# Molecular and Hormonal Mechanisms Regulating Fleshy Fruit Ripening
## 6 The Identification and Function of Major Regulator TFs
### The Rin Mutation (RIN-MC)  
The tomato _rin_ mutation causes a severely inhibited ripening phenotype, involving the loss of the respiratory climacter and the major ripening-associated increase in ethylene synthesis, and a severe reduction in pigment accumulation, flavour production and softening [266]. Hundreds of genes involved in many aspects of ripening-related pathways, such as ethylene synthesis (_ACS2_ and _ACS4_) [129], cell wall modification (_PG_, _TBG4_ and _EXP1_) [129] and volatile production (_LoxC_) [267] are direct RIN targets, and transcriptome, proteome and metabolome analyses confirmed the conclusion that RIN is a global tomato fruit-ripening regulator [268]. Recent research, however, has led to a re-evaluation of this conclusion. The _rin_ mutation was known to involve a deletion and fusion of parts of two adjacent _MADS_ genes (_RIN_ and _MC_) located on tomato chromosome 5. The resulting _RIN-MC_ gene fusion was originally believed to generate a loss of function mutation [266] (Figure 5). However, [PERSON] et al. [18] and [PERSON] et al. [15; 17] showed that there were major discrepancies between the severe ripening-inhibited phenotypes of the _rin_ mutant fruit and the phenotype of RIN-deficient fruit, which were able to undergo partial ripening. The _RIN-MC_ fusion transcript was further shown to accumulate in significant quantities in _rin_ mutant fruit and demonstrated to be translated into an active nuclear-localised TF, which has a strong repressor function [17]. It was concluded that _rin_ was actually a gain of function mutation, cause by part of the _MC_ sequence in the _RIN-MC_ fusion that conferred the ability to repress the expression of some ripening genes. It is this repressor activity that severely inhibits the change in colour, flavour, texture, aroma and ethylene synthesis required for normal ripening to occur (Figure 5). This was confirmed by comparative transcriptome analysis of _rin_ and _rin-355_:_RIN-MC_ RNAi, and AC and AC-355_:_RIN_ RNAi fruits [269]. Greatly reducing _RIN-MC_ transcripts by RNAi, deleting _RIN-MC_ from the _rin_ mutant, or deleting _RINfrom WT plants using CRISPR/Cas9 gene editing generated much less severe ripening phenotypes than _rin_[15, 17, 18]. Another important difference between _rin_ fruit (expressing _RIN-MC_) and RIN-deficient fruit (obtained by CRISPR/Cas9) is that the latter soften extensively. Transcripts of some softening enzyme genes including _XTH5_ and _XTH8_ were expressed at higher levels compared to WT, which might explain why RIN-deficient fruits softened similarly to WT fruits at early ripening stages. At later stages, _MAN1_, _Mside7_, _MAN4a_, _TBG4_, _PG_ and _PME2.1_ transcripts were significantly more abundant than in WT, and the fruit softened even more than WT, which may indicate that, when present, RIN can actually repress their expression, but this needs experimental confirmation [15]. Further experiments with these RIN-deficient fruits, together with other information concerning ripening behaviour discussed in Section 5, indicated that ethylene is necessary and sufficient to initiate tomato fruit ripening and RIN is not required for this. RIN is, however, absolutely required for full progression of ripening [15, 17, 18]. This is perhaps not surprising, given that WT RIN has been shown to have hundreds of gene targets and RIN-deficient fruits have greatly reduced levels of transcripts for some carotenoid  
Figure 5: Re-evaluation of the roles of MADS-RIN and NAC-NOR. The structure of _rin_ and _nor_ mutant genes and their mutant phenotypes and differences compared to the corresponding CRISPR/Cas9 MADS-RIN and NAC-NOR mutants are shown. Redrawn from [PERSON] et al. [269] and [PERSON] et al. [14]. (**a**) The _rin_ mutation is the result of a partial deletion and fusion of 2 adjacent genes, _RIN_ and _MC_. (**b**) Translated amino acids predicted for RIN and RIN-MC proteins. The colour key shows MADS-box, protein binding, I and K-region for protein structure formation, C-terminal for activation.