Fig 1.
Expression of the rinG2 allele in tomato.
A. Ripe fruits with homozygous or heterozygous mutant alleles in the rin locus. Fruits with the indicated genotypes were harvested at 7 days after the breaker stage. B. The rinG2 mutation generates an early stop codon. “A” in red represents the base inserted by genome editing. C. Schematic diagrams of the proteins encoded by the rin and rinG2 alleles. The rin allele produces a protein encoded by a fusion mRNA composed of RIN lacking the last exon and MC lacking the first exon [9]. The rinG2 mutation generates an early stop codon, which removes the C-terminus including the transcriptional activator domain of RIN [23]. D. Accumulation of mutant proteins in heterozygous fruits. In rin- and rinG2-heterozygous fruits, the mutant proteins (indicated as rin and rinG2, respectively) accumulated simultaneously with wild-type protein (RIN). Proteins from nuclei of fruits harvested at 4 days after the breaker stage were separated and subjected to an immunoblotting assay with RIN-antibodies [15]. We did not measure the detection efficiencies of the allelic proteins in extracts from heterozygous lines, and therefore, we cannot conclusively state whether the differences in signal intensities between the allelic proteins reflect differences in their abundances in the cells.
Fig 2.
Heterozygous effects of rinG2 on fruit shelf life.
Fruits were harvested at 7 days after the breaker stage (0 W) and stored at 25°C.
Fig 3.
Fruit softening, carotenoid accumulation, and ethylene biosynthesis in rinG2/+.
A. Firmness of fruits with mutations in the rin locus. Fruits harvested just before the breaker stage were examined as 0 week samples. Breaker stage fruits were harvested and examined at 1, 2, and 4 weeks after harvest. Data represent the means ± SE of five biological replicates. B. Lycopene and β-carotene contents of fruits with mutations in the rin locus. Fruits were harvested at 10 days after the breaker stage. Data represent the means ± SE of five or six biological replicates. C. Ethylene biosynthesis in fruits with mutations in the rin locus. Fruits just before the breaker stage were harvested and examined daily. Data represent the means ± SE of at least four biological replicates.
Fig 4.
The expression of ripening-associated genes in rinG2/+ fruit.
mRNA levels were measured by qRT-PCR. mRNA was prepared from fruits at the green-coloring stage just before the initiation of ripening (G) and fruits during ripening (4 and 7days after the breaker stage; B+4 and B+7). A primer set for the RIN alleles anneals to a common sequence of the wild type, rin, and rinG2 alleles. Data represent the means ± SE of three biological replicates.