A modulatory role of CG methylation on gene expression in soybean implicates its potential utility in breeding.

Cytosine methylation (mCG) is an important heritable epigenetic modification, yet its functions remain to be fully defined in important crops. This study investigates mCG in soybean following the loss-of-function mutation of two GmMET1 genes. We generated knockout mutants of GmMET1s by CRISPR-Cas9 and conducted comprehensive methylome and transcriptome analyses. Our findings unravel the functional redundancy of the two GmMET1s, with GmMET1b being more critically involved in maintaining mCG levels, and complete knockout of both copies is lethal. We establish that genome-wide mCG levels scale with aggregated expression of GmMET1s. We identify a set of mCG-regulated genes whose expression levels were quantitatively modulated by upstream, body, or downstream mCG. Moreover, we find genes that were negatively regulated by upstream or body mCG are enriched in specific biological processes such as that of jasmonic acid metabolism. Notably, >80% of the differentially methylated genes (DMGs) in the mutants also exist as DMGs in natural soybean populations. Phenotypically, mutants that are heterozygous for GmMET1a and homozygous for GmMET1b knockouts (GmMET1aGmMET1b) exhibited early flowering, which was inherited by their selfed progeny (GmMET1aGmMET1b) with otherwise normal growth and development. Moreover, mutation of either GmMET1s, with slight reduction of mCG levels and similar phenotypes compared to the wild type under normal conditions, showed enhanced tolerance to cold and drought stresses. Together, our results underscore highly orchestrated regulatory effects of mCG on gene expression in soybean, which dictates growth, development and stress responses, implicating its utility in the improvement of soybean for better adaptability and higher yield.