Heterologous Expression of Platycodon grandiflorus PgF35H Modifies Flower Color Pigmentation in Tobacco

Flavonoid-3 ',5 '-hydroxylase (F3 ' 5 ' H) is the key enzyme for the biosynthesis of delphinidin-based anthocyanins, which are generally required for purple or blue flowers. Previously, we isolated a full-length cDNA of PgF3 ' 5 ' H from Platycodon grandiflorus, which shared the highest homology with Campanula medium F3 ' 5 ' H. In this study, PgF3 ' 5 ' H was subcloned into a plant over-expression vector and transformed into tobacco via Agrobacterium tumefaciens to investigate its catalytic function. Positive transgenic tobacco T-0 plants were obtained by hygromycin resistance screening and PCR detection. PgF3 ' 5 ' H showed a higher expression level in all PgF3 ' 5 ' H transgenic tobacco plants than in control plants. Under the drive of the cauliflower mosaic virus (CaMV) 35S promoter, the over-expressed PgF3 ' 5 ' H produced dihydromyricetin (DHM) and some new anthocyanin pigments (including delphinidin, petunidin, peonidin, and malvidin derivatives), and increased dihydrokaempferol (DHK), taxifolin, tridactyl, cyanidin derivatives, and pelargonidin derivatives in PgF3 ' 5 ' H transgenic tobacco plants by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis, resulting in a dramatic color alteration from light pink to magenta. These results indicate that PgF3 ' 5 ' H products have F3 ' 5 ' H enzyme activity. In addition, PgF3 ' 5 ' H transfer alters flavonoid pigment synthesis and accumulation in tobacco. Thus, PgF3 ' 5 ' H may be considered a candidate gene for gene engineering to enhance anthocyanin accumulation and the molecular breeding project for blue flowers.