CRISPR/Cas9 gene editing uncovers the roles of CONSTITUTIVE TRIPLE RESPONSE 1 and REPRESSOR OF SILENCING 1 in melon fruit ripening and epigenetic regulation.

Melon (Cucumis melo) has emerged as an alternative model to tomato for studying fruit ripening due to the coexistence of climacteric and non-climacteric varieties. Previous characterization of a major quantitative trait locus (QTL), ETHQV8.1, that is able to trigger climacteric ripening in a non-climacteric background resulted in the identification of a negative regulator of ripening CTR1-like (MELO3C024518) and a putative DNA demethylase ROS1 (MELO3C024516) that is the orthologue of DML2, a DNA demethylase that regulates fruit ripening in tomato. To understand the role of these genes in climacteric ripening, in this study we generated homozygous CRISPR knockout mutants of CTR1-like and ROS1 in a climacteric genetic background. The climacteric behavior was altered in both loss-of-function mutants in two growing seasons with an earlier ethylene production profile being observed compared to the climacteric wild type, suggesting a role of both genes in climacteric ripening in melon. Single-cytosine methylome analyses of the ROS1-knockout mutant revealed changes in DNA methylation in the promoter regions of the key ripening genes such as ACS1, ETR1, and ACO1, and in transcription factors associated with ripening including NAC-NOR, RIN, and CNR, suggesting the importance of ROS1-mediated DNA demethylation for triggering fruit ripening in melon.