Li Chuang, Wu Jun, Hu Kang-Di, Wei Shu-Wei, Sun Hong-Ye, Hu Lan-Ying, Han Zhuo, Yao Gai-Fang, Zhang Hua
1School of Food and Biological Engineering, Hefei University of Technology, 230009 Hefei, China.
2Centre of Pear Engineering Technology Research, State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, 210095 Nanjing, China.
Hortic Res. 2020 Mar 15;7:37. doi: 10.1038/s41438-020-0254-z. eCollection 2020.
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 and were screened as candidate genes for controlling anthocyanin biosynthesis by transcriptome data and bioinformatics analysis. The effect of anthocyanin accumulations after cotransformation of or with its partners , , and 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 , , and in anthocyanin biosynthesis and in anthocyanin transport instead of the and . Furthermore, the cotransformed and could bind to the promoter, and directly activated the transcription of . 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 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.
红皮梨因其鲜亮的外观和丰富的花青素而受到青睐。花青素的生物合成由形成调控复合物的转录因子（TFs）控制。在红皮梨中，WRKY转录因子与花青素生物合成有显著关系，但WRKY转录因子参与调控红皮梨颜色形成的分子机制尚不清楚。在本研究中，通过转录组数据和生物信息学分析筛选出TFs 和 作为控制花青素生物合成的候选基因。通过瞬时表达系统在烟草叶片和草莓花托中验证了 或 与其伙伴 、 和 共转化后花青素积累的效果。RT-qPCR分析和双荧光素酶报告系统进一步证实，这种共转化激活了花青素生物合成中 、 和 的表达以及花青素转运中 的表达，而不是 和 的表达。此外，共转化的 和 可以与 启动子结合，并且 直接激活 的转录。此外，萤火虫荧光素酶互补和酵母双杂交（Y2H）试验证实，转录因子PyWRKY26可以与PybHLH3相互作用。这些结果表明，PyWRKY26和PybHLH3的相互作用可以共同靶向 启动子，从而导致红皮梨中花青素的积累。本研究进一步加深了对花青素积累调控机制的理解，并有助于改善红皮梨的外观。
