Heterologous Expression of Platycodon grandiflorus PgF3′5′H Modifies Flower Color Pigmentation in Tobacco

Abstract

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 T0 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.