AUTHOR=Lafferty Declan J. , Espley Richard V. , Deng Cecilia H. , Dare Andrew P. , Günther Catrin S. , Jaakola Laura , Karppinen Katja , Boase Murray R. , Wang Lei , Luo Henry , Allan Andrew C. , Albert Nick W. 

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

JOURNAL=Frontiers in Plant Science

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2022.910155

DOI=10.3389/fpls.2022.910155

ISSN=1664-462X

ABSTRACT=<p><italic>Vaccinium</italic> berries are regarded as “superfoods” owing to their high concentrations of anthocyanins, flavonoid metabolites that provide pigmentation and positively affect human health. Anthocyanin localization differs between the fruit of cultivated highbush blueberry (<italic>V. corymbosum</italic>) and wild bilberry (<italic>V. myrtillus</italic>), with the latter having deep red flesh coloration. Analysis of comparative transcriptomics across a developmental series of blueberry and bilberry fruit skin and flesh identified candidate anthocyanin regulators responsible for this distinction. This included multiple activator and repressor transcription factors (TFs) that correlated strongly with anthocyanin production and had minimal expression in blueberry (non-pigmented) flesh. R2R3 MYB TFs appeared key to the presence and absence of anthocyanin-based pigmentation; <italic>MYBA1</italic> and <italic>MYBPA1.1</italic> co-activated the pathway while <italic>MYBC2.1</italic> repressed it. Transient overexpression of <italic>MYBA1</italic> in <italic>Nicotiana benthamiana</italic> strongly induced anthocyanins, but this was substantially reduced when co-infiltrated with <italic>MYBC2.1</italic>. Co-infiltration of <italic>MYBC2.1</italic> with <italic>MYBA1</italic> also reduced activation of <italic>DFR</italic> and <italic>UFGT</italic>, key anthocyanin biosynthesis genes, in promoter activation studies. We demonstrated that these TFs operate within a regulatory hierarchy where <italic>MYBA1</italic> activated the promoters of <italic>MYBC2.1</italic> and <italic>bHLH2.</italic> Stable overexpression of <italic>VcMYBA1</italic> in blueberry elevated anthocyanin content in transgenic plants, indicating that <italic>MYBA1</italic> is sufficient to upregulate the TF module and activate the pathway. Our findings identify TF activators and repressors that are hierarchically regulated by SG6 <italic>MYBA1,</italic> and fine-tune anthocyanin production in <italic>Vaccinium.</italic> The lack of this TF module in blueberry flesh results in an absence of anthocyanins.</p>