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 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.
Highlights
Pear (Pyrus L.) is one of the most common and popular fruits in the world, and red pears are favored by consumers for their beautiful appearance and abundant anthocyanins[1]
PyWRKY26 or PyWRKY31 with PyMYB114 and PybHLH3 were cotransformed into tobacco leaves and strawberry receptacles by a transient expression system
The results indicated that the gene Pbr013092.1 has the most similar predicted protein sequences with homologous genes among all anthocyanin-related genes by a detailed phylogenetic analysis, which included the transcription factors (TFs) AtWRKY4, FvWRKY44, NtWRKY2, PhPH3, PtWRKY1, and Pbr000122.1, which have high homology with AtWRKY6 (Fig. 1b)
Summary
Pear (Pyrus L.) is one of the most common and popular fruits in the world, and red pears are favored by consumers for their beautiful appearance and abundant anthocyanins[1]. Anthocyanins are widely present; these molecules are important flavonoids with multiple physiological functions that aid pollination, seed dispersal, and resistance to adverse environmental conditions[2]. Anthocyanins have significant antioxidant activity and potential benefits for human health, such as reducing the risk of cancer, inflammation, and coronary arteriosclerosis[3,4,5]. Anthocyanin biosynthesis is coordinately regulated by structural genes and TFs in many species. The structural genes consist of seven enzyme genes, from phenylalanine ammonia-lyase (PAL) to UDP-glucose: flavonoid 3-O-. Synthetic anthocyanins are transported and stored in vacuoles
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