Characterization and analysis of anthocyanin-related genes in wild-type blueberry and the pink-fruited mutant cultivar 'Pink Lemonade': New insights into anthocyanin biosynthesis

Authors: DIE, JOSE - University Of Cordoba; Jones, Richard; Ogden, Elizabeth; Ehlenfeldt, Mark; Rowland, Lisa

Submitted to: Agronomy
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/27/2020
Publication Date: 9/1/2020
Publication URL: https://handle.nal.usda.gov/10113/7116014
Citation: Die, J.V., Jones, R.W., Ogden, E.L., Ehlenfeldt, M.K., Rowland, L.J. 2020. Characterization and analysis of anthocyanin-related genes in wild-type blueberry and the pink-fruited mutant cultivar 'Pink Lemonade': New insights into anthocyanin biosynthesis. Agronomy. https://doi.org/10.3390/agronomy10091296.
DOI: https://doi.org/10.3390/agronomy10091296

Interpretive Summary: Anthocyanins are plant pigments with important benefits to human health. They are highly abundant in some fruits and vegetables, like blueberries, for example. In blueberry, anthocyanins are responsible for the blue color of the fruit. Many breeding programs are interested in improving nutritional quality and in generating ornamental varieties through manipulation of the plant's own pathway for synthesizing anthocyanins. In this study, we used a mutant ornamental variety of blueberry, ‘Pink Lemonade’, which produces pink fruit, to further our understanding of anthocyanin synthesis in blueberry and identify the affected gene in this variety. We compared expression of eight genes of the anthocyanin pathway in ‘Pink Lemonade’ and wild-type blue-fruited varieties. Significantly lower expression of nearly all the genes of the pathway suggested that a regulator of the pathway was probably affected in 'Pink Lemonade'. Expression of a well known regulator of the pathway (from work done in other plants), MYB1, was then compared and found to be significantly lower in ‘Pink Lemonade’ than in wild-type. In addition, over-expressing Myb1 in 'Pink Lemonade' resulted in enhanced anthocyanin production. By examining the gene sequence, significant differences were also found between the pink-fruited and blue-fruited varieties. Altogether, this work suggests that Myb1 is the affected gene responsible for the pink colored fruit of 'Pink Lemonade'.

Technical Abstract: Blueberries are one of the richest sources of antioxidants, such as anthocyanins, among fruits and vegetables. Anthocyanin mutants, like the pink-fruited cultivar ‘Pink Lemonade’, are valuable resources for investigating anthocyanin biosynthesis in blueberry. In this study, we examined expression of flavonoid pathway genes during fruit development in wild-type, blue-fruited blueberry using quantitative real-time PCR and found expression was correlated with anthocyanin accumulation. Expression was also compared between wild-type and the pink-fruited ‘Pink Lemonade’. Significantly lower expression in ‘Pink Lemonade’ than in wild-type of nearly all the structural genes examined suggested that a transcriptional regulator of the pathway was affected. Hence, we compared expression of three known regulatory genes and found the gene encoding the transcription factor MYB1 was expressed at a significantly lower level in ‘Pink Lemonade’ than in wild-type. To validate the capacity of this MYB1 to regulate transcription of anthocyanin genes in blueberry, a transient expression assay was conducted. Results indicated MYB1 over-expression enhanced anthocyanin production. Comparative sequence analysis between wild-type and mutant MYB1 variants found differences in highly conserved features suggesting a mechanistic explanation for the mutant phenotype. Collectively, results presented here contribute to a better understanding of mechanisms regulating anthocyanin biosynthesis in Vaccinium.