The Rosaceae is an economically important group of cultivated plants, which includes fruit-producing genera such as Malus (apples), Pyrus (pears), Prunus (e.g. peach, plums, apricots), Fragaria (strawberries), and Rubus (raspberry, blackberry, boysenberry), as well as ornamental plants such as Rosa (rose). In these fruits and flowers, colour is a key quality trait and is often caused by anthocyanin. Anthocyanins are water-soluble pigments that belong to the flavonoid family of compounds giving red, blue and purple colours in a range of flowers, fruits, foliage, seeds and roots . Anthocyanins are involved in a wide range of functions, such as the attraction of pollinators, seed dispersal, protection against UV light damage, and pathogen attack [2–5]. Recently, research on anthocyanins has intensified because of their potential benefits to human health, including protection against cancer, inflammation, coronary heart diseases and other age-related diseases [6–11].
In plants, the structural genes of the flavonoid biosynthetic pathway are largely regulated at the level of transcription. In all species studied to date, the regulation of the expression of anthocyanin biosynthetic genes are through a complex of MYB transcription factors (TF), basic helix-loop-helix (bHLH) TFs and WD-repeat proteins (the MYB-bHLH-WD40 "MBW" complex; ). A model has been proposed for the activation of structural pigmentation genes, with regulators interacting with each other to form transcriptional complexes in conjunction with the promoters of structural genes . For example, the R2R3 MYB C1 protein, that regulates the anthocyanin pathway in maize, interacts with a bHLH TF (either of the genes termed B or R) to activate the promoter of dihydroflavonol reductase (DFR). In contrast, the R2R3 MYB P protein, which regulates the phlobaphene pathway in maize, can activate the same promoter without a bHLH TF .
MYB TFs can be classified into three subfamilies based on the number of highly conserved imperfect repeats in the DNA-binding domain including R3 MYB (MYB1R) with one repeat, R2R3 MYB with two repeats, and R1R2R3 MYB (MYB3R) with three repeats [15, 16]. Among these MYB transcription factors, R2R3-MYBs constitute the largest TF gene family in plants, with 126 R2R3 MYB genes identified in Arabidopsis . Those associated with up-regulation of the anthocyanin pathway are R2R3 MYBs. Over-expression of the AtPAP1 gene (AtMYB75, At1 g56650) results in the accumulation of anthocyanins in Arabidopsis . Several repressors of the phenylpropanoid pathway, and perhaps anthocyanins specifically, are also MYB TFs, including an R2R3 MYB repressor from strawberry FaMYB1 , Arabidopsis AtMYB6, 4, and 3 , Antirrhinum AmMYB308 , and a one repeat MYB in Arabidopsis, AtMYBL2 [22, 23]. How the repressor MYBs interact with the MBW transcriptional complex is beginning to be elucidated [22, 23].
Based on the phylogenetic relationship between Arabidopsis R2R3 MYB TFs and anthocyanin-related MYBs of other species, it appears that anthocyanin-regulating R2R3 MYBs fall into one or two clades [17, 24, 25]. Anthocyanin-regulating MYBs have been isolated from many species, including Arabidopsis AtMYB75 or PAP1, AtMYB90 or PAP2, AtMYB113 and AtMYB114 , Solanum lycopersicum ANT1 , Petunia hybrida AN2 , Capsicum annuum A , Vitis vinifera VvMYB1a , Zea mays P , Oryza saliva C1 , Ipomoea batatas IbMYB1 , Anitirrhinum majus ROSEA1, ROSEA2 and VENOSA , Gerbera hybrid GhMYB10 , Picea mariana MBF1 , Garcinia mangostana GmMYB10 , Malus × domestica MdMYB10, MdMYB1/MdMYBA [24, 38, 39], and Gentian GtMYB3 .
For rosaceous species, MYBs that regulate the genes of the anthocyanin pathway have been examined in apple and strawberry. In apple (Malus × domestica) MYB10 was isolated from red-fleshed apple 'Red Field' , and showed a strong correlation between the expression of MYB10 and apple anthocyanin levels during fruit development. Transgenic apple lines constitutively expressing MYB10 produced highly pigmented shoots. Two more apple TFs, MYB1 and MYBA, were also reported to regulate genes in the anthocyanin pathway in red-skinned fruit [38, 39]. Both MYB1 and MYBA share identical sequences , while MYB10 and MYB1 genes are located at very similar positions on linkage group 9 of the apple genetic map . In strawberry (Fragaria × ananassa), the R2R3 MYB TF FaMYB1 plays a key role in down-regulating the biosynthesis of anthocyanins and flavonols .
In this current study, we used an allele-specific PCR primer approach to show that MdMYB1/MdMYBA/MdMYB10 are highly likely to be allelic in the apple genome. We then isolated genes with high sequence similarity to MYB10 from 20 species within the Rosaceae. Sequence and functional characterization of these genes provides insight into the evolution of this TF, within a plant family where higher levels of pigmentation has been selected for during the process of domestication. Expression analysis during the fruit development, and functional testing using transient assays and transgenic plants suggest that these R2R3 MYBs are responsible for controlling anthocyanin biosynthesis in these crops.