Single and Double Mutations in Tomato Ripening Transcription Factors Have Distinct Effects on Fruit Development and Quality Traits

Spontaneous mutations associated with the tomato transcription factors COLORLESS NON-RIPENING (SPL-CNR), NON-RIPENING (NAC-NOR), and RIPENING-INHIBITOR (MADS-RIN) result in fruit that do not undergo the normal hallmarks of ripening but are phenotypically distinguishable. Here, we expanded knowledge of the physiological, molecular, and genetic impacts of the ripening mutations on fruit development beyond ripening. We demonstrated through phenotypic and transcriptome analyses that Cnr fruit exhibit a broad range of developmental defects before the onset of fruit ripening, but fruit still undergo some ripening changes similar to wild type. Thus, Cnr should be considered as a fruit developmental mutant and not just a ripening mutant. Additionally, we showed that some ripening processes occur during senescence in the nor and rin mutant fruit, indicating that while some ripening processes are inhibited in these mutants, others are merely delayed. Through gene expression analysis and direct measurement of hormones, we found that Cnr, nor, and rin have alterations in the metabolism and signaling of plant hormones. Cnr mutants produce more than basal levels of ethylene, while nor and rin accumulate high concentrations of abscisic acid. To determine genetic interactions between the mutations, we created for the first time homozygous double mutants. Phenotypic analyses of the double ripening mutants revealed that Cnr has a strong influence on fruit traits and that combining nor and rin leads to an intermediate ripening mutant phenotype. However, we found that the genetic interactions between the mutations are more complex than anticipated, as the Cnr/nor double mutant fruit has a Cnr phenotype but displayed inhibition of ripening-related gene expression just like nor fruit. Our reevaluation of the Cnr, nor, and rin mutants provides new insights into the utilization of the mutants for studying fruit development and their implications in breeding for tomato fruit quality.

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

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.

Light and ripening-regulated BBX protein-encoding genes in Solanum lycopersicum

Light controls several aspects of plant development through a complex signalling cascade. Several B-box domain containing proteins (BBX) were identified as regulators of Arabidopsis thaliana seedling photomorphogenesis. However, the knowledge about the role of this protein family in other physiological processes and species remains scarce. To fill this gap, here BBX protein encoding genes in tomato genome were characterised. The robust phylogeny obtained revealed how the domain diversity in this protein family evolved in Viridiplantae and allowed the precise identification of 31 tomato SlBBX proteins. The mRNA profiling in different organs revealed that SlBBX genes are regulated by light and their transcripts accumulation is directly affected by the chloroplast maturation status in both vegetative and fruit tissues. As tomato fruits develops, three SlBBXs were found to be upregulated in the early stages, controlled by the proper chloroplast differentiation and by the PHYTOCHROME (PHY)-dependent light perception. Upon ripening, other three SlBBXs were transcriptionally induced by RIPENING INHIBITOR master transcriptional factor, as well as by PHY-mediated signalling and proper plastid biogenesis. Altogether, the results obtained revealed a conserved role of SlBBX gene family in the light signalling cascade and identified putative members affecting tomato fruit development and ripening.

Inhibition of Droplet Growth in Model Beverage Emulsions Stabilized Using Poly (ethylene glycol) Alkyl Ether Surfactants Having Various Hydrophilic Head Sizes: Impact of Ester Gum

The effect of ester gum, a widely used weighting agent, on Ostwald ripening in model beverage emulsions formulated using different food-grade surfactants was examined. A microfluidizer was used to prepare 5% orange oil-in-water emulsions stabilized by a series of ethylene glycol alkyl ether surfactants. Emulsions prepared using only orange oil exhibited an appreciable increase in droplet size during a 14-day storage, independent of surfactant type or concentration. Incorporation of ester gum into the oil phase of the emulsions effectively inhibited droplet growth at concentrations ≥20%. The inhibition of droplet growth by ester gum depended on the surfactant type (hydrophilic group size) and concentration. Overall, ester gum stabilized the emulsions by acting as an Ostwald ripening inhibitor, as well as a weighting agent.