Abstract

The tomato (Solanum lycopersicum) ripening inhibitor (rin) mutation is known to completely repress fruit ripening. The heterozygous (RIN/rin) fruits have extended shelf life, ripen normally, but have poor taste and flavour. Even the CRISPR/Cas9-generated rin alleles have these undesirable attributes associated with the rin mutation. To address this, we used genome editing to generate newer alleles of RIN (rinCR) by targeting the K domain, which is essential for the oligomerization of MADS-box transcription factors. Unlike previously reported CRISPR alleles, the rinCRalleles displayed delayed onset of ripening, suggesting that the mutated K domain represses the onset of ripening. The rinCR fruits had extended shelf life and accumulated carotenoids at an intermediate level between rin and wild-type parent. Besides, the metabolites and hormonal levels in rinCR fruits were more akin to rin. To overcome the negative attributes of rin, we crossed the rinCR alleles with Nps1, which enhances carotenoid levels in tomato fruits. Nps1 harbours a dominant-negative mutation in the plant photoreceptor phototropin1. The resulting Nps1/rinCR hybrids had extended shelf life and 4.4-7.1-fold higher carotenoid levels than the wild-type parent. The Nps1/rinCR fruits had higher auxin and reduced ABA levels, which are reportedly linked with slower ripening. The metabolome of Nps1/rinCRfruits revealed higher sucrose, malate, and volatiles associated with tomato taste and flavour. Notably, the boosted volatile levels in Nps1/rinCR were only observed in fruits bearing the homozygous Nps1 (Nps1/Nps1) mutation. Our findings suggest that the Nps1 introgression into tomato ripening mutants provides a promising strategy for developing tomato cultivars with extended shelf life, improved taste, and flavour.