-IR with a synthetic RSM1 transgene (synRSM1) resistant to RSM1-IR–induced hypermethylation.