Molecular and Enzymatic Characterization of Flavonoid 3′-Hydroxylase of Malus × domestica

Malus × domestica (apple) accumulates particularly high amounts of dihydrochalcones in various tissues, with phloridzin (phloretin 2′-O-glucoside) being prevalent, although small amounts of 3-hydroxyphloretin and 3-hydroxyphloridzin are also constitutively present. The latter was shown to correlate with increased disease resistance of transgenic M. × domestica plants. Two types of enzymes could be involved in 3-hydroxylation of dihydrochalcones: polyphenol oxidases or the flavonoid 3′-hydroxylase (F3′H), which catalyzes B-ring hydroxylation of flavonoids. We isolated two F3′H cDNA clones from apple leaves and tested recombinant Malus F3′Hs for their substrate specificity. From the two isolated cDNA clones, only F3′HII encoded a functionally active enzyme. In the F3′HI sequence, we identified two putatively relevant amino acids that were exchanged in comparison to that of a previously published F3′HI. Site directed mutagenesis, which exchanged an isoleucine into methionine in position 211 restored the functional activity, which is probably because it is located in an area involved in interaction with the substrate. In contrast to high activity with various flavonoid substrates, the recombinant enzymes did not accept phloretin under assay conditions, making an involvement in the dihydrochalcone biosynthesis unlikely.

Poplar MYB117 promotes anthocyanin synthesis and enhances flavonoid B-ring hydroxylation by upregulating the flavonoid 3’,5’-hydroxylase gene

Flavonoids such as anthocyanins, proanthocyanidins and flavonols, are widespread plant secondary metabolites and important for plant adaptation to diverse abiotic and biotic stresses. Flavonoids can be variously hydroxylated and decorated; their biological activity is partly dependent on the degree of hydroxylation of the B-ring. Flavonoid biosynthesis is regulated by MYB transcription factors, which have been identified and characterized in a diversity of plants. Here we characterize a new MYB activator, MYB117, in hybrid poplar (Populus tremula x tremuloides). When overexpressed in transgenic poplar plants, MYB117 enhanced anthocyanin accumulation in all tissues. Transcriptome analysis of MYB117-overexpressing poplars confirmed the upregulation of flavonoid and anthocyanin biosynthesis genes, as well as two flavonoid 3’,5’-hydroxylase (F3'5'H) genes. We also identified upregulated cytochrome b5 genes, required for full activity of F3'5'H. Phytochemical analysis demonstrated a corresponding increase in B-ring hydroxylation of anthocyanins, proanthocyanidins and flavonols in these transgenics. Similarly, overexpression of F3'5'H1 directly in hybrid poplar also resulted in increased B-ring hydroxylation, but without affecting overall flavonoid content. However, the overexpression of cytochrome b5 gene in F3'5'H1-overexpressing plants did not further increase B-ring hydroxylation. Our data indicate that MYB117 regulates the biosynthesis of anthocyanins in poplar, but also enhances B-ring hydroxylation by upregulating F3'5'H1.