Characterization of an apple TT2-type R2R3 MYB transcription factor functionally similar to the poplar proanthocyanidin regulator PtMYB134

Abstract

The apple MdMYB9 gene encodes a positive regulator of proanthocyanidin synthesis that activates anthocyanidin reductase promoters from apple and poplar via interaction with basic helix-loop-helix proteins. The regulation of proanthocyanidins (PAs, condensed tannins) is of great importance in food plants due to the many benefits of PAs in the human diet. Two candidate flavonoid MYB regulators, MdMYB9 and MdMYB11, were cloned from apple (Malus × domestica) based on their similarity to known MYB PA regulators. Transcript accumulation of both MdMYB9 and MdMYB11 was induced by high light and wounding, similar to the poplar (Populus spp) PA regulator PtMYB134. In transient activation assays with various basic helix-loop-helix (bHLH) co-regulators, MdMYB9 activated apple and poplar anthocyanidin reductase (ANR) promoters, while MdMYB11 showed no activity. Potential transcription factor binding elements were found within several ANR promoters, and the importance of the bHLH binding site (E-box) on ANR promoter activation was demonstrated via mutational analysis. The ability of MdMYB9 and PtMYB134 to reciprocally activate ANR promoters from both apple and poplar and to partner with heterologous bHLH co-factors from these plants confirms the high degree of conservation of PA regulatory complexes across species. The similarity in apple and poplar PA regulation suggests that regulatory genes from poplar could be effectively employed for metabolic engineering of the PA pathway in apple.

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

“…MYB134 and a GL3-like bHLH cofactor (bHLH079; see below) were not affected by MYB182 overexpression. However, MYB115, the MYBPA1-type positive PA regulator, and bHLH131, a PA synthesis cofactor of the TT8/DEL/ JAF13 group (Gesell et al, 2014), were affected in the MYB182-overexpressing hairy roots, and both showed reduced expression (Fig. 2D).…”

“…We had shown previously that the poplar ANR1 promoter is activated by MYB134 when cotransformed with the poplar bHLH131 cofactor into a transient expression system (Zifkin et al, 2012;Gesell et al, 2014). To show negative regulation by MYB182, a positive regulator such as MYB134 was used in combination with the MYB182 repressor construct, and the reduction in activation was measured.…”

“…1) and thus are predicted to interact directly with bHLH cofactors. To probe this interaction, we assayed repressor and activator constructs together with two poplar bHLHs belonging to different subclades: poplar bHLH131, which is part of the TT8 subclade (Gesell et al, 2014), and poplar bHLH079, which belongs to the JAF13/GL3 subclade (Supplemental Fig. S7).…”

“…3, A-E). While the light levels in such an experimental setup are difficult to control, the intensity of the light was not excessive, and we have used this treatment previously to reliably stimulate flavonoid metabolism (Mellway et al, 2009;Gesell et al, 2014). However, the transition from greenhouse conditions with no UV-B light penetration and lower intensity visible light to natural sunlight likely constitutes a mild stress treatment (see "Materials and Methods").…”

“…MYB134 is an activator of PA biosynthesis and stimulates the poplar ANR1 promoter in transient expression assays in Arabidopsis (Zifkin et al, 2012;Gesell et al, 2014). Transgenic poplar plants overexpressing MYB134 accumulate up to 50 times more PAs in their leaves, yet they have normal anthocyanin levels and only slightly elevated flavonol contents, suggesting that MYB134 regulates PAs specifically (Mellway et al, 2009).…”