Introgression of a dominant phototropin1 mutant superenhances carotenoids and boosts flavor-related volatiles in genome-edited tomatoRINmutants

Abstract

AbstractThe 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-generatedrinalleles have these undesirable attributes associated with therinmutation. To address this, we used genome editing to generate newer alleles ofRIN(rinCR) by targeting the K domain, which is essential for the oligomerization of MADS-box transcription factors. Unlike previously reported CRISPR alleles, therinCRalleles displayed delayed onset of ripening, suggesting that the mutated K domain represses the onset of ripening. TherinCRfruits had extended shelf life and accumulated carotenoids at an intermediate level betweenrinand wild-type parent. Besides, the metabolites and hormonal levels inrinCRfruits were more akin torin. To overcome the negative attributes ofrin, we crossed therinCRalleles withNps1, which enhances carotenoid levels in tomato fruits.Nps1harbours a dominant-negative mutation in the plant photoreceptor phototropin1. The resultingNps1/rinCRhybrids had extended shelf life and 4.4-7.1-fold higher carotenoid levels than the wild-type parent. TheNps1/rinCRfruits had higher auxin and reduced ABA levels, which are reportedly linked with slower ripening. The metabolome ofNps1/rinCRfruits revealed higher sucrose, malate, and volatiles associated with tomato taste and flavour. Notably, the boosted volatile levels inNps1/rinCRwere only observed in fruits bearing the homozygousNps1(Nps1/Nps1) mutation. Our findings suggest that theNps1introgression into tomato ripening mutants provides a promising strategy for developing tomato cultivars with extended shelf life, improved taste, and flavour.