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.

中文翻译：

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MdBBX22–miR858–MdMYB9/11/12 模块调节苹果皮中原花青素的生物合成

原花青素 (PA) 具有抗氧化特性，对人体健康有益。苹果 ( Malus  ×  domestica Borkh.) 的果实，尤其是果皮，富含多种类黄酮，如 PAs，是膳食抗氧化剂的重要来源。以往对苹果PAs调控的研究主要集中在转录水平，而在转录后水平的研究相对较少。在本研究中，我们研究了苹果果皮中 mdm-miR858（一种在植物发育中具有多种功能的 miRNA）的功能。我们发现 mdm-miR858 通过靶向MdMYB9 / 11 / 12负调控 PA 积累在果皮中。在果实发育过程中，mdm-miR858表达与MdMYB9 / 11 / 12表达和PA积累呈负相关。5'-RACE 实验、GUS 染色测定和瞬时发光测定表明 mdm-miR858 切割并抑制MdMYB9 / 11 / 12的表达。苹果愈伤组织、烟草和拟南芥中 mdm-miR858 的过表达减少了由MdMYB9 / 11 / 12过表达诱导的 PA 的积累。此外，我们发现 MdBBX22 与 mdm-miR858 启动子结合并诱导其表达。MdBBX22的过表达诱导mdm-miR858的表达抑制过表达MdMYB9 / 11 / 12的苹果愈伤组织中PAs的积累。在光胁迫下，MdBBX22 诱导 mdm-miR858 表达以抑制 PA 积累，从而间接增强果皮中花青素的合成。目前的结果表明，MdBBX22–miR858– MdMYB9 / 11 / 12模块调节苹果中 PA 的积累。该研究结果为进一步研究PA积累的调控机制以及PAs与花青素的关系提供了参考。