We included fruit at the T and OR stages in the validation experiment to capture the gene expression dynamics across fruit ripening and senescence. In Cnr MG fruit, SlACS4 was significantly lower expressed than WT, like the other mutants, but SlACS2 showed no significant difference. Interestingly, Cnr MG fruit had higher gene expression of four ACC oxidases than WT MG fruit, including SlACO3, which is involved in System 1 of ethylene biosynthesis. The increased ACC oxidase expression in Cnr MG fruit could explain the high ethylene levels detected in these fruit . At RR, four ACO genes had significantly higher expression than WT, except for SlACO1 that showed no significant differences in RT-qPCR relative expression shown in Supplementary Table 6. Ethylene signaling and response genes, including ETHYLENE INSENSITIVE 3 and EIN3-BINDING F-BOX homologs, were generally higher expressed in Cnr than WT at MG and RR stages. Nor and rin displayed the opposite trend, with generally lower expression than WT at both stages in these genes . These patterns were also reflected in significant enrichments of ethylene signaling and response genes at the RR stage . Interestingly, ethylene receptor encoding genes were lower expressed across all genotypes and stages compared to WT. In contrast, ethylene response TFs were generally higher expressed in all genotypes at the RR stage. A decrease of ABA during ripening was found in WT, consistent with previous reports .

This pattern was also present in Cnr fruit. However, in nor fruit, ABA remained at the same level across both stages, large pots plastic and rin showed a significant increase at the RR stage. ABA biosynthesis was significantly enriched among mutation-related DEGs in nor and rin RR fruit, consistent with the high ABA levels observed . We looked at specific ABA biosynthesis genes enriched in nor and rin that were also down-regulated in WT at the RR stage and found SlNCED1, encoding the 9-cis-epoxycarotenoid dioxygenase that catalyzes the rate-limiting step in ABA biosynthesis . We validated the expression of SlNCED1 with independent samples and additional stages using RT-qPCR . We also confirmed the expression of an upstream biosynthesis gene, SlZEP, encoding a zeaxanthin epoxidase, which was also significantly up-regulated in nor at the RR stage and in rin at the T stage . While Cnr accumulated ABA, signaling and response genes were altered in at MG and RR fruit, including higher expression in ABRE-binding protein /ABRE binding factors at both stages compared to WT. Nor and rin showed alterations in signaling and response at the RR stage, such as lower expression of receptor protein genes and higher expression of the PP2C phosphatase . We observed changes in biosynthesis and signaling of other plant hormones implicated in fruit development, such as auxins, cytokinins, jasmonic acid, and brassinosteroids . Cnr MG fruit had alterations in all hormone pathways examined, further supporting the differences present in Cnr phenotype before ripening begins. At the RR stage, all mutants presented multiple defects in hormone metabolism compared to WT. Ripening-related DEGs with hormone functions displayed a similar expression pattern in WT and Cnr fruit, whereas nor and rin displayed low numbers of ripening-related DEGs from these categories.

Another way in which the mutations in the CNR, RIN, and NOR may affect gene expression of ripening processes is through direct or indirect interactions with each other. We performed RT-qPCR on fruit from the MG, T, RR, and OR stages in each genotype for each of the genes encoding the ripening TFs . In WT, each TF follows a ripening pattern, peaking in expression at the T stage. Mutations in any of the three TFs led to a decrease or delay in the expression of the other TFs compared to WT. For example, RIN expression does not begin to show an increase until the OR stage for nor and Cnr. A similar pattern was exhibited in CNR expression for nor and rin and NOR expression in rin and Cnr. The Cnr fruit displayed the most dramatic decreases in expression across the TFs, while the nor fruit showed the most delays.The changes in gene expression of CNR, NOR, and RIN in the ripening mutants indicate that the genes are interconnected during fruit development. In addition, Cnr consistently showed earlier defects in fruit traits, gene expression, and hormone pathways. To characterize the combined genetic effects of the mutations on tomato fruit, we generated homozygous double mutants through reciprocal crosses of the single mutants. We then phenotyped the double mutants for fruit traits and ethylene production . Because the reciprocal crosses produced fruit indistinguishable from each other, we report them as only one double mutant . Fruit of nor/rin double mutants were almost indistinguishable from both nor and rin fruit in appearance and external color. Fruit resulting from any cross with Cnr as a parent presented similar visual characteristics . Wealso performed a PCA of the color measurements to compare the double mutants to their parental lines at the RR stage and confirmed this observation . Based on these observations and our earlier phenotypic and transcriptional data, we confirmed that the Cnr mutation affects early fruit development. In contrast, the nor and rin mutations act during fruit ripening. If defects in Cnr occur earlier in fruit development than those caused by nor or rin, we expected the Cnr/rin and Cnr/nor double mutants to behave similarly to Cnr and display similar phenotypes . Cnr/rin fruit were significantly less firm than either parent at the MG stage but performed most similarly to Cnr at the RR stage. Cnr/nor fruit was not distinguishable from either parent in firmness at MG but was firmer than Cnr RR fruit. Interestingly, Cnr/nor fruit exhibited high ethylene production at the MG stage like the Cnr fruit.

At the RR stage, Cnr/nor showed a less pronounced decrease in ethylene production, resulting in higher hormone levels than either parent. Although some phenotypic differences were detected, we verified that Cnr/rin and Cnr/nor resembled the Cnr parent for most of the fruit traits measured. If nor and rin act synergistically during ripening, the rin/nor double mutants would have a more extreme phenotype than either on their own. At the MG stage, rin/nor fruit firmness was statistically similar to rin but became an intermediate phenotype at the RR stage. For ethylene, rin/nor fruit produced less than either parent at both stages, although not significant, suggesting a combined effect of both mutations.The strong effect of Cnr in the double mutant phenotypes led us to investigate if gene expression in the fruit was altered in a similar way. We selected the Cnr/nor double mutant to perform an RNAseq experiment of fruit at MG and RR stages and assessed the overall transcriptional changes resulting from the two mutations combined. We conducted a PCA of total mapped reads for MG and RR fruit of Cnr/nor and the single mutant parents . In this analysis, Cnr/nor expression appeared more similar to Cnr than nor in PC1 , plant plastic pot but PC2 accounted for differences between Cnr and Cnr/nor. We analyzed mutation-related DEGs in the Cnr/nor fruit by comparing the gene expression patterns of the double mutant against WT at both MG and RR stages. We then determine which of these mutation-related DEGs were also differentially expressed between the single mutant parents and WT . Similar to Cnr, Cnr/nor fruit started with a high number of mutation-related DEGs at the MG stage, showing defects in development before the initiation of ripening. However, Cnr/nor MG and RR fruit showed more mutation related DEGs than either nor or Cnr fruit, including 634 unique DEGs at the MG stage and 948 at the RR stage. These data indicate that the Cnr/nor fruit present additional defects than Cnr fruit prior to ripening.The spontaneous ripening mutants, Cnr, nor, and rin, are essential genetic tools to untangle the complexity of climacteric fruit ripening and to breed for extended shelf-life or field harvest traits in tomato . However, thorough phenotyping of the fruit traits affected by these mutants using plants grown under field conditions has been neglected. Here, we produced an extensive quantitative study of fruit quality in the tomato ripening mutants and corroborated it across multiple field seasons. We were able to carefully describe physiological and molecular differences between the mutants by sampling large numbers of fruit and surveying distinct stages through ripening in ways not feasible with greenhouse experiments.We determined that some ripening events in the mutants nor and rin were not completely blocked but severely delayed. By examining the OR stage, we found that the mutation in nor may strongly affect firmness and taste while pigment accumulation was only delayed and slightly perturbed . These phenotypes were supported by higher expression of carotenoid biosynthesis genes in nor RR than WT and an increase in SlPSY1 between the MG and RR stages . The accumulation of pigments in nor fruit, particularly at late stages in development, has gone unnoticed in previous studies, but it partially resembles the CRISPR-NOR mutants . In contrast, rin fruit showed strong inhibition of pigment accumulation but less dramatic alterations to fruit taste-related traits, only delaying the accumulation of sugars and decrease in acidity . The lack of upregulation of SlPSY1 in rin appears to contribute to the color defects, consistent with evidence that RIN directly regulates this gene . Both nor and rin exhibited severe delays or inhibition of ripening-related gene expression changes. While highly similar to WT at the MG stage, nor and rin fruit showed large deviations from WT at the RR stage. In fact, the gene expression profiles of nor and rin RR fruit remained similar to those from WT MG fruit. The physiological data generated in this study show nor and rin mutations have different impacts on fruit quality traits. Soluble solids and acid accumulation are negatively impacted in both mutants, but more dramatically in nor fruit. In addition, previous reports have demonstrated a similar pattern among volatile profiles of the mutants at the red ripe stage, with rin again showing more similarity to WT in flavor related traits . This suggests rin fruit are less likely to hinder flavor profiles than nor fruit when breeding for fresh-market hybrid varieties with extended shelf-life. Although nor showed lower quality flavor attributes, its coloration at overripe stages was most similar to WT compared to rin; and thus, it can be useful in breeding hybrid varieties when coloration is a critical fruit trait, such as in the case of processing tomato varieties. Overall, this knowledge will provide valuable information on these tradeoffs of using either loci for breeding programs. Because the Cnr, nor, and rin mutants never acquire equivalent colorations to WT, their ripening stages have been determined based on the fruit’s age expressed as days after anthesis or days after the breaker stage. Sometimes described as BR + 7 days, the RR stage has been the primary developmental time employed for studying the ripening mutants. As we showed here, the OR stage could provide better comparisons against WT RR fruit for mutants with delayed ripening phenotypes. We demonstrated that in the nor fruit, the RIN and CNR genes only begin to increase in expression in a way comparable to WT at the OR stage . This observation corresponds to over a 10-day delay for some of the ripening processes to begin. The delayed ripening events observed in the OR fruit have not been described before in the spontaneous nor mutant.Although the Cnr mutant has been assumed to have normal fruit development before ripening , there have been indications that the Cnr mutant displays defects that are not ripening-specific, such as earlier chlorophyll degradation and altered expression of CWDE . We showed that the Cnr mutation causes substantial defects in fruit prior to ripening as seen through statistically significant deviations in fruit size, color, firmness, and TA, ethylene production, and gene expression at the MG stage . Therefore we propose Cnr may be more accurately described as a developmental mutant and not exclusively a ripening mutant. Further complementing these results, the Cnr fruit displayed large transcriptional deviations from WT that can be traced back as far as 7 dpa . These early development defects are likely a result of reduced CNR expression in the mutant, which is typically expressed in locular tissue before fruit maturity .