Identification and functional characterization of a new flavonoid synthase gene MdFLS1 from apple

Main conclusion The flavonoid synthase gene MdFLS1 from apple, which possibly plays an important role in anthocyanin synthesis, accumulates in the purple-red branches of Malus 'Pink spire'. Flavonoid metabolism serves an important function in plant growth and development. In this study, we selected 20 varieties of apple lines, 10 green and ten red branches, from the plant nursery of Qingdao Agriculture Academy. Metabolite analysis revealed that large amounts of anthocyanins accumulated in the purple-red branches of M. 'Pink spire'. Real-time polymerase chain reaction showed that the expression of the flavonol synthase gene MdFLS1 was over 1500-fold higher in M. 'Pink spire' than in the other varieties. A single base A was inserted at the first three bases of the active binding site of MdFLS1 to prove that the purple-red colour of apple leaves and stems in M. 'Pink spire' may be caused by the inactivation of MdFLS1 protein. The results of in vitro enzymatic reaction revealed that the MdFLS1 protein lost its activity. MdFLS1 was expressed in Arabidopsis thaliana to explore further its functions. High-expression wild-type strains (OE1 and OE2) and high-expression strains of A-base insertion (A-OE1 and A-OE2) were obtained. Compared with the wild-type strains, the overexpression lines showed lighter tissue colour and less accumulation of anthocyanins. However, A-OE1 and A-OE2 showed no difference in colouration. In conclusion, we speculated that the MdFLS1 gene in M. 'Pink spire' cannot bind flavonoids, triggering the synthesis of anthocyanins in another branch of the flavonoid metabolic pathway and resulting in the purple-red colouration of apple leaves and stems. These results suggest that MdLS1 is a potential genetic target for breeding high-flavonoid apples in future cultivar development.