Reactivation of a loss-of-function SlGLK2 allele by frame-restoring genome editing in tomato

Plant domestication and improvement processes have inadvertently led to the loss of gene functions that contribute to crop quality. In widely-cultivated tomato ( Solanum lycopersicum ) varieties, a uniform ripening ( u ) mutation, a loss-of-function allele of the Solanum lycopersicum Golden2-like 2 ( SlGLK2 ) gene, has been selected to improve fruit appearance. However, this selection is associated with reduced nutritional quality of the fruit, lowering the levels of sugars, carotenoids, and tocopherols due to impaired plastid development. In this study, the function of SlGLK2 was restored by introducing an additional mutation. A frameshift was introduced via genome editing using the temperature-tolerant Lachnospiraceae bacterium ND 2006 Cas12a ( Lb Cas12a) system. A 13-bp deletion in the linker region of the SlGLK2 protein in the Slglk2-4 line corrected the reading frame and led to enhanced plastid development in the basal part of the fruit. Biochemical and transcriptomic analyses confirmed the functional restoration of SlGLK2 in the Slglk2-4 line. This study provides a model Micro-Tom line for studying plastid biogenesis in tomato fruits and demonstrates the potential of genome editing to revive the latent functions of pseudogenized genes in modern crops, offering a new approach for recovering traits lost during domestication.