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, plastic nursery plant pot while nor and rin fruit have moderate to minimal changes between the MG and RR stages.
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 reportthem 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 . We also 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 anddisplay 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 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, seedling starter pot 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 normutant.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 . Our analysis of ripening-related gene expression in Cnr showed striking similarities to WT in the number and functions of genes changing between stages. Moreover, 69.5% of ripening-related DEGs in Cnr were shared with WT . These results further support the hypothesis that Cnr is not exclusively a ripening mutant. Instead, Cnr fruit undergoes gene expression changes consistent with WT “ripening.” However, the ripening related changes in gene expression that occur in Cnr are not enough to compensate for the large defects accumulated in the fruit during growth and maturation. In a recent report, a knockout mutation to the gene body of CNR yielded little visible effects on fruit development and ripening , which suggests that the Cnr mutant phenotype may result from more than just a reduced expression of the CNR gene as previously reported . It has also been demonstrated that Cnr fruit have genome-wide methylation changes that inhibit ripening-related gene expression . The developmental defects observed in Cnr are likely caused by these methylation changes, directly or indirectly caused by the Cnr mutation . Thus, to better understand the Cnr mutation, more physiological data at earlier stages of development needs to be analyzed and complemented with more in-depth functional analysis of gene expression alterations at the corresponding stages. In addition, further molecular and genetic studies need to be performed and compared against complete CNR knockout mutants.Previous reports have shown ethylene levels to be very low or even undetectable in the ripening mutants . Our data support that the mutants never produce a burst in ethylene production, even at the OR stage where more ripening phenotypes are observed . The orange-red pigmentation in nor OR fruit and the similarities of OR fruit in texture and taste-related attributes to WT RR fruit occur independently of an ethylene burst. These observations evidence that other regulatory mechanisms exist to initiate ripening events outside of ethylene . Unlike previous reports, our data consistently showed that Cnr presented increased ethylene levels at the MG stage compared to WT . Interestingly, Cnr fruit produced more of the ethylene precursor ACC than WT at the RR stage. Also, rin made equivalent levels to WT fruit. Ethylene biosynthesis is divided into two programs: System 1 produces basal levels of the hormone during development, and System 2 generates the climacteric rise in ethylene during ripening . Each of these systems is catalyzed by a different set of ethylene bio-synthetic enzymes . It is clear that all mutants show defects to System 2 of ethylene biosynthesis, but they also appear to have alterations specific to System 1. For example, we observed that SlACO3, a System 1-specific ACC oxidase, was higher expressed in Cnr fruit than WT .The role of ABA in climacteric ripening is not as well explored but has been reported to be complementary to ethylene . Previous reports in WT fruit have shown that ABA increases until the breaker stage, just before the ethylene burst . ABA has also been shown to induce ethylene production and linked to the NOR transcription factor . We found that nor and rin fruit did not show decreases in ABA concentration during ripening like WT did . For nor, the constant levels of ABA between MG and RR stages are another example of how fruit ripening events are delayed or inhibited. RIN and ABA have been demonstrated to have an inverse relationship where RIN expression is repressed with the induction of ABA . The significant increase of ABA accumulation in rin during ripening suggests that ABA biosynthesis and metabolism are misregulated in this mutant. rin fruit appear to present a delayed peak in ABA levels compared to WT fruit. More developmental stages, genetic manipulations, and exogenous hormone treatments are needed to investigate further the trends of ABA accumulation seen in the ripening mutants.The interactions between the CNR, NOR, and RIN in ripening have been debated in the literature . The TF RIN directly interacts with NOR and CNR, binding to their respective promoters, and therefore has been proposed to be the most upstream TF among the three regulators . Here we provided evidence that the three TFs display at least indirect effects on each other. We have argued that the Cnr mutant shows a wide breadth of defects across fruit development before ripening begins, and thus, we propose the Cnr mutation is acting before NOR or RIN. This further supports the hypothesis made in Wang et al. that Cnr acts epistatically to nor and rin .