MYBA From Blueberry (Vaccinium Section Cyanococcus) Is a Subgroup 6 Type R2R3MYB Transcription Factor That Activates Anthocyanin Production

Abstract

The <i>Vaccinium</i> genus in the family <i>Ericaceae</i> comprises many species, including the fruit-bearing blueberry, bilberry, cranberry, huckleberry, and lingonberry. Commercially, the most important are the blueberries (<i>Vaccinium</i> section <i>Cyanococcus</i>), such as <i>Vaccinium corymbosum</i> (northern highbush blueberry), <i>Vaccinium virgatum</i> (rabbiteye blueberry), and <i>Vaccinium angustifolium</i> (lowbush blueberry). The rising popularity of blueberries can partly be attributed to their "superfood" status, with an increasing body of evidence around human health benefits resulting from the fruit metabolites, particularly products of the phenylpropanoid pathway such as anthocyanins. Activation of anthocyanin production by R2R3-MYB transcription factors (TFs) has been characterized in many species, but despite recent studies on blueberry, cranberry, and bilberry, no MYB anthocyanin regulators have been reported for <i>Vaccinium</i>. Indeed, there has been conjecture that at least in bilberry, MYB TFs divergent to the usual type are involved. We report identification of <i>MYBA</i> from blueberry, and show through sequence analysis and functional studies that it is homologous to known anthocyanin-promoting R2R3-MYBs of subgroup 6 of the MYB superfamily. In transient assays, MYBA complemented an anthocyanin MYB mutant of <i>Antirrhinum majus</i> and, together with a heterologous bHLH anthocyanin regulator, activated anthocyanin production in <i>Nicotiana benthamiana</i>. Furthermore anthocyanin accumulation and anthocyanin structural gene expression (assayed by qPCR and RNA-seq analyses) correlated with <i>MYBA</i> expression, and <i>MYBA</i> was able to transactivate the <i>DFR</i> promoter from blueberry and other species. The RNA-seq data also revealed a range of other candidate genes involved in the regulation of anthocyanin production in blueberry fruit. The identification of <i>MYBA</i> will help to resolve the regulatory mechanism for anthocyanin pigmentation in the <i>Vaccinium</i> genus. The sequence information should also prove useful in developing tools for the accelerated breeding of new <i>Vaccinium</i> cultivars.