Long-term methylome changes after experimental seed demethylation and their interaction with recurrent water stress in Erodium cicutarium (Geraniaceae)

O_LIThe frequency and length of drought periods are increasing in subtropical and temperate regions worldwide. Epigenetic responses to water stress could be key for plant resilience to this largely unpredictable challenge. Experimental DNA demethylation together with application of a stress factor stands as a suitable strategy to uncover the contribution of epigenetics to plant responses to stress. C_LIO_LIWe analysed leaf cytosine methylation changes in adult plants of the Mediterranean weed, Erodium cicutarium, after seed demethylation with 5-Azacytidine and/or recurrent water stress in a greenhouse. We used bisulfite RADseq (BsRADseq) and a newly reported reference genome for E. cicutarium to characterize methylation changes in a 2x2 factorial design, controlling for plant relatedness. C_LIO_LIIn 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 level 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%. C_LIO_LISeed demethylation and recurrent drought exhibited a highly significant interaction in terms of global and context-specific cytosine methylation supporting an epigenetic contribution in response to stress at molecular level. C_LI