Molecular characterization of a flavanone 3-hydroxylase gene from citrus fruit reveals its crucial roles in anthocyanin accumulation.

Background: Flavanone 3-hydroxylase (F3H), a key enzyme in the flavonoid biosynthetic pathway, plays an important role in the regulation of flavonols and anthocyanidins accumulation. Citrus fruit is a rich source of flavonoids with varied flavonoid compositions among different varieties. To date, the study on F3H is limited in citrus, and its roles in regulating flavonoid accumulation in citrus fruit are still unclear.

Results: In this study, we isolated a CitF3H from three different citrus varieties, Satsuma mandarin (Citrus unshiu Marc.), Ponkan mandarin (C. reticulata Blanco) and blood orange 'Moro' (C. sinensis Osbeck). Functional analysis showed that CitF3H encoded a functional flavanone 3-hydroxylase. It catalyzed the hydroxylation of naringenin to yield dihydrokaempferol, which was a precursor of anthocyanins in flavonoid biosynthetic pathway. In the juice sacs, CitF3H was differentially expressed among the three citrus varieties, and its expression level was positively correlated with the accumulation of anthocyanins during the ripening process. In the juice sacs of Satsuma mandarin and Ponkan mandarin the expression of CitF3H kept constant at an extremely low level, and no anthocyanin was accumulated during the ripening process. In contrast, the expression of CitF3H increased rapidly along with the accumulation of anthocyanin in the juice sacs of blood orange 'Moro' during the ripening process. In addition, we found that blue light irradiation was effective to up-regulate the expression of CitF3H and improve anthocyanin accumulation in the juice sacs of blood orange 'Moro' in vitro.

Conclusion: CitF3H was a key gene regulating anthocyanin accumulation in the juice sacs of citrus fruit. The results presented in this study will contribute to elucidating anthocyanin biosynthesis in citrus fruit, and provide new strategies to improve the nutritional and commercial values of citrus fruit.