Abstract
The tomato (Solanum lycopersicum) ripening inhibitor (rin) mutation completely represses fruit ripening, as rin fruits fail to express ripening-associated genes and remain green and firm. Moreover, heterozygous rin fruits (rin/+) ripen normally but have extended shelf life, an important consideration for this perishable fruit crop; therefore, heterozygous rin has been widely used to breed varieties that produce red tomatoes with improved shelf life. We previously used CRISPR/Cas9 to produce novel alleles at the rin locus. The wild-type allele RIN encodes a MADS-box transcription factor and the novel allele, named as rinG2, generates an early stop codon, resulting in C-terminal truncation of the transcription factor. Like rin fruits, rinG2 fruits exhibit extended shelf life, but unlike rin fruits, which remain yellow-green even after long-term storage, rinG2 fruits turn orange due to ripening-associated carotenoid production. Here, to explore the potential of the rinG2 mutation for breeding, we characterized the effects of rinG2 in the heterozygous state (rinG2/+) compared to the effects of rin/+. The softening of rinG2/+ fruits was delayed compared to the wild type but to a lesser degree than rin/+ fruits. Lycopene and β-carotene levels in rinG2/+ fruits were similar to those of the wild type, whereas rin/+ fruits accumulated half the amount of β-carotene compared to the wild type. The rinG2/+ fruits produced lower levels of ethylene than wild-type and rin/+ fruits. Expression analysis revealed that in rinG2/+ fruits, the rinG2 mutation (like rin) partially inhibited the expression of ripening-associated genes. The small differences in the inhibitory effects of rinG2 vs. rin coincided with small differences in phenotypes, such as ethylene production, softening, and carotenoid accumulation. Therefore, rinG2 represents a promising genetic resource for developing tomato cultivars with extended shelf life.
Highlights
Extending the shelf life of agricultural products is important for supplying fresh, nutritious foods and reducing food losses
To examine the possible use of the rinG2 mutation as a practical method for breeding tomatoes with extended shelf life, we investigated the heterozygous effects of rinG2 by developing F1 hybrid plants with the rinG2/+ genotype
Genome editing resulted in a one-base insertion at position 596 from the translation start position in the mRNA, which caused a frame-shift producing an early stop codon just after the insertion (Fig 1B). This mutation modified the wildtype RIN (242 amino acid residues) into a peptide with 199 amino acid residues composed of 198 amino acids from the original RIN protein and an additional amino acid produced by the frame shift (Fig 1C)
Summary
Extending the shelf life of agricultural products is important for supplying fresh, nutritious foods and reducing food losses. Storing fruits and vegetables at low temperature and maintaining an appropriate atmosphere during transport and storage can preserve the quality of fresh vegetables and fruits. Developing cultivars that produce fruits with resistance to over-ripening. A novel rin allele affects tomato ripening is another effective way to maintain fruit quality [1]. Tomato (Solanum lycopersicum), a major vegetable consumed all over the world, is a typical fruit species that is often wasted due to over-ripening-associated quality losses, such as cracking, wilting, and infection [2, 3]. Extending shelf life has been an important objective for tomato breeding programs [4,5,6]
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