Fruit development and ripening is regulated by genetic and environmental factors and is of critical importance for seed dispersal, reproduction and fruit quality. Tomato rin mutant fruit have a classic ripening-inhibited phenotype, which is attributed to a genomic DNA deletion resulting in the fusion of two truncated transcription factors, RIN and MC. In wild-type fruit, RIN, a MADS-box transcription factor, is a key regulator of the ripening gene expression network with hundreds of gene targets controlling changes in color, flavor, texture and taste during tomato fruit ripening; MC, on the other hand, has low expression in fruit, and the potential functions of the RIN-MC fusion gene in ripening remain unclear. Here, over-expression of RIN-MC in transgenic wild-type AC tomato fruits impaired several ripening processes, and down-regulating RIN-MC expression in the rin mutant was found to stimulate the normal yellow mutant fruit to produce a weak red color, suggesting a distinct negative role for RIN-MC in tomato fruit ripening. By comparative transcriptome analysis of rin and rin 35S::RIN-MC RNAi fruits, a total of 1168 and 1234 genes were identified as potential targets of RIN-MC activation and inhibition. Further, the RIN-MC fusion gene was shown to be translated into a chimeric transcription factor that was localized to the nucleus and was capable of protein interactions with other MADS-box factors. These results indicated that RIN-MC is functional and controls expression of many ripening genes, thereby contributing to the rin mutant phenotype.

Fruit ripening is a complex developmental process that depends on a coordinated regulation of numerous genes, including ripening-related transcription factors (TFs), fruit-related microRNAs, DNA methylation and chromatin remodeling. It is known that various TFs, such as MADS-domain, MYB, AP2/ERF and SBP/SPL family proteins play key roles in modulating ripening. However, little attention has been given to members of the large NF-Y TF family in this regard, although genes in this family are known to have important functions in regulating plant growth, development, and abiotic or biotic stress responses. In this study, the evolutionary relationship between Arabidopsis thaliana and tomato (Solanum lycopersicum) NF-Y genes was examined to predict similarities in function. Furthermore, through gene expression analysis, 13 tomato NF-Y genes were identified as candidate regulators of fruit ripening. Functional studies involving suppression of NF-Y gene expression using virus induced gene silencing (VIGS) indicated that five NF-Y genes, including two members of the NF-YB subgroup (Solyc06g069310, Solyc07g065500) and three members of the NF-YA subgroup (Solyc01g087240, Solyc08g062210, Solyc11g065700), influence ripening. In addition, subcellular localization analyses using NF-Y proteins fused to a green fluorescent protein (GFP) reporter showed that the three NF-YA proteins accumulated in the nucleus, while the two NF-YB proteins were observed in both the nucleus and cytoplasm. In this study, we identified tomato NF-Y genes by analyzing the tomato genome sequence using bioinformatics approaches, and characterized their chromosomal distribution, gene structures, phylogenetic relationship and expression patterns. We also examined their biological functions in regulating tomato fruit via VIGS and subcellular localization analyses. The results indicated that five NF-Y transcription factors play roles in tomato fruit ripening. This information provides a platform for further investigation of their biological functions.