Abstract
Non-CG DNA methylation, a plant-specific epigenetic mark mainly regulated by chromomethylase (CMT), is known to play important roles in Arabidopsis thaliana. However, whether and to what extent non-CG DNA methylation modulates agronomic traits in crops remain to be explored. Here, we describe the consequences of non-CG DNA hypomethylation on development, seed composition, and yield in soybean (Glycine max). We created a Gmcmt mutant line lacking function of all four CMT genes. This line exhibited substantial hypomethylation of non-CG (CHG and CHH) sites. Non-CG hypomethylation enhanced chromatin accessibility and promoted or repressed the expression of hundreds of functionally relevant genes, including upregulation of GOLDEN-LIKE 10 (GmGLK10), which led to enhanced photosynthesis and, unexpectedly, improved nitrogen fixation efficiency. The Gmcmt line produced larger seeds with increased protein content. This study provides insights into the mechanisms of non-CG methylation-based epigenetic regulation of soybean development and suggests viable epigenetic strategies for improving soybean yield and nutritional value.