Understanding epigenetic regulation in nonmodel plants: transcriptomic responses to seed demethylation in leaves and roots of the annual herb Erodium cicutarium (Geraniaceae).

Epigenetic regulation, and particularly modifications in DNA methylation, plays critical roles in plant adaptation. DNA methylation inhibitors have been used to investigate the relationship between DNA methylation and plastic plant phenotypes. However, their effect in gene expression regulation throughout the lifetime remains understudied in nonmodel plants. Here, we analyze the effects of seed exposure to the hypomethylating agent 5-azacytidine (5-azaC) in plant transcriptome. Seeds of Erodium cicutarium were soaked in either a solution of 5-azaC in DMSO or water with DMSO (control, hereafter) before sowing. Subsequently, RNA was extracted from juvenile roots, juvenile leaves, and adult leaves, and their transcriptomes were sequenced. Differential gene expression analysis was performed between control and treated samples for all tissues together and separately, presuming that changes between treatments will be substantially weaker than among tissues and developmental stages. Beforehand, a draft genome of E. cicutarium was assembled as reference for the transcriptome analysis. We found that 5-azaC upregulated chromomethylase CMT1 across all treated samples, and domain rearranged DNA methyltransferase DRM2 in juvenile roots. Furthermore, adult leaves showed more differentially expressed genes between control and 5-azaC-treated samples compared to juvenile leaves. Finally, gene co-expression network analysis revealed a module of co-expressed genes with differential gene expression linked to 5-azaC treatment in juvenile roots, pointing toward activation of genes associated with transposable elements. These results show how experimental treatment with 5-azaC at seed stage has both short and long-term effects in the plant transcriptome, potentially broadening phenotype variation due to nondirectional effects of 5-azaC on gene expression.