Regulation of anthocyanin accumulation in apple by the transcription factor MdMYB10

Abstract

Anthocyanin concentration is an important determinant of the colour of many fruits. In apple (Malus x domestica), centuries of breeding have produced numerous varieties in which levels of anthocyanin pigment vary widely and change differently in response to environmental and developmental stimuli. The apple fruit cortex is usually colourless, although germplasm does exist where the cortex is highly pigmented due to the accumulation of anthocyanins, carotenoids or chlorophylls. From studies in a diverse array of plants species, it is apparent that anthocyanin biosynthesis is controlled at the level of transcription. In this thesis the transcript levels of the anthocyanin biosynthetic genes in a red-fleshed apple are compared with a white-fleshed cultivar. A MYB transcription factor (TF), MdMYB10, was isolated and is shown to be similar in sequence to known anthocyanin regulators in other species. Further, this TF is shown to induce anthocyanin accumulation in both heterologous and homologous systems, generating pigmented patches in transient assays in tobacco leaves and highly pigmented apple plants following stable transformation with constitutively expressed MdMYB10. Efficient induction of anthocyanin biosynthesis in transient assays by MdMYB10 was dependent on the co-expression of two bHLH proteins from apple, MdbHLH3 and MdbHLH33. A strong correlation between expression of MdMYB10 and apple anthocyanin levels during fruit development suggests that this TF is responsible for controlling anthocyanin biosynthesis in apple fruit; in the red-fleshed cultivar and in the skin of other varieties, there is an induction of MdMYB10 expression concurrent with colour formation during development. Mutations in the genes encoding for either the biosynthetic or transcriptional regulation of the anthocyanin pathway have been linked to colour phenotypes. Generally this is a loss of function resulting in a reduction or a change in the distribution of anthocyanin. The upstream regulatory region of MdMYB10 was investigated and found to contain a rearrangement. This modification is responsible for increasing the level of anthocyanin throughout the plant to produce a striking phenotype that includes red foliage and red fruit flesh. It consists of a series of multiple repeats, forming a minisatellite-like structure that comprises five direct tandem repeats of a 23 base pair sequence. This MdMYB10 rearrangement is present in all the red foliage apple varieties and species tested, but in none of the white fleshed varieties. Transient assays demonstrated that the 23 bp sequence motif is a target of the MdMYB10 protein itself, and the number of repeat units correlates with an increase in transactivation by MdMYB10 protein. The repeat motif is capable of binding MdMYB10 protein in electrophoretic mobility shift assays. Taken together, these results indicate that a rearrangement in the promoter of MdMYB10 has generated an autoregulatory locus and this autoregulation is sufficient to account for the increase in MdMYB10 transcript levels and subsequent ectopic accumulation of anthocyanins throughout the plant. Characterisation of MdMYB10 and the rearrangement in the promoter region has implications for the development of new varieties through classical breeding or a biotechnological approach. Understanding whether this mutation is simply an allele of other recently published apple MYB TFs, or if this is the only R2R3 MYB TF involved in apple anthocyanin response, is a challenge for future research.