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

Abstract

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 <i>PyWRKY31</i> and <i>PyWRKY26</i> were screened as candidate genes for controlling anthocyanin biosynthesis by transcriptome data and bioinformatics analysis. The effect of anthocyanin accumulations after cotransformation of <i>PyWRKY31</i> or <i>PyWRKY26</i> with its partners <i>PyMYB10</i>, <i>PyMYB114</i>, and <i>PybHLH3</i> 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 <i>PyDFR</i>, <i>PyANS</i>, and <i>PyUFGT</i> in anthocyanin biosynthesis and <i>PyGST</i> in anthocyanin transport instead of the <i>PyABC transporter</i> and <i>PyAVP</i>. Furthermore, the cotransformed <i>PyWRKY26</i> and <i>PybHLH3</i> could bind to the <i>PyMYB114</i> promoter, and <i>PyWRKY26</i> directly activated the transcription of <i>PyMYB114</i>. 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 <i>PyMYB114</i> 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.