PyHAT5 regulates anthocyanin biosynthesis in red-fleshed pear and undergoes ubiquitination and degradation by PyAIP2

Red fruit flesh is a rare occurrence in pears (Pyrus spp.), yet it is an attractive trait as anthocyanin enrichment would add nutritional value and novelty for consumers. Previous research has focused on red-skinned phenotypes in pear fruit, but the potential regulatory mechanisms controlling red flesh remain unclear. Here, we identified the Homeodomain leucine zipper (HD-ZIP) family transcription factor PyHAT5 as a controlling factor using transcriptome analysis of pear fruit flesh at 3 different developmental stages in a hybrid population of red-fleshed and white-fleshed pears. The expression level of PyHAT5 was significantly negatively correlated (correlation coefficient of -0.94) with anthocyanin content in the flesh. Overexpression of PyHAT5 inhibited anthocyanin accumulation in pear tissues (skin and callus), peach (Prunus persica) flesh, and tobacco (Nicotiana tabacum) leaves, while virus-induced silencing of PyHAT5 promoted intense coloration of the pear skin. Further analysis found that PyHAT5 protein interacts with PyMYB10, thereby blocking the formation and transcriptional activity of the PyMYB10-PybHLH3 complex. Additionally, we identified through yeast 2-hybrid screening that an E3 ubiquitin ligase, PyAIP2, interacts with PyHAT5 and may promote its ubiquitination and degradation. Overexpression of PyAIP2 promoted anthocyanin biosynthesis in the pear skin, while knockdown of PyAIP2 had the opposite effect. Our findings reveal a regulatory module, PyAIP2-PyHAT5-PyMYB10, that plays a critical role in regulating anthocyanin biosynthesis in red-fleshed pear. These findings also advance our understanding of the regulation of anthocyanin biosynthesis in other tissues and provide genetic knowledge for advancing breeding in pear.