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.

In non-climacteric fruits, the respiratory increase is absent and no phytohormone is appearing to be critical for their ripening process. They must remain on the parent plant to enable full ripening and be picked at or near the fully ripe stage to obtain the best eating quality. However, huge losses often occur for their quick post-harvest senescence. To understanding the complex mechanism of non-climacteric fruits post-harvest senescence, we constructed two small RNA libraries and one degradome from strawberry fruit stored at 20°C for 0 and 24 h. A total of 88 known and 1224 new candidatemiRNAs, and 103 targets cleaved by 19 known miRNAs families and 55 new candidatemiRNAs were obtained. These targets were associated with development, metabolism, defense response, signaling transduction and transcriptional regulation. Among them, 14 targets, including NAC transcription factor, Auxin response factors (ARF) and Myb transcription factors, cleaved by 6 known miRNA families and 6 predicted candidates, were found to be involved in regulating fruit senescence. The present study provided valuable information for understanding the quick senescence of strawberry fruit, and offered a foundation for studying the miRNA-mediated senescence of non-climacteric fruits.

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