To determine the impact of Cnr, nor, and rin mutations on the key fruit traits, we measured external color, firmness, total soluble solids , and titratable acidity at multiple ripening stages. Fruit from the isogenic mutants Cnr, nor, and rin, were harvested alongside WT from an experimental field at selected ripening stages, mature green , turning , red ripe , and overripe . We captured field variability through large sample sizes and validated across two to four independent field seasons. A summary of all seasons is displayed in Figure 1 while a breakdown of the data by field season can be found in Supplementary Table 2. As expected, between the MG and RR stages WT fruit turned red internally and externally, reduced firmness, accumulated TSS, and became less acidic during ripening. Cnr fruit showed visual differences compared to all of the genotypes at the MG stage and continuing through subsequent stages, including significantly smaller size and its characteristic colorless flesh, marked by an opaque yellow coloration of the pericarp . Statistical analyses performed for color and size confirmed Cnr exhibited significant differences consistently across each field season, as reported in Supplementary Table 2. Fruit of nor and rin displayed a distinct absence of any red coloration compared to WT at the RR stage; instead, these fruit began to turn yellow externally. Fruit of all ripening mutants were significantly firmer across all stages than the WT, tall plastic pots though this difference was especially pronounced in Cnr . Overall, Cnr was consistently different from the other genotypes before and during ripening, while nor and rin remained similar to WT MG fruit.
The OR stage was selected to investigate if the ripening mutants displayed phenotypic changes at later time points that could be associated with a delay in fruit development. At this stage, nor fruit started to turn orange-red externally and red internally, similar to WT fruit. The nor OR fruit resembled WT fruit between the T and RR stages. We performed a PCA of the color data to compare the genotypes at the RR and OR stage, and found nor OR measured closely with WT RR in external coloration . A summary ofthe color data and statistical analyses performed can be found in Supplementary Table 2. While nor OR fruit visually looked most similar to WT RR fruit, rin OR fruit consistently measured similarly to WT fruit at the RR and OR stages in the tasterelated traits of TSS and TA. These phenotypes were especially noticeable in the OR stage, suggesting that rin exhibits a delayin these traits. In contrast, Cnr remained distinct from WT and the other mutants at the OR stage in all measurements . Thus, in the OR stage, nor and rin behaved more similar to WT, suggesting they display more ripening phenotypes after the RR stage. The distinct phenotypic differences observed between the ripening mutants indicate that each mutation has a unique impact on fruit molecular processes at specific developmental stages. We performed an RNAseq study of WT, Cnr, nor, and rin fruit at the MG and RR stages to gain insights into the observed phenotypes. A principal component analysis was performed using mapped normalized reads to the tomato predicted transcriptome from WT and mutant samples at MG and RR stages . The PCA revealed that the genotypes were mainly separated by ripening stage and that Cnr was distinct from WT and the other mutants . Remarkably, Cnr displayed the most similar pattern to WT across PC1 than any other mutant.
Like their phenotypes suggested, nor and rin transcriptomic profiles showed little change between the MG and RR stage and clustered with the WT MG fruit. The separation driven by PC2 supported our observations that Cnr fruit was phenotypically different from other genotypes.Because Cnr showed deviation from WT at the MG stage in both phenotype and transcriptional profiles, we hypothesized that gene expression across the genome was affected prior to the MG stage. To determine when the transcriptional profile of Cnr began to diverge from WT and other mutants, we obtained and reanalyzed raw RNAseq data from all genotypes at four early stages of fruit growth and development . We performed a PCA for each developmental stage and found that Cnr was separated from other genotypes as early as 7 dpa in fruit development, while nor and rin were similar to WT throughout early development . When evaluating differentially expressed genes between Cnr and WT fruit at 7 dpa, we detected 1,320 mutation-related DEGs while nor and rin had only 173 and 392, respectively . These results suggest that Cnr fruit have different gene expression profiles from WT throughout fruit growth and maturation, even before ripening begins.We determined DEGs from the MG and RR stages to identify specific molecular functions altered in Cnr, nor, and rin fruit. First, we compared the ripening mutants to the WT at each stage and obtained a total of 16,085 mutation related DEGs across all comparisons . Like the PCA suggested , Cnr MG fruit presented the largest amount of mutation-related DEGs , while nor and rin MG had considerably fewer DEGs when compared to the WT counterpart . At the RR stage, large differences between each mutant and WT were observed, with Cnr RR fruit displaying once again the largest differences in the amount of mutation-related DEGs . The large number of mutation-related DEGs shown by Cnr fruit further supports our hypothesis that the Cnr mutation more broadly affects fruit development and that nor and rin appear to be more ripening-specific mutations.
We examined molecular functions based on KEGG annotations that were significantly enriched among the mutation-related DEGs for each Cnr, nor, and rin fruit at MG and RR . Large differences in enriched functions were detected in the Cnr MG fruit, which mainly corresponded to alterations in carbohydrate and amino acid metabolism, chlorophyll, and carotenoid biosynthesis,and interestingly many processes related to DNA replication and repair. The lack of green color in Cnr MG fruit could be explained by lower expression of photosynthesis and carbon fixation genes. The nor MG and rin MG fruit showed few alterations compared to WT and were mainly noted in amino acid metabolism and plant hormone signal transduction. In contrast, at the RR stage, the three ripening mutants showed significant alterations across multiple molecular pathways that range from primary and secondary metabolism to transcription, translation, and signaling processes. We proceeded to mine the mutation-related DEGs for key genes known to affect the fruit traits evaluated in the ripening mutants: color, firmness, TSS, and acidity. We selected five carotenoid biosynthesis genes involved in fruit pigmentation, six genes encoding cell wall degrading enzymes that promote fruit softening, four genes related to sugar accumulation and transport that impact the fruit’s TSS, and one gene that regulates the levels of citric acid then affecting the fruit’s acidity . At the MG stage, large plastic pots we observed that Cnr fruit showed significantly lower expression than WT for several of these key genes, consistent with our phenotypic data , including firmness related enzymes and carotenoid biosynthesis genes. MG fruit from the three ripening mutants showed significantly lower gene expression in an important invertase in fruit , which may contribute to the lower levels of TSS observed in all the mutants . At the RR stage, most of the fruit trait-associated genes surveyed in the ripening mutants had a significantly lower expression than WT, in support of the phenotypic data and reinforced by the numerous functional enrichments among the mutation related DEGs . The critical carotenoid biosynthesis gene that encodes PHYTOENE SYNTHASE 1 was significantly lower expressed than WT in the mutant fruit across all stages, accounting for the lack of red pigmentation at the RR stage. Also, downstream genes in the pathway encoding Lycopene β-cyclases were highly expressed in the mutants at the RR stage, suggesting that not only was less lycopene being produced but more was being metabolized. CWDEs were negatively affected across all genotypes, with Cnr having the most mutation-related DEGs in this category. We were interested in examining if the Cnr, nor, and rin mutant fruit displayed altered ripening progression or if they were completely inhibited or delayed in ripening events. We performed another set of differential expression analyses comparing RR against MG fruit for WT and each of the mutants to reveal ripening-related DEGs. As anticipated, WT had the largest number of ripening-related DEGs , while nor showed almost no change between the two ripening stages with only 89 DEGs detected . Cnr and rin had fewer ripening-related DEGs compared to WT but still exhibited significant changes during the transition between stages with 5,788 and 2,799 DEGs, respectively. Although Cnr showed the most differences from WT in mutation-related DEGs , it had the largest number of ripening-related DEGs in common with WT fruit . Cnr also displayed similar functional enrichments to WT among their respective ripening-related DEGs, including photosynthesis-related pathways, carbohydrate, and amino acid metabolism, and plant hormone signal transduction . Compared to Cnr, rin shared a smaller number of ripening-related DEGs and functional enrichments with WT fruit .
The number of ripening-related DEGs shared between nor and WT fruit was negligent, and no functional enrichments were detected in this set of DEGs. Similar to our previous analysis, we mined the ripening related DEGs to determine the patterns of expression of key genes involved in fruit quality traits . We observed that Cnr and WT showed similar gene expression of SlPSY1, SlLCY1, POLYGALACTURONASE 2A , pectate lyase , PECTIN METHYLESTERASE 1 , and ACTINATE HYDRATASE . Fruit from nor and rin did not have similar ripening expression patterns to WT fruit for those genes, except for the SlPG2A and SlPME1 in rin. Altogether, these data indicate that Cnr fruit undergo the most similar ripening progression to WT fruit, while nor and rin fruit have moderate to minimal changes between the MG and RR stages.Alterations in transcriptional and hormone control likely cause the extensive gene expression differences that lead to the pleiotropic ripening defects in the mutants. Our transcriptional data pointed out that both mutation-related and ripening-related DEGs were significantly enriched in functions related to hormone regulation . Thus, we decided to look closer at defects in hormone biosynthesis and signaling in the mutant fruit, with a particular focus on ethylene and ABA as they are known to promote tomato ripening . It has been reported multiple times that ethylene production is negatively affected in the Cnr, nor, and rin mutants . We confirmed that the three ripening mutants do not present the ethylene burst associated with climacteric fruit ripening at any of the stages evaluated, MG, RR, and OR . However, in a oneway ANOVA and Tukey HSD test comparing all genotypes at the MG stage we noted that Cnr fruit produced significantly more ethylene at the MG stage than WT MG fruit and the other mutants at the equivalent stages. The results from each season can be found in Supplementary Table 2. To give a sense if ethylene was inhibited at an early step in biosynthesis in the mutants, we measured the accumulation of the immediate ethylene precursor ACC at the MG and RR stages. ACC accumulates typically at the RR stage in WT fruit, reflecting the increase in ethylene biosynthesis and ethylene production. Surprisingly, ACC concentrations also increased in Cnr and rin fruit during ripening, reaching values similar to WT fruit ; yet the fruit did not produce normal ethylene levels. Moreover, the ACC accumulation in Cnr RR fruit was the highest across all genotypes and ripening stages, significantly more than WT RR fruit. These results suggest that the low levels of ethylenein Cnr and rin RR fruit may be partially explained by inhibition of the final enzymatic step in ethylene biosynthesis. We found ethylene biosynthesis significantly enriched among mutation-related and ripening-related DEGs in several of the mutants . At the MG stage, nor and rin fruit had significantly lower expression of the primary ripening ACC synthases and ACC oxidases . At RR, this pattern was maintained except for SlACO4, which was higher than WT for both mutants. SlACS2 was significantly down-regulated across all mutants and stages compared to WT. We validated the expression patterns of SlACS2 by RT-qPCR experiment using independent samples from WT and the mutant fruit obtained from another field season .