Identification of key amino acids for the evolution of promoter target specificity of anthocyanin and proanthocyanidin regulating MYB factors

Abstract

A complex of R2R3-MYB and bHLH transcription factors, stabilized by WD40 repeat proteins, regulates gene transcription for plant cell pigmentation and epidermal cell morphology. It is the MYB component of this complex which specifies promoter target activation. The Arabidopsis MYB TT2 regulates proanthocyanidin (PA) biosynthesis by activating the expression of ANR (anthocyanidin reductase), the gene product of which catalyzes the first committed step of this pathway. Conversely the closely related MYB PAP4 (AtMYB114) regulates the anthocyanin pathway and specifically activates UFGT (UDP-glucose:flavonoid-3-O-glucosyltransferase), encoding the first enzyme of the anthocyanin pathway. Both at the level of structural and regulatory genes, evolution of PA biosynthesis proceeded anthocyanin biosynthesis and we have identified key residues in these MYB transcription factors for the evolution of target promoter specificity. Using chimeric and point mutated variants of TT2 and PAP4 we found that exchange of a single amino acid, Gly/Arg(39) in the R2 domain combined with an exchange of a four amino acid motif in the R3 domain, could swap the pathway selection of TT2 and PAP4, thereby converting in planta specificity of the PA towards the anthocyanin pathway and vice versa. The general importance of these amino acids for target specificity was also shown for the grapevine transcription factors VvMYBPA2 and VvMYBA2 which regulate PAs and anthocyanins, respectively. These results provide an insight into the evolution of the different flavonoid regulators from a common ancestral gene.

The Transcriptional Repressor MYB2 Regulates Both Spatial and Temporal Patterns of Proanthocyandin and Anthocyanin Pigmentation inMedicago truncatula

“…changes are found between these two classes as have been reported in comparisons between anthocyanin and PA activators (Supplemental Figure 5; Heppel et al, 2013).…”

“…In addition to the spatial and temporal regulation of transcription, as shown with seed coat-specific expression of TT2 (Gonzalez et al, 2009), key amino acids in MYB transcription factors for target promoter specificity were found between TT2-type and PAP4-type MYB proteins (Heppel et al, 2013). The switching mechanism of a dimerization domain in a bHLH factor to permit distinct configurations of a regulatory complex for binding on different promoters was also reported (Kong et al, 2012).…”

The MYB182 Protein Down-Regulates Proanthocyanidin and Anthocyanin Biosynthesis in Poplar by Repressing Both Structural and Regulatory Flavonoid Genes

“…1A). At positions 90 to 93 of the C-terminal end of the R3 repeat, a short stretch of amino acid residues was observed by Heppel et al (2013) to be different in the PA MYBs (Asp-Asn-Glu-Ile) from the anthocyanin MYBs (Ala-Asn-Asp-Val). In MYB182, this region has residues characteristic of both the PA and anthocyanin regulators (Asp-Asn-Glu-Val), which could reflect MYB182's capacity to repress both pathways.…”

“…An effect of MYB182 on PA, anthocyanin, and core flavonoid genes is consistent with several features of the MYB182 protein sequence, which contains amino acid motifs shared by other flavonoid regulators. A recent study by Heppel et al (2013) identified two regions of conserved amino acid residues in the R2 and R3 domains of activator MYB transcription factors and proposed that these residues are important for the specific regulation of the PA and anthocyanin pathway. The MYB182 sequence contains a Gly at position 49 in the R2 domain, a conserved feature in all PA regulators and in the other R2R3-MYB repressors (Fig.…”

The Phenylpropanoid Pathway Is Controlled at Different Branches by a Set of R2R3-MYB C2 Repressors in Grapevine

“…C2 repressors showed differences in one of two elements that, in MYB activators, mediate the specificity for either the anthocyanin or PA pathway. This conserved element, named A2 box (Stracke et al, 2001) or element 3 (Heppel et al, 2013), possesses the signature ANDV (or SNDV in fewer cases) within anthocyanin regulators belonging to more than 15 different plant species (Lin-Wang et al, 2010). Instead, if the sequence for this element is exchanged to DNEI, the specificity is redirected to the regulation of PA synthesis (Heppel et al, 2013).…”