The MdBBX22-miR858-MdMYB9/11/12 module regulates proanthocyanidin biosynthesis in apple peel.

Proanthocyanidins (PAs) have antioxidant properties and are beneficial to human health. The fruit of apple (Malus × domestica Borkh.), especially the peel, is rich in various flavonoids, such as PAs, and thus is an important source of dietary antioxidants. Previous research on the regulation of PAs in apple has mainly focussed on the transcription level, whereas studies conducted at the post-transcriptional level are relatively rare. In this study, we investigated the function of mdm-miR858, a miRNA with multiple functions in plant development, in the peel of apple fruit. We showed that mdm-miR858 negatively regulated PA accumulation by targeting MdMYB9/11/12 in the peel. During fruit development, mdm-miR858 expression was negatively correlated with MdMYB9/11/12 expression and PA accumulation. A 5'-RACE experiment, GUS staining assays and transient luminescent assays indicated that mdm-miR858 cleaved and inhibited the expression of MdMYB9/11/12. Overexpression of mdm-miR858 in apple calli, tobacco and Arabidopsis reduced the accumulation of PAs induced by overexpression of MdMYB9/11/12. Furthermore, we found that MdBBX22 bound to the mdm-miR858 promoter and induced its expression. Overexpression of MdBBX22 induced the expression of mdm-miR858 to inhibit the accumulation of PAs in apple calli overexpressing MdMYB9/11/12. Under light stress, MdBBX22 induced mdm-miR858 expression to inhibit PA accumulation and thereby indirectly enhanced anthocyanin synthesis in the peel. The present results revealed that the MdBBX22-miR858-MdMYB9/11/12 module regulates PA accumulation in apple. The findings provide a reference for further studies of the regulatory mechanism of PA accumulation and the relationship between PAs and anthocyanins.

MdBBX21, a B-Box Protein, Positively Regulates Light-Induced Anthocyanin Accumulation in Apple Peel.

The red coloration of apple (Malus × domestica Borkh.) is due to the accumulation of anthocyanins in the fruit peel. Light is essential for anthocyanin biosynthesis in apple. In this study, we performed a transcriptome sequencing (RNA-seq) analysis of apple fruit exposed to light after unbagging. The identified differentially expressed genes included MdBBX21, which is homologous to Arabidopsis BBX21, suggesting it may be involved in light-induced anthocyanin biosynthesis. Additionally, MdBBX21 was localized in the nucleus and its gene was expressed earlier than MdMYB1 in apple peel treated with light. Overexpressing MdBBX21 in Arabidopsis and apple calli under light increased anthocyanin accumulation. Dual-luciferase and yeast one-hybrid assays confirmed that MdBBX21 binds to the MdHY5, MdBBX20, and MdBBX22-1/2 promoters and induces expression. At the same time, MdHY5 can also activate the expression of MdBBX21. Furthermore, bimolecular fluorescence complementation and yeast two-hybrid assays demonstrated that MdBBX21 can interact with MdHY5. This interaction can significantly enhance MdMYB1 promoter activity. These findings clarify the molecular mechanism by which MdBBX21 positively regulates light-induced anthocyanin accumulation in apple.

mdm-miR828 Participates in the Feedback Loop to Regulate Anthocyanin Accumulation in Apple Peel.

Anthocyanins are responsible for the red pigmentation in the peel of apple (Malus × domestica Borkh.) fruit. Relatively few studies have investigated anthocyanins at the posttranscriptional level. MicroRNAs play an important role in plant growth and development by regulating gene expression at the posttranscriptional level. In this study, mdm-miR828 showed a relatively low expression level during the rapid fruit coloration period. However, the mdm-miR828 expression level increased in the late fruit coloration stage. Overexpression of mdm-miR828 inhibited anthocyanin synthesis in apple and Arabidopsis. Dual-luciferase and yeast one-hybrid assays showed that MdMYB1 is capable of binding to the promoter of mdm-MIR828b to promote its expression. The results indicate that mdm-miR828 is involved in a feedback regulatory mechanism associated with anthocyanin accumulation in apple. In addition, mdm-miR828 is involved in the inhibition of anthocyanin accumulation in response to high temperature.