Allelic mutations in the Ripening-inhibitor (RIN) locus generate extensive variation in tomato ripening

RIPENING INHIBITOR (RIN) is a transcription factor with transcriptional activator activity that plays a major role in regulating fruit ripening in tomato (Solanum lycopersicum). Recent studies have revealed that: (i) RIN is indispensable for full ripening but not for the induction of ripening and (ii) the rin mutation, which produces non-ripening fruits that never turn red or soften, is not a null mutation but instead converts the encoded transcriptional activator into a repressor. Here, we have uncovered aspects of RIN function by characterizing a series of allelic mutations within this locus that were produced by CRISPR/Cas9. Fruits of RIN-knockout plants, which showed partial ripening and low levels of lycopene but never turned fully red, showed excess flesh softening compared to the wild type. The knockout mutant fruits also showed accelerated cell wall degradation, suggesting that contrary to the conventional view, RIN represses over-ripening in addition to facilitating ripening. A C-terminal domain-truncated RIN protein, encoded by another allele of the RIN locus (rinG2), did not activate transcription but formed transcription factor complexes that bound to target genomic regions in a similar manner to wild-type RIN protein. Fruits expressing this truncated RIN protein exhibited extended shelf life, but unlike rin fruits, accumulated lycopene and appeared orange. The diverse ripening properties of the RIN allelic mutants suggest that substantial phenotypic variation can be produced by tuning the activity of a transcription factor.