An increase in DNA methylation contributes to anthocyanin biosynthesis in the pericarp as litchi (Litchi chinensis Sonn.) fruit ripens

INTRODUCTION: Significant progress has been made in uncovering the role of DNA methylation in the development and ripening of fruit. However, whether DNA methylation is involved in litchi fruit ripening and in the corresponding mechanisms remain unknown. OBJECTIVES: The goal of this study was to explore the role of DNA methylation in the ripening, senescence, and the regulatory mechanism of litchi as well as to provide new perspectives for controlling litchi pericarp browning after harvest. METHODS: Here, single-base resolution maps of the DNA methylome during litchi fruit ripening and senescence was generated using whole genome bisulfite sequencing (WGBS). Association analysis of WGBS and RNA-sequencing was performed to reveal the potential regulatory effects of different regional methylation sites on gene expression in litchi in different fruit ripening and postharvest stages. A DNA methylation inhibitor, 5-azacytidine, treatment was also conducted. Meanwhile, the LcDML1 silencing mutant (TRV-LcDML1) was obtained using virus-induced gene silencing to confirm the regulatory role of LcDML1 in litchi ripening. RESULTS: Results showed that about 55%, 34%, and 11% of CG, CHG and CHH sites were methylated, respectively. Genome-wide DNA methylation increased during litchi fruit ripening and exhibited a regulatory role in gene expression during ripening but had limited influence during senescence. Furthermore, WGBS and RNA-sequencing analysis indicated that LcDML1 contributed to changes in genome-wide DNA methylation during litchi maturation. A 5-azacytidine treatment inhibited anthocyanin synthesis in litchi pericarp. In addition, LcDML1 silencing induced early coloration of litchi pericarp and upregulation of anthocyanin synthesis-related genes but downregulation of potential negative regulator of anthocyanin synthesis genes (NRAs). McrBC-PCR and dual-luciferase reporter assay suggested methylation regulation of NRAs promoters regulated the maturation and coloration of litchi fruit. CONCLUSION: LcDML1 is an important factor modulating the change of genome-wide DNA methylation as well as anthocyanin synthesis and participates in regulating litchi fruit ripening.