AaMYB3 interacts with AabHLH1 to regulate proanthocyanidin accumulation in Anthurium andraeanum (Hort.)-another strategy to modulate pigmentation

Proanthocyanidins (PAs), also known as condensed tannins, are colorless metabolites produced through the flavonoid pathway that are involved in stress resistance in plants. Because PAs are involved in the anthocyanin biosynthetic pathway, they play a role in the modification of pigmentation conferred by anthocyanins in ornamental organs. In this study, we isolated the gene and functionally characterized an R2R3-MYB transcription factor (TF), AaMYB3, and a basic helix-loop-helix TF, AabHLH1, from Anthurium andraeanum (Hort.), a typical tropical flower. AaMYB3 is primarily expressed in the spathe and negatively correlates with anthocyanin accumulation. A complementation test in an Arabidopsis tt8 mutant showed that AabHLH1 successfully restores the PA-deficient seed coat phenotype. The ectopic overexpression of AaMYB3 alone or its coexpression with AabHLH1 in transgenic tobacco resulted in light pink or even pale-pink corolla limbs by reducing their anthocyanin levels and greatly enhancing their accumulation of PAs. This overexpression of the anthurium TF genes upregulated the late anthocyanin enzyme-encoding genes (NtDFR and NtANS) and the key PA genes (NtLAR and NtANR) in transgenic tobacco. The interaction between AaMYB3 and the AabHLH1 protein was confirmed using yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. In the developing red spathes of the cultivars Vitara and Tropical, the expression of AaMYB3 was closely linked to PA accumulation, and AaMYB3 was coexpressed with AaCHS, AaF3H, AaDFR, AGANS, AaLAR, and AaANR. The expression pattern of AabHLH1 was similar to that of AaF3'H. Our results suggest that AaMYB3 and AabHLH1 are involved in the regulation of PA biosynthesis in anthurium and could potentially be used to metabolically engineer PA biosynthesis in plants.