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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  AabHLH 1 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 ,  AaANS ,  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.

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