Expansion and subfunctionalisation of flavonoid 3',5'-hydroxylases in the grapevine lineage

Abstract

Background: Flavonoid 3',5'-hydroxylases (F3'5'Hs) and flavonoid 3'-hydroxylases (F3'Hs) competitively control the synthesis of delphinidin and cyanidin, the precursors of blue and red anthocyanins. In most plants,F3'5'Hgenes are present in low-copy number, but in grapevine they are highly redundant.Results: The first increase inF3'5'Hcopy number occurred in the progenitor of the eudicot clade at the time of the γ triplication. Further proliferation ofF3'5'Hs has occurred in one of the paleologous loci after the separation of Vitaceae from other eurosids, giving rise to 15 paralogues within 650 kb. Twelve reside in 9 tandem blocks of ~35-55 kb that share 91-99% identity. The second paleologousF3'5'Hhas been maintained as an orphan gene in grapevines, and lacks orthologues in other plants. DuplicateF3'5'Hs have spatially and temporally partitioned expression profiles in grapevine. The orphanF3'5'Hcopy is highly expressed in vegetative organs. More recent duplicateF3'5'Hs are predominately expressed in berry skins. They differ only slightly in the coding region, but are distinguished in the structure of the promoter. Differences incis-regulatory sequences of promoter regions are paralleled by temporal specialisation of gene transcription during fruit ripening. Variation in anthocyanin profiles consistently reflects changes in theF3'5'HmRNA pool across different cultivars. MoreF3'5'Hcopies are expressed at high levels in grapevine varieties with 93-94% of 3'5'-OH anthocyanins. In grapevines depleted in 3'5'-OH anthocyanins (15-45%), fewerF3'5'Hcopies are transcribed, and at lower levels. Conversely, only two copies of the gene encoding the competing F3'H enzyme are present in the grape genome; one copy is expressed in both vegetative and reproductive organs at comparable levels among cultivars, while the other is transcriptionally silent.Conclusions: These results suggest that expansion and subfunctionalisation ofF3'5'Hs have increased the complexity and diversification of the fruit colour phenotype among red grape varieties.