Regulation of DNA Methylation in Peanut Leaves and Roots: Uncovering the Molecular Mechanisms for Increased Yield After Single‐Seed Sowing

ABSTRACT Cytosine methylation is a crucial epigenetic modification that responds to various environmental cues, yet the specific mechanisms influencing planting patterns remain incompletely understood. This study reveals significant growth differences between single‐seed (SS) precision sowing and double‐seed (DS) sowing observed 42 days after germination under controlled indoor conditions. These differences were eliminated by the application of the DNA methylation inhibitor 5‐azacytidine (5‐aza), highlighting the role of DNA methylation in these processes. To further investigate the role of DNA methylation in planting pattern, we generated DNA methylation profiles of peanut leaves and roots under both DS and SS planting patterns. The analysis revealed increased CHH methylation in both tissues, caused by the RNA‐directed DNA methylation (RdDM) pathway. Further analysis, including differential methylation, transposable element (TE) analysis and methylation‐related gene analysis, demonstrated tissue‐specific epigenetic responses to planting patterns. Integrating methylome and transcriptome data, we found that DS was associated with hyper‐CHH methylation in WRKY gene promoters in leaves, accelerating leaf senescence. Meanwhile, SS reduced CHH methylation in promoters in roots, upregulating genes involved in flavonoid biosynthesis. This upregulation enhanced root nodule formation and improved stress resistance, resulting in increased concentrations of nitrogen and phosphorus in the roots, as confirmed by metagenomic functional analysis. This research provides novel insights into the epigenetic regulation of plant growth and development.