Abstract
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.
Highlights
Fleshy fruit gain most of their quality traits, such as color, texture, flavor, and nutritional value, as a result of physiological and biochemical changes associated with ripening
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
The ACC accumulation in Cnr red ripe (RR) fruit was the highest across all genotypes and ripening stages, significantly more than wild type (WT) RR fruit. These results suggest that the low levels of ethylene in Cnr and rin RR fruit may be partially explained by inhibition of the final enzymatic step in ethylene biosynthesis
Summary
Fleshy fruit gain most of their quality traits, such as color, texture, flavor, and nutritional value, as a result of physiological and biochemical changes associated with ripening. Much of this research has been done in the model for fleshy fruit ripening, tomato (Solanum lycopersicum), and has utilized the spontaneous single ripening mutants Cnr (Colorless non-ripening), nor (nonripening), and rin (ripening inhibitor) (Robinson and Tomes, 1968; Tigchelaar et al, 1973; Thompson et al, 1999; Giovannoni et al, 2004; Manning et al, 2006) Each of these mutations produces pleiotropic defects to ripening and occur in or near genes encoding the transcription factors (TFs) SPL-CNR, NACNOR, and MADS-RIN, belonging to the SQUAMOSA promoter binding protein-like (SPL), NAM, ATAF1/2, CUC2 (NAC) and, MCM1, AG, DEF, SRF (MADS) TF families, respectively. These TFs were regarded as master regulators of ripening; given the new information about the nature of the mutations in Cnr, nor, and rin, it is less clear the precise roles the TFs are playing in ripening (Giovannoni et al, 2017; Wang et al, 2020a)
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