The MYB transcription factors play important roles in the regulation of many secondary metabolites at the transcriptional level. biosynthetic reactions, and genes for transcription factors that regulate the expression of these structural genes and the accumulation of flavonoid metabolites. Transcription factors can act as activators or repressors of gene expression, and mediate either increases or decreases in transcription through sequence-specific DNA binding and protein-protein interactions (Broun 2004). MYB proteins have been identified in a large number of eukaryotes, including fungi, plants and vertebrates (Ohi et al. 1994; Martin and Paz-Ares 1997; Slamon et al. 1986), and have roles in a wide array of cellular processes. These include the regulation of secondary metabolism, signal transduction, cell division, and responses to plant diseases and various forms of stress (UV-B light, cold and buy 571170-77-9 drought) (Larkin et al. 1994; Borevitz et al. 2000; Stracke et al. 2001; Vailleau et al. 2002; Cominelli et al. 2005). The R2R3-MYB-related proteins activate the transcription of structural genes that function in different branches of phenylpropanoid metabolism (Martin and Paz-Ares 1997). The promoters of these structural genes each contain potential MYB protein recognition motifs and also bind with the basic helix-loop-helix (bHLH) domain and conserved WD40 repeat proteins (Park et al. 2007a, b; Martin and Paz-Ares 1997; Winkel-Shirley 2001). The classification of MYB transcription factors is based on the strong conservation of imperfect repeats in the MYB DNA-binding domain of the member proteins (Martin and Paz-Ares 1997). In contrast to animals (R1R2R3), the MYB domain transcription factors found in plants are characterized by the R2R3-type MYB domain, comprising a DNA-binding domain and an activation/repression domain. Moreover, the balance between activators and repressors in this transcription factor family in plants may provide extra flexibility in terms of transcriptional control (Jin et al. 2000). There have been 126 R2R3 MYB genes identified in based on available information indicating that these genes are induced by environmental stress (UV-B irradiation) (Kranz et al. 1998). However, they do not have an informative phenotype, because of the structural and functional redundancy among these factors (Bouche and Bouchez 2006). Furthermore, although there have been other studies of anthocyanin accumulation in lettuce, one of the most popular and commercially important vegetables in the world, this process has not been characterized before at the level of transcriptional regulation. The regulation of anthocyanin accumulation in plants is also a key buy 571170-77-9 issue in our understanding of the regulation of leaf color. The identification of the factors that exert this control will provide valuable tools to moderate the extent and distribution of anthocyanin-derived pigmentation in plant tissues. In this study, we overexpressed the gene in lettuce plants (Jinjachuckmyun cultivar) that are highly pigmented with a red color throughout their leaf tissues. Because the loss of these pigments is easily detected, the activity and repression of the MYB-type transcription factors that affect these pathways can be investigated. In the leaves of lettuce, coordinated transcriptional buy 571170-77-9 regulation controls virtually each step of the anthocyanin biosynthetic pathway (Park et al. 2007a). We compared the expression of the major anthocyanin biosynthetic genes and the AtMYB60 gene by RT-PCR. We show that this transcription factor is functionally active in repressing anthocyanin accumulation and can thus generate green lettuce leaves. We also show, by functionally repressing the accumulation of this compound, that plays a significant role in controlling anthocyanin biosynthesis via the LRP1 inhibition of a key target gene encoding dihydroflavonol reductase (DFR). Thus, we report here for the first time that anthocyanin biosynthesis and the transcription of the gene are repressed through the overexpression of R2R3-MYB transcription factors in transgenic lettuce plants. We also discuss the function of transcriptional regulators in the control of the expression of the structural protein that are required for anthocyanin biosynthesis in lettuce. Materials and methods Plant materials and growth condition To investigate the effects of exogenous AtMYB proteins on anthocyanin biosynthesis in lettuce leaves at the molecular level, Jinjachuckmyun plants, supplied by Syngenta (Seoul, Korea), were used. The plants were grown at the National Institute of Agricultural Biotechnology in Suwon, South Korea. Fresh.