DNA Methylation Participates in Drought Stress Memory and Response to Drought in Medicago ruthenica

Background: Drought is currently a global environmental problem, which inhibits plant growth and development and seriously restricts crop yields. Many plants exposed to drought stress can generate stress memory, which provides some advantages for resisting recurrent drought. DNA methylation is a mechanism involved in 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 epigenetic molecular mechanisms underlying this process have not been described in this species. Methods: We conducted methylome and transcriptome sequencing to identify gene methylation and expression changes in plants with a history of two drought stress exposures. Results: Methylation analysis showed that drought stress resulted in an approximately 4.41% decrease in M. ruthenica genome methylation levels. The highest methylation levels were in CG dinucleotide contexts, 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 methylation within CHH motifs in gene promoter regions. Genes were divided into four categories according to the relationship between methylation and gene expression; the up-regulation of hypo-methylated gene expression accounted for the vast majority (692 genes) and included genes encoding factors key for abscisic acid (ABA) and proline synthesis. The hypo-methylation of the promoter and body regions of these two gene groups induced increased gene transcription levels. Conclusions: In conclusion, DNA methylation may contribute to drought stress memory formation and maintenance in M. ruthenica by increasing the transcription levels of genes key for ABA and proline biosynthesis.