Why Is Pink Lemonade Pink? New Insights into Anthocyanin Biosynthesis in Blueberry

Anthocyanins are plant pigments with important benefits to human health. They are produced through the flavonoid biosynthetic pathway, and are highly abundant in some fruits and vegetables, blueberries for example. Many breeding programs are interested in improving nutritional quality and in generating ornamental varieties through manipulation of this pathway. In this study, we used a mutant ornamental cultivar of blueberry, ‘Pink Lemonade’, which produces pink fruit, to further our understanding of anthocyanin biosynthesis in blueberry and identify the affected gene in this genotype. Through real-time qPCR experiments, we compared expression of eight structural genes of the flavonoid biosynthesis pathway in ‘Pink Lemonade’ and wild-type genotypes. Significantly lower expression of nearly all the genes suggested that a transcriptional regulator of the pathway was affected. Expression of the transcription factor VvMYB1 (MYB1 of Vaccinium virgatum) was then compared and found to be significantly lower in ‘Pink Lemonade’ than in wild-type. In addition, transient expression assays were performed on ripe fruit of ‘Pink Lemonade’ using cDNA constructs of the VvMYB1 gene from wild-type and ‘Pink Lemonade’, as well as cDNA constructs of two wild-type structural genes of the pathway. Only the construct using the wild-type VvMYB1 gene appeared to partially complement the mutation and result in development of purple fruit. Currently, MYB1-cDNA clones have been isolated and sequenced from ‘Pink Lemonade’ and a wild-type genotype. Genomic clones will be isolated as well and used to identify the mutations in the ‘Pink Lemonade’ MYB1 alleles.