# Molecular and Hormonal Mechanisms Regulating Fleshy Fruit Ripening
## 6 The Identification and Function of Major Regulator TFs
### Cnr (SPL-CNR) Methylation and Function  
Plants carrying the _Cnr_ mutation appear normal, but the rip fruits have a yellow skin with colourless pericarp and show a loss of cell-to-cell adhesion, which affects the fruit texture. Map-based cloning and sequencing identified CNR as an SBP-box protein (SPL-CNR) [261], a TF with two zinc-finger motifs in the C-terminal SBP-box domain. The _Cnr_ mutation does not affect the _SPL-CNR_ coding sequence itself, but a 286 bp DNA region of the gene promoter, approximately 2.4 kb upstream of the coding sequence, is hypermethylated in the mutant. VIGS of _SPL-CNR_ in WT tomato, using a Potato Virus X-based vector, led to development of non-ripening fruit sectors.  
Further analysis showed that the _Cnr_ epigenome is hypermethylated [197; 262]. These additional methylation marks are not normally found in WT, which might be due to lack of expression of tomato _DEMETER-Like DNA demethylylase 2_ (_DML2_) [198]. [PERSON] et al. [263] used a Cucumber Mosaic Virus-based vector containing double stranded RNA homologous to the _Cnr_ promoter to induce promoter-silencing in WT plants. This caused hypermethylation of the targeted _Cnr_ promoter and partially suppressed ripening. Inhibiting the accumulation of tomato _DML2_ by RNAi also inhibited ripening by causing DNA hypermethylation, which repressed the genes encoding TFs and their downstream gene targets required for ripening [198]. [PERSON] et al. [262] showed, using VIGS, that CHROMOMETHYLASE3 (SICMT3) and other methyltransferases are required for maintenance of the _Cnr_ phenotype.  
These results pointed to the conclusion that SPL-CNR plays an important role in ripening control and that the _Cnr_ mutation affects epigenomic methylation that inhibits normal expression of SPL-CNR. When CRISPR/Cas9 was used to generate a SPL-CNR deletion mutant of tomato, however, surprisingly, the fruit ripened more fully. RNA-seq analysis of the _Cnr_ mutant and _CNR_-deficient fruits showed that the expression of ripening genes such as _ACS2_, _ACO1_, _PSY1_, _PG_ and _EXP_ are partially expressed in the CRISPR/Cas9 lines but repressed in the natural mutants [262]. These experiments raised serious doubts about the importance of _Cnr_ in the control of tomato ripening [14; 16; 264; 265]. The question remains how to reconcile the differences in phenotypes between the analysis of the CRISPR and natural _Cnr_ mutants. It is known that the _Cnr_ mutation is located in a 13 kb DNA region in the tomato genome [239].The mutant phenotype must be due to a change in this 13 kb region, possibly caused by new methylation mark(s), and it is possible, therefore, that DNA hypermethylation in nearby regions might contribute to the _Cnr_ non-ripening phenotype [262].  
It seems that CNR plays a less critical role in ripening than previously thought, and its role needs further investigation. It seems possible that the _Cnr_ mutation is a gain of function caused by some methylation change. If so, understanding the phenotype is best addressed by investigations on the mutant itself, rather than focusing on the WT alone. It is possible that other yet to be discovered structural or epigenetic changes in this 13 kb region may provide an explanation for the discrepancies between the phenotype of the naturally occurring mutant and the CRISPR/Cas9 _Cnr_ mutations.