Plant polyphenolics exhibit a broad spectrum of health-promoting effects when consumed as part of the diet, and there is considerable interest in enhancing the levels of these bioactive molecules in plants used as foods. AtMYB12 was originally identified as a flavonol-specific transcriptional activator in Arabidopsis thaliana, and this has been confirmed by ectopic expression in tobacco. AtMYB12 is able to induce the expression of additional target genes in tobacco, leading to the accumulation of very high levels of flavonols. When expressed in a tissue-specific manner in tomato, AtMYB12 activates the caffeoyl quinic acid biosynthetic pathway, in addition to the flavonol biosynthetic pathway, an activity which probably mirrors that of the orthologous MYB12-like protein in tomato. As a result of its broad specificity for transcriptional activation in tomato, AtMYB12 can be used to produce fruit with extremely high levels of multiple polyphenolic anti-oxidants. Our data indicate that transcription factors may have different specificities for target genes in different plants, which is of significance when designing strategies to improve metabolite accumulation and the anti-oxidant capacity of foods.

The color of tomato fruit is mainly determined by carotenoids and flavonoids. Phenotypic analysis of an introgression line (IL) population derived from a cross between Solanum lycopersicum 'Moneyberg' and the wild species Solanum chmielewskii revealed three ILs with a pink fruit color. These lines had a homozygous S. chmielewskii introgression on the short arm of chromosome 1, consistent with the position of the y (yellow) mutation known to result in colorless epidermis, and hence pink-colored fruit, when combined with a red flesh. Metabolic analysis showed that pink fruit lack the ripening-dependent accumulation of the yellow-colored flavonoid naringenin chalcone in the fruit peel, while carotenoid levels are not affected. The expression of all genes encoding biosynthetic enzymes involved in the production of the flavonol rutin from naringenin chalcone was down-regulated in pink fruit, suggesting that the candidate gene underlying the pink phenotype encodes a regulatory protein such as a transcription factor rather than a biosynthetic enzyme. Of 26 MYB and basic helix-loop-helix transcription factors putatively involved in regulating transcription of genes in the phenylpropanoid and/or flavonoid pathway, only the expression level of the MYB12 gene correlated well with the decrease in the expression of structural flavonoid genes in peel samples of pink- and red-fruited genotypes during ripening. Genetic mapping and segregation analysis showed that MYB12 is located on chromosome 1 and segregates perfectly with the characteristic pink fruit color. Virus-induced gene silencing of SlMYB12 resulted in a decrease in the accumulation of naringenin chalcone, a phenotype consistent with the pink-colored tomato fruit of IL1b. In conclusion, biochemical and molecular data, gene mapping, segregation analysis, and virus-induced gene silencing experiments demonstrate that the MYB12 transcription factor plays an important role in regulating the flavonoid pathway in tomato fruit and suggest strongly that SlMYB12 is a likely candidate for the y mutation.

The cuticle covering plants' aerial surfaces is a unique structure that plays a key role in organ development and protection against diverse stress conditions. A detailed analysis of the tomato colorless-peel y mutant was carried out in the framework of studying the outer surface of reproductive organs. The y mutant peel lacks the yellow flavonoid pigment naringenin chalcone, which has been suggested to influence the characteristics and function of the cuticular layer. Large-scale metabolic and transcript profiling revealed broad effects on both primary and secondary metabolism, related mostly to the biosynthesis of phenylpropanoids, particularly flavonoids. These were not restricted to the fruit or to a specific stage of its development and indicated that the y mutant phenotype is due to a mutation in a regulatory gene. Indeed, expression analyses specified three R2R3-MYB-type transcription factors that were significantly down-regulated in the y mutant fruit peel. One of these, SlMYB12, was mapped to the genomic region on tomato chromosome 1 previously shown to harbor the y mutation. Identification of an additional mutant allele that co-segregates with the colorless-peel trait, specific down-regulation of SlMYB12 and rescue of the y phenotype by overexpression of SlMYB12 on the mutant background, confirmed that a lesion in this regulator underlies the y phenotype. Hence, this work provides novel insight to the study of fleshy fruit cuticular structure and paves the way for the elucidation of the regulatory network that controls flavonoid accumulation in tomato fruit cuticle.

The R2R3MYB proteins comprise one of the largest families of transcription factors and play regulatory roles in developmental processes and defense responses in plants. However, there has been relatively little effort to systematically carry out comprehensive genomic and functional analyses of these genes in tomato (Solanum lycopersicum L.), a reference species for Solanaceae plants, and the model plant for fruit development. In this study, a total of 121 R2R3MYB genes were identified in the tomato genome released recently and further classified into 29 subgroups based on the phylogenetic analysis of the complete protein sequences. Phylogenetic comparison of the members of this superfamily among tomato, Arabidopsis, grape, rice, poplar, soybean, cucumber and apple revealed that the putative functions of some tomato R2R3MYB proteins were clustered into the Arabidopsis functional clades. The chromosome distribution pattern revealed that tomato R2R3MYB genes were enriched on several chromosomes and 52 % of the family members were tandemly duplicated genes. Tissue specificity or different expression levels of SlR2R3MYBs in different tissues suggested differential regulation of tissue development as well as metabolic regulation. The transcript abundance level analysis during abiotic conditions identified a group of R2R3MYB genes that responded to one or more treatments suggesting that the SlR2R3MYBs played major roles in the plant response to abiotic conditions and involved in signal transduction pathways. This study not only provides a solid foundation for further functional dissection of tomato R2R3MYB family genes, but may also be profitable for, in the future, the improvement of tomato stress tolerance and fruit quality.