Long‐term methylome changes after experimental seed demethylation and their interaction with recurrent water stress in <i>Erodium cicutarium</i> (Geraniaceae)

The frequencies and lengths of drought periods are increasing in subtropical and temperate regions worldwide. Epigenetic responses to water stress could be key for plant resilience to these largely unpredictable challenges. Experimental DNA demethylation, together with application of a stress factor is an appropriate strategy to reveal the contribution of epigenetics to plant responses to stress. We analysed leaf cytosine methylation changes in adult plants of the annual Mediterranean herb, Erodium cicutarium, in a greenhouse, after seed demethylation with 5-Azacytidine and/or recurrent water stress. We used bisulfite RADseq (BsRADseq) and a newly reported reference genome for E. cicutarium to characterize methylation changes in a 2 × 2 factorial design, controlling for plant relatedness. In the long term, 5-Azacytidine treatment alone caused both hypo- and hyper-methylation at individual cytosines, with substantial hypomethylation in CG contexts. In control conditions, drought resulted in a decrease in methylation in all but CHH contexts. In contrast, the genome of plants that experienced recurrent water stress and had been treated with 5-Azacytidine increased DNA methylation level by ca. 5%. Seed demethylation and recurrent drought produced a highly significant interaction in terms of global and context-specific cytosine methylation. Most methylation changes occurred around genic regions and within Transposable Elements. The annotation of these Differentially Methylated Regions associated with genes included several with a potential role in stress responses (e.g., PAL, CDKC, and ABCF), confirming an epigenetic contribution in response to stress at the molecular level.