Hierarchical regulation of MYBPA1 by anthocyanin- and proanthocyanidin-related MYB proteins is conserved in Vaccinium species

Abstract

Members of the Vaccinium genus bear fruits rich in anthocyanins, a class of red-purple flavonoid pigments that provide human health benefits, although the localization and concentrations of anthocyanins differ between species: blueberry (V. corymbosum) has white flesh, while bilberry (V. myrtillus) has red flesh. Comparative transcriptomics between blueberry and bilberry revealed that MYBPA1.1 and MYBA1 strongly correlated with the presence of anthocyanins, but were absent or weakly expressed in blueberry flesh. MYBPA1.1 had a biphasic expression profile, correlating with both proanthocyanidin biosynthesis early during fruit development and anthocyanin biosynthesis during berry ripening. MYBPA1.1 was unable to induce anthocyanin or proanthocyanidin accumulation in Nicotiana benthamiana, but activated promoters of flavonoid biosynthesis genes. The MYBPA1.1 promoter is directly activated by MYBA1 and MYBPA2 proteins, which regulate anthocyanins and proanthocyanidins, respectively. Our findings suggest that the lack of VcMYBA1 expression in blueberry flesh results in an absence of VcMYBPA1.1 expression, which are both required for anthocyanin regulation. In contrast, VmMYBA1 is well expressed in bilberry flesh, up-regulating VmMYBPA1.1, allowing coordinated regulation of flavonoid biosynthesis genes and anthocyanin accumulation. The hierarchal model described here for Vaccinium may also occur in a wider group of plants as a means to co-regulate different branches of the flavonoid pathway.

The Coordinated Action of MYB Activators and Repressors Controls Proanthocyanidin and Anthocyanin Biosynthesis in Vaccinium

“…Northern Highbush blueberry (V. corymbosum) 'Nui' fruit and wild bilberry fruit were obtained as described previously, representing a developmental series from stage 4 to 8 (Günther et al, 2020;Lafferty et al, 2022). Additionally, stage 1-8 whole berry samples from "Nui" blueberry were sampled for qRT-PCR (Lafferty et al, 2022).…”

“…Blueberries (Vaccinium spp., mainly Vaccinium corymbosum and Vaccinium virgatum) are one of the most well-known and commercially cultivated Vaccinium species, while there are numerous species such as bilberry (Vaccinium myrtillus), which are harvested from the wild. The anthocyanin localization in the fruit of these species differs, with blueberries having a pale, colorless flesh while bilberries have a red, anthocyanin-rich flesh (Lafferty et al, 2022).…”

“…Hierarchy between activator TFs also occurs, with activator MYBs upregulating bHLH genes in Arabidopsis, kiwifruit, clover, Antirrhinum, tobacco and petunia (Baudry et al, 2006;Xu et al, 2013;Albert et al, 2014Albert, 2015;Montefiori et al, 2015;Liu et al, 2021). Recently, the hierarchical regulation of blueberry MYBPA1.1 by both SG5 and SG6 MYB activators was reported (Lafferty et al, 2022). This was the first report of a SG6 MYB regulating MYBPA1-type MYBs, while their regulation by SG5 MYBs had been seen in apple, grape and poplar (Terrier et al, 2009;James et al, 2017;Wang et al, 2018).…”

“…The MYB transcription factor (TF) determines the specificity of the complex for the different biosynthetic gene promoters. Subgroup (SG) 5 MYBs typically regulate PA biosynthesis, and have been characterized in a number of fruiting species, including blueberry, bilberry, grape and strawberry (Terrier et al, 2009;Schaart et al, 2013;Karppinen et al, 2021;Lafferty et al, 2022). These MYBs drive MBW activation of the PA specific ANR and LAR genes.…”

“…These are key regulators for controlling anthocyanin production in fruit (Albert and Allan, 2021), including blueberry and bilberry (Plunkett et al, 2018;Karppinen et al, 2021). An additional class of MYBs, MYBPA1-type, are dual regulators of PA and anthocyanin biosynthesis in fruit and are required for full activation of both pathways (Karppinen et al, 2021;Lafferty et al, 2022). These activate genes common to PA and anthocyanin biosynthesis, but are not sufficient to activate these pathways, and require the SG5 and SG6 MYBs to activate the additional pathway-specific genes for metabolite production.…”