Functional Analysis of Cytochrome b5 in Regulating Anthocyanin Biosynthesis in Malus domestica

Cytochrome b5 (CB5), a small heme-binding protein, plays an important role in plant biotic and abiotic stress. Anthocyanin is a critical determinant for fruit coloration, however, whether CB5 is involved in regulating anthocyanin biosynthesis has not yet been investigated in apple fruit (Malus domestica). In this study, we determined that MdCYB5, an apple CB5 gene, was a positive regulator for anthocyanin biosynthesis in apple fruit. We first found that MdCYB5 showed a high sequence and structural similarity with Arabidopsis cytochrome b5 isoform E (CB5E) at the protein level. Quantitative reverse transcription PCR (qRT-PCR) analysis showed that MdCYB5 responds to light signals. Subcellular localization showed that MdCYB5 is localized to the cytoplasmin inthe epidermal cells of Nicotiana benthamiana leaves. Further investigation revealed that overexpressing MdCYB5 promoted anthocyanin biosynthesis in both apple calli and tissue-cultured apple seedlings. Furthermore, results of transient expression assay showed that overexpressing MdCYB5 promoted anthocyanin accumulation and fruit coloration in apple fruit. Taken together, this study suggests that MdCYB5 has a positive regulatory effect on anthocyanin biosynthesis in apple fruit.