Summary: 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.

Peel browning is a natural phenomenon that adversely affects the appearance of fruits. Research on the regulation of browning in apples (Malus × domestica Borkh.) has mainly focused on post-harvest storage, while studies at the pre-harvest stage are relatively rare. Apple is an economically important horticultural crop prone to peel browning during growth, especially when the fruits are bagged (dark conditions). The present study’s integrated transcriptomics and metabolomics analysis revealed that pre-harvest apple peel browning was primarily due to changes in phenolics and flavonoids. The detailed analysis identified MdLAC7’s (laccase 7) role in the pre-harvest apple peel browning process. Transient injection, overexpression, and CRISPR/Cas9 knockout of the MdLAC7 gene in apple fruit and calli identified vallinic acid, anthocyanidin, tannic acid, sinapic acid, and catechinic acid as its catalytic substrates. In addition, yeast one-hybrid (Y1H) assay, electrophoretic mobility shift assay (EMSA), luciferase (LUC) reporter assay, and ChIP-PCR analysis revealed that MdWRKY31 binds to the promoter of MdLAC7 and positively regulates its activity to promote peel browning of bagged fruits (dark conditions). Interestingly, upon light exposure, the light-responsive transcription factor MdHY5 (ELONGATED HYPOCOTYL 5) bound to the promoter of MdWRKY31 and inhibited the gene’s expression, thereby indirectly inhibiting the function of MdLAC7. Subsequent analysis showed that MdHY5 binds to the MdLAC7 promoter at the G-box1/2 site and directly inhibits its expression in vivo. Thus, the study revealed the MdLAC7-mediated mechanism regulating pre-harvest apple peel browning and demonstrated the role of light in inhibiting MdLAC7 activity and subsequently reducing peel browning. These results provide theoretical guidance for producing high-quality apple fruits.

β-galactosidase (β-Gal), one of the cell wall modifying enzymes, plays an important role in fruit ripening and softening. However, its role in apple fruit texture remains unclear. In this study, the role of β-Gal was analyzed in two apple cultivars, ‘Fuji’ and ‘Qinguan,’ which are characterized by different fruit texture types, during fruit development and ripening. The firmness and pectin content of the fruits rapidly decreased and were much lower in ‘Fuji’ than in ‘Qinguan’ from 105 days after full bloom (DAFB). Transmission electron microscopy showed that the pectin-rich middle lamella was substantially degraded from 105 to 180 DAFB in the two apple cultivars. However, the degradation was more severe in ‘Fuji’ than in ‘Qinguan.’ Subcellular localization analysis showed that the Mdβ-Gal1, Mdβ-Gal2, and Mdβ-Gal5 proteins were located in the cell wall. β-Gal activity continuously increased during all fruit developmental stages and was much higher in the mature fruits of ‘Fuji’ than in those of ‘Qinguan,’ indicating that pectin was degraded by β-Gal. Consistent with the enzyme activities, expression levels of β-Gal genes (Mdβ-Gal1, Mdβ-Gal2, and Mdβ-Gal5) showed only slight changes from 60 to 105 DAFB but then dramatically increased until fruit ripening, with higher values in ‘Fuji’ than in ‘Qinguan.’ Furthermore, we found that activities of deletion derivatives in the Mdβ-Gal2 promoter and transcript level of Mdβ-Gal2 were induced by the treatment with methyl jasmonate (MeJA) and ethylene (ETH) hormones. Two ETH and one MeJA hormone-responsive elements were identified by analyzing the promoter sequence. These results suggest that β-Gals, induced by ETH and MeJA, are involved in different fruit texture types of apple cultivars by influencing the degradation of pectin during the mature fruit stage.