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

Abstract

Vaccinium 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 (V. corymbosum) and wild bilberry (V. myrtillus), 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; MYBA1 and MYBPA1.1 co-activated the pathway while MYBC2.1 repressed it. Transient overexpression of MYBA1 in Nicotiana benthamiana strongly induced anthocyanins, but this was substantially reduced when co-infiltrated with MYBC2.1. Co-infiltration of MYBC2.1 with MYBA1 also reduced activation of DFR and UFGT, key anthocyanin biosynthesis genes, in promoter activation studies. We demonstrated that these TFs operate within a regulatory hierarchy where MYBA1 activated the promoters of MYBC2.1 and bHLH2. Stable overexpression of VcMYBA1 in blueberry elevated anthocyanin content in transgenic plants, indicating that MYBA1 is sufficient to upregulate the TF module and activate the pathway. Our findings identify TF activators and repressors that are hierarchically regulated by SG6 MYBA1, and fine-tune anthocyanin production in Vaccinium. The lack of this TF module in blueberry flesh results in an absence of anthocyanins.

Two genes, ANS and UFGT2, from Vaccinium spp. are key steps for modulating anthocyanin production

“…In our assay, co-infiltration with low amounts of VcMYBA1 was required to initiate anthocyanin production. VcMYBA1 has previously been found to control the activation of anthocyanin structural genes, such as DFR , ANS and UFGT ( Plunkett et al., 2018 ; Lafferty et al., 2022a ).…”

“…The ANS phylogeny consisted of 51 sequences from 41 species. LDOX sequences from grape, tobacco, apple, kiwifruit and blueberry were also included in this analysis because of their high similarity in sequence and function of LDOX and ANS (Jun et al, 2018;Lafferty et al, 2022a). There were 62 sequences from 52 species included in the UFGT phylogenetic tree.…”

Identification of R2R3-MYB family in blueberry and its potential involvement of anthocyanin biosynthesis in fruits

“…More importantly, VcMYBPA1.1 gene expression profiles show peaks during early and late stages of development, when proanthocyanidins (PA) biosynthesis occurs and anthocyanins accumulate, respectively [ 42 ]. Furthermore, VcMYBA1 and VcMYBPA1 positively co-regulate the accumulations of PAs and anthocyanins by activating VcbHLH2 and VcMYBC2.1 [ 43 , 47 , 54 ]. The protein sequences of VcMYBA1 and VcMYBPA1 were highly homology to VcMYB437 and VcMYB336, respectively, in this study (Fig.…”

“…Moreover, VcMYBA and VcMYB1 have been proved as important roles correlated with the expression of anthocyanin accumulation related structural gene, meanwhile they are able to transactivate the DFR promoter in blueberry fruit [ 1 , 14 ]. Differently, MYBC2.1 and MYBR3.1, activated by MYBPA1, are demonstrated as two distinct MYB repressors of both PA and anthocyanin biosynthesis [ 43 ]. Nonetheless, in order to understand how the R2R3-MYB regulators are involved in anthocyanin biosynthesis, a comprehensive functional characterization is needed.…”

The miR156/SPL12 module orchestrates fruit colour change through directly regulating ethylene production pathway in blueberry

“…The colour change accompanied by anthocyanin accumulation is a critical process for the quality formation of blueberry fruit, making it an ideal material for studying fruit colouration (Zifkin et al ., 2012 ). While several transcriptional regulators and phytohormones have been documented to be implicated in regulating anthocyanin biosynthesis in blueberry fruit, knowledge regarding the regulatory network underlying the entire process of colour change is still limited (Farneti et al ., 2022 ; Jaakola et al ., 2010 ; Karppinen et al ., 2021 ; Lafferty et al ., 2022a , b ; Wang et al ., 2022b ; Xie et al ., 2022 ; Zifkin et al ., 2012 ). In a previous study, we demonstrated that VcMIR156a can influence anthocyanin and chlorophyll accumulation through its direct target VcSPL12 in tomato and Arabidopsis (Li et al ., 2020b ).…”