Requirement of CHROMOMETHYLASE3 for somatic inheritance of the spontaneous tomato epimutation Colourless non-ripening

Naturally-occurring epimutants are rare and have mainly been described in plants. However how these mutants maintain their epigenetic marks and how they are inherited remain unknown. Here we report that CHROMOMETHYLASE3 (SlCMT3) and other methyltransferases are required for maintenance of a spontaneous epimutation and its cognate Colourless non-ripening (Cnr) phenotype in tomato. We screened a series of DNA methylation-related genes that could rescue the hypermethylated Cnr mutant. Silencing of the developmentally-regulated SlCMT3 gene results in increased expression of LeSPL-CNR, the gene encodes the SBP-box transcription factor residing at the Cnr locus and triggers Cnr fruits to ripen normally. Expression of other key ripening-genes was also up-regulated. Targeted and whole-genome bisulfite sequencing showed that the induced ripening of Cnr fruits is associated with reduction of methylation at CHG sites in a 286-bp region of the LeSPL-CNR promoter and a decrease of DNA methylation in differentially-methylated regions associated with the LeMADS-RIN binding sites. Our results indicate that there is likely a concerted effect of different methyltransferases at the Cnr locus and the plant-specific SlCMT3 is essential for sustaining Cnr epi-allele. Maintenance of DNA methylation dynamics is critical for the somatic stability of Cnr epimutation and for the inheritance of tomato non-ripening phenotype.