DNA methylation participates in drought stress memory and response to drought in Medicago ruthenica

Drought is a serious environmental problem, affecting plant growth and development and resulting in severe yield loss. Many plants exposed to drought stress can generate stress memory, which provides some advantages for resisting recurrent drought. DNA methylation is a mechanism of stress memory formation, and many plants can alter methylation levels to form stress memories; however, it remains unclear whether Medicago ruthenica exhibits drought stress memory, as the molecular mechanisms underlying this process have not been described in this species. Here, we conducted methylome and transcriptome sequencing to identify gene methylation and expression changes in plants with a history of two drought stress exposures (D2). Methylation analysis showed that drought stress resulted in an approximately 4.41% decrease in M. ruthenica genome methylation levels. CG dinucleotide contexts had the highest methylation levels, followed by CHG contexts, with CHH contexts having the lowest levels. Analysis of associations between methylation and transcript levels showed that most DNA methylation was negatively correlated with gene expression, except for methylation within CHH in gene promoter regions. Genes were divided into four categories according to correlation between methylation and gene expression; demethylated genes expression upregulation accounted for the vast majority (n = 692 genes), and included genes encoding key factors for abscisic acid (ABA) and proline synthesis. Demethylation of the promoter and body regions of these two genes induced increased gene expression levels. In conclusion, DNA methylation may contribute to drought stress memory formation and maintenance in M. ruthenica by increasing transcription levels of genes key for ABA and proline biosynthesis.