Genome-Wide Reinforcement of DNA Methylation Occurs during Somatic Embryogenesis in Soybean

Abstract

Somatic embryogenesis is an important tissue culture technique that sometimes leads to phenotypic variation via genetic and/or epigenetic changes. To understand the genomic and epigenomic impacts of somatic embryogenesis, we characterized soybean (Glycine max) epigenomes sampled from embryos at 10 different stages ranging from 6 weeks to 13 years of continuous culture. We identified genome-wide increases in DNA methylation from cultured samples, especially at CHH sites. The hypermethylation almost exclusively occurred in regions previously possessing non-CG methylation and was accompanied by increases in the expression of genes encoding the RNA-directed DNA methylation (RdDM) machinery. The epigenomic changes were similar between somatic and zygotic embryogenesis. Following the initial global wave of hypermethylation, rare decay events of maintenance methylation were observed, and the extent of the decay increased with time in culture. These losses in DNA methylation were accompanied by downregulation of genes encoding the RdDM machinery and transcriptome reprogramming reminiscent of transcriptomes during late-stage seed development. These results reveal a process for reinforcing already silenced regions to maintain genome integrity during somatic embryogenesis over the short term, which eventually decays at certain loci over longer time scales.

Embryo CHH hypermethylation is mediated by RdDM and is autonomously directed inBrassica rapa

“…CG methylation was largely unchanged across the tissues, with the exception of endosperm, which was demethylated in CG and CHG contexts, consistent with observations in Arabidopsis and rice [32][33][34]. As in Arabidopsis, soybean, and chickpea [22][23][24][25][26][27] we also observed elevated global CHH methylation in B. rapa mature embryos, with moderately increased CHH methylation in torpedo-stage embryos ( Figure 1A). The increased CHH methylation in torpedo-stage embryos was correlated with CHH hypermethylation in mature embryos ( Figure 1B, correlation coefficient = 0.6), indicating that hypermethylation is a gradual process during embryogenesis.…”

“…With the exception of Arabidopsis, which has only a small reduction in seed size, loss of RdDM in most species results in disruption of reproductive development, indicating that RdDM is necessary for successful sexual reproduction [17][18][19][20][21]. Mature embryos accumulate high levels of CHH methylation in Arabidopsis, soybean, and chickpea, suggesting that RdDM might enable reproduction through hypermethylation of the mature embryo [22][23][24][25][26][27]. In Arabidopsis, the developing endosperm is demethylated at sequences that show hypermethylation in the embryo, leading to the hypothesis that siRNAs produced in the endosperm might move to the embryo to direct methylation [22,[28][29][30].…”

“…Movement of siRNAs between the maternal integuments and the filial tissues has also been proposed [31]. However, embryos produced through somatic embryogenesis also display hypermethylation, despite lack association with either endosperm or maternal integuments [27].…”

Epigenetic Regulation of Auxin-Induced Somatic Embryogenesis in Plants

“…In a pioneering work on DNA methylation in SE induction, a treatment with auxins but not cytokinins was found to increase the level of global DNA methylation in embryogenic cell cultures of Daucus carota [119]. The impact of 2,4-D on DNA methylation has also been confirmed in embryogenic cultures of Picea omorika [120], Malus xiaojinensis [121], and Glycine max [122]. The changes in global 5mC content in the auxin-induced embryogenic cultures of different plants have been associated with a differential gene expression [123], and an extensive modulation of the genes encoding both the methylases and demethylases of DNA has been attributed to the auxin-induced SE-transcriptomes of different plants, i.e., Populus trichocarpa, Arabidopsis, G. hirsutum, and G. max [122,[124][125][126].…”

“…The impact of 2,4-D on DNA methylation has also been confirmed in embryogenic cultures of Picea omorika [120], Malus xiaojinensis [121], and Glycine max [122]. The changes in global 5mC content in the auxin-induced embryogenic cultures of different plants have been associated with a differential gene expression [123], and an extensive modulation of the genes encoding both the methylases and demethylases of DNA has been attributed to the auxin-induced SE-transcriptomes of different plants, i.e., Populus trichocarpa, Arabidopsis, G. hirsutum, and G. max [122,[124][125][126]. In Arabidopsis, DOMAINS REARRANGED METHYLTRANSFERASE1-2 (DRM1-2) are required for de novo methylation via RNA-directed DNA methylation (RdDM) pathway, while METHYLTRANSFERASE1 (MET1) and CHROMOMETHYLASE1-3 (CMT1-3) maintain the methylation pattern during DNA replication.…”