Temporal expression of flavonoid biosynthesis-related genes regulates flower pigmentation in gentian plants

The cDNA clones encoding homologues of flavanone 3-hydroxylase (F3H), anthocyanidin synthase (ANS), flavonoid 3′-hydroxylase (F3′H) and flavone synthase II (FSII) genes were isolated from petals of Gentiana triflora. Deduced amino acid sequences exhibited 60-80% identities with the corresponding sequences from other dicotyledonous species. Southern blot analysis showed that they were present as multiple copies in the gentian genome, and Northern blot analysis showed that the flavonoid biosynthesis-related genes could be classified into three groups by their temporal expression patterns during gentian flower development. The first included chalcone synthase (CHS) and chalcone isomerase (CHI) expressing during all flower development stages; the second included F3′H and FSII expressing at flower early developmental stages; and the third included F3H, flavonoid 3′,5′-hydroxylase (F3′,5′H), dihydroflavonol 4-reductase (DFR), ANS, UDP-glucose:flavonoid 3-O-glucosyltransferase (3GT) and anthocyanin 5-aromatic acyltransferase (5AT) expressing at flower late developmental stages. In general, low or undetectable levels of expression were observed in both the leaves and stems. High performance liquid chromatography (HPLC) analysis revealed that flavone accumulates from the early flower bud stage, but anthocyanin accumulation peaked at the later flower anthesis stage. A significant correlation between gene expression and pigment accumulation has been found, indicating that flavonoid biosynthesis during gentian flower development is regulated by temporal expression of these genes.