5mC DNA methylation modification-mediated regulation in tissue functional differentiation and important flavor substance synthesis of tea plant (Camellia sinensis L.)

In plants,5mC DNA methylation is an important and conserved epistatic mark involving genomic stability,gene transcriptional regulation,developmental regulation,abiotic stress response,metabolite synthesis,etc.However,the roles of 5mC DNA methylation modification(5mC methylation)in tea plant growth and development(in pre-harvest processing)and flavor substance synthesis in pre-and post-harvest processing are unknown.We therefore conducted a comprehensive methylation analysis of four key pre-harvest tissues(root,leaf,flower,and fruit)and two processed leaves during oolong tea post-harvest processing.We found that differential 5mC methylation among four key tissues is closely related to tissue functional differentiation and that genes expressed tissue-specifically,responsible for tissue-specific functions,maintain relatively low 5mC methylation levels relative to non-tissue-specifically expressed genes.Importantly,hypomethylation modifications of CsAlaDC and TS/GS genes in roots provided the molecular basis for the dominant synthesis of theanine in roots.In addition,integration of 5mC DNA methylationomics,metabolomics,and transcriptomics of post-harvest leaves revealed that content changes in flavor metabolites during oolong tea processing were closely associated with transcription level changes in corresponding metabolite synthesis genes,and changes in transcript levels of these important synthesis genes were strictly regulated by 5mC methylation.We further report that some key genes during processing are regulated by 5mC methylation,which can effectively explain the content changes of important aroma metabolites,includingα-farnesene,nerolidol,lipids,and taste substances such as catechins.Our results not only highlight the key roles of 5mC methylation in important flavor substance synthesis in pre-and post-harvest processing,but also provide epimutation-related gene targets for future improvement of tea quality or breeding of whole-tissue high-theanine varieties.