PyMYB10 and PyMYB10.1 Interact with bHLH to Enhance Anthocyanin Accumulation in Pears

Color is an important agronomic trait of pears, and the anthocyanin content of fruit is immensely significant for pear coloring. In this study, an anthocyanin-activating R2R3-MYB transcription factor gene, PyMYB10.1, was isolated from fruits of red sand pear (Pyrus pyrifolia cv. Aoguan). Alignments of the nucleotide and amino acid sequences suggested that PyMYB10.1 was involved in anthocyanin regulation. Similar to PyMYB10, PyMYB10.1 was predominantly expressed in red tissues, including the skin, leaf and flower, but it was minimally expressed in non-red fruit flesh. The expression of this gene could be induced by light. Dual-luciferase assays indicated that both PyMYB10 and PyMYB10.1 activated the AtDFR promoter. The activation of AtDFR increased to a greater extent when combined with a bHLH co-factor, such as PybHLH, MrbHLH1, MrbHLH2, or AtbHLH2. However, the response of this activation depended on the protein complex formed. PyMYB10-AtbHLH2 activated the AtDFR promoter to a greater extent than other combinations of proteins. PyMYB10-AtbHLH2 also induced the highest anthocyanin accumulation in tobacco transient-expression assays. Moreover, PybHLH interacted with PyMYB10 and PyMYB10.1. These results suggest that both PyMYB10 and PyMYB10.1 are positive anthocyanin biosynthesis regulators in pears that act via the formation of a ternary complex with PybHLH. The functional characterization of PyMYB10 and PyMYB10.1 will aid further understanding of the anthocyanin regulation in pears.

色泽是梨重要的农艺性状之一，果实花青素（anthocyanin）含量对梨果着色至关重要。本研究从红皮沙梨（*Pyrus pyrifolia* cv. Aoguan）的果实中克隆得到一个激活花青素合成的R2R3-MYB类转录因子（R2R3-MYB transcription factor）基因PyMYB10.1。核苷酸与氨基酸序列比对分析表明，PyMYB10.1参与花青素合成调控。与PyMYB10类似，PyMYB10.1主要在红色组织（果皮、叶片和花瓣）中表达，而在非红色的果肉中表达量极低。该基因的表达可被光诱导。双荧光素酶报告实验（dual-luciferase assays）结果显示，PyMYB10与PyMYB10.1均可激活AtDFR启动子。当与bHLH类辅因子（bHLH co-factor，如PybHLH、MrbHLH1、MrbHLH2或AtbHLH2）联合作用时，对AtDFR启动子的激活效果会显著增强。不过，该激活效应依赖于所形成的蛋白复合物类型。相较于其他蛋白组合，PyMYB10与AtbHLH2的组合对AtDFR启动子的激活效果最强。在烟草瞬时表达实验（tobacco transient-expression assays）中，PyMYB10-AtbHLH2组合也能诱导出最高的花青素积累量。此外，PybHLH可分别与PyMYB10和PyMYB10.1发生互作。上述结果表明，PyMYB10与PyMYB10.1均为梨中花青素生物合成的正向调控因子，其调控功能通过与PybHLH形成三元复合物得以实现。对PyMYB10与PyMYB10.1的功能解析，将有助于进一步解析梨中花青素合成的调控机制。