Allelic Mutations in the <i>Ripening</i><i>-Inhibitor</i> Locus Generate Extensive Variation in Tomato Ripening

Abstract

RIPENING INHIBITOR (RIN) is a transcription factor with transcriptional activator activity that plays a major role in regulating fruit ripening in tomato (<i>Solanum lycopersicum</i>). Recent studies have revealed that (1) <i>RIN</i> is indispensable for full ripening but not for the induction of ripening; and (2) the <i>rin</i> mutation, which produces nonripening 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 <i>RIN</i> function by characterizing a series of allelic mutations within this locus that were produced by CRISPR/Cas9. Fruits of <i>RIN</i>-knockout plants, which are characterized by partial ripening and low levels of lycopene but never turn 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 <i>RIN</i> locus (<i>rinG2</i>), did not activate transcription but formed transcription factor complexes that bound to target genomic regions in a manner similar to that observed for wild-type RIN protein. Fruits expressing this truncated RIN protein exhibited extended shelf life, but unlike <i>rin</i> fruits, they accumulated lycopene and appeared orange. The diverse ripening properties of the <i>RIN</i> allelic mutants suggest that substantial phenotypic variation can be produced by tuning the activity of a transcription factor.