PyWRKY26 and PybHLH3 cotargeted the PyMYB114 promoter to regulate anthocyanin biosynthesis and transport in red-skinned pears

Red pear is favored because of its bright appearance and abundant anthocyanins. Anthocyanin biosynthesis is controlled by transcription factors (TFs) forming regulatory complexes. In red-skinned pears, the WRKY TFs have a significant relationship with anthocyanin biosynthesis, but the molecular mechanism of the WRKY TFs involved in regulating color formation in red-skinned pear is unclear. In this study, the TFs PyWRKY31 and PyWRKY26 were screened as candidate genes for controlling anthocyanin biosynthesis by transcriptome data and bioinformatics analysis. The effect of anthocyanin accumulations after cotransformation of PyWRKY31 or PyWRKY26 with its partners PyMYB10, PyMYB114, and PybHLH3 was verified in tobacco leaves and strawberry receptacles by a transient expression system. RT-qPCR analysis and a dual-luciferase reporter system further confirmed that this cotransformation activated the expression of PyDFR, PyANS, and PyUFGT in anthocyanin biosynthesis and PyGST in anthocyanin transport instead of the PyABC transporter and PyAVP. Furthermore, the cotransformed PyWRKY26 and PybHLH3 could bind to the PyMYB114 promoter, and PyWRKY26 directly activated the transcription of PyMYB114. In addition, the TF PyWRKY26 could interact with PybHLH3, as confirmed by firefly luciferase complementation and yeast two-hybrid (Y2H) assays. These results showed that the interaction of PyWRKY26 and PybHLH3 could cotarget the PyMYB114 promoter, which resulted in anthocyanin accumulation in red-skinned pear. This study further strengthened the understanding of the regulatory mechanism of anthocyanin accumulation and contributed to improving the appearance of red-skinned pears.