Semi-dominant effects of a novel ripening inhibitor (rin) locus allele on tomato fruit ripening

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.

A single MYB transcription factor with multiple functions during flower development

“…The conclusions are the following: (1) EOB2 is a multifunctional protein, (2) EOB2 transcriptional activity drives flower maturation, and (3) the R2R3‐MYB domain is, in addition to its DNA‐binding role in transcription, involved in the regulation of indirect senescence‐related targets through an unknown mechanism. Cases of nonfunctional vs partially functional gene product showing different phenotypes are rare in the literature, an example is the tomato MADS‐box gene RIN (for ripening inhibitor ) where the rin‐knockout (Ito et al ., 2017) and semi‐dominant rinG2 (Ito et al ., 2021) alleles were leading to different tomato fruit phenotypes. Proper conclusions about the function(s) of the protein require a careful analysis of the protein structure before targeting a TF gene by mutagenesis.…”

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

“…Unlike knockout mutants, the immunoblots of rinG2 showed a truncated RIN protein (Ito et al, 2021). Though the rinG2 allele had extended fruit shelf life, it had negative attributes of the rin mutant and had reduced lycopene levels similar to the knockout mutants (Ito et al, 2020).…”

“…However, homozygous CYCB knockouts were lethal, so in rinG2, only heterozygous CYCB knockouts could be raised, which had moderately increased lycopene levels (Ito et al, 2020). In contrast, the crossing of rinG2 with the wild-type parent restored the lycopene nearly to the wild-type levels while maintaining the extended shelf life of the fruits (Ito et al, 2021).…”

Introgression of a dominant phototropin1 mutant enhances carotenoids and boosts flavour‐related volatiles in genome‐edited tomato RIN mutants

“…Another gene‐edited RIN allele, rinG2 , partially truncated in the C domain of RIN (199 aa), exhibited a ripening phenotype distinct from the null mutants. Unlike knockout mutants, the immunoblots of rinG2 showed a truncated RIN protein (Ito et al ., 2020, 2021). Though the rinG2 allele had extended fruit shelf life, it had negative attributes of the rin mutant and had reduced lycopene levels like knockout mutants.…”

“…However, homozygous CYCB knockouts were lethal, so only heterozygous CYCB knockouts could be raised in rinG2 , which had moderately increased lycopene levels (Ito et al ., 2020). By contrast, the crossing rinG2 with the WT parent restored lycopene levels close to the WT levels while maintaining the extended fruit's shelf life (Ito et al ., 2021).…”