School of Food and Biological Engineering, Hefei University of Technology, Hefei 230009, China.
Shandong Institute of Pomology, Tai'an 271000, China.
Plant Physiol. 2023 Jul 3;192(3):1892-1912. doi: 10.1093/plphys/kiad068.
Red-skinned pears (Pyrus L.) are preferred to consumers for their attractive color and abundant anthocyanins. Pyrus ETHYLENE RESPONSE FACTOR 3 (PyERF3) positively regulates anthocyanin biosynthesis through interacting with Pyrus myeloblastosis family 114 (PyMYB114) and Pyrus basic helix-loop-helix 3 (PybHLH3) in red-skinned pears. However, the role of APETALA2/ethylene response factors (AP2/ERFs), which negatively regulate anthocyanin biosynthesis, remains unclear in red-skinned pears. Here, we validated that 2 AP2/ERFs, PyERF4.1 and PyERF4.2, screened from the transcriptome data of 'Starkrimson' pear (Pyrus communis L.) and its green mutant, inhibit anthocyanin biosynthesis in transgenic pear calli, as well as in overexpression and gene-edited tomato (Solanum lycopersicum) fruits. Meanwhile, the co-transformation of PyERF4.1/PyERF4.2 with PyERF3-PyMYB114-PybHLH3 inhibited anthocyanin biosynthesis in pear fruits and strawberry (Fragaria vesca) receptacles. Further assays showed that PyMYB114 activated the transcription of PyERF4.1/PyERF4.2; PyERF4.1/PyERF4.2 then interacted with PyERF3 to affect the stability of the PyERF3-PyMYB114-PybHLH3 complex, thereby inhibiting the transcription of the anthocyanin biosynthesis gene Pyrus anthocyanidin synthase (PyANS). Furthermore, deletion of the ERF-associated-amphiphilic repression (EAR) motif eliminated the inhibitory effect of PyERF4.1/PyERF4.2 on anthocyanin biosynthesis, and a mutation of the PyERF4.2-EAR motif (LxLxM to LxLxL) strengthened the inhibitory effect, demonstrating that the EAR motif is indispensable for the inhibitory effect of PyERF4.1/PyERF4.2 on anthocyanin biosynthesis in pears. Our study has shed light on a feedback regulatory loop mechanism that balances the excessive accumulation of anthocyanins in red-skinned pears, providing insights into the regulatory mechanism of anthocyanin biosynthesis and the regulatory network of coloration in red-skinned pears.
红皮梨（Pyrus L.）因其诱人的颜色和丰富的花青素而受到消费者的喜爱。在红皮梨中，梨乙烯响应因子 3（PyERF3）通过与梨髓细胞瘤家族 114（PyMYB114）和梨碱性螺旋-环-螺旋 3（PybHLH3）相互作用，正向调控花青素生物合成。然而，APETALA2/乙烯响应因子（AP2/ERFs）在红皮梨中对花青素生物合成的负调控作用尚不清楚。在这里，我们从‘Starkrimson’梨（Pyrus communis L.）及其绿色突变体的转录组数据中筛选出 2 个 AP2/ERFs，PyERF4.1 和 PyERF4.2，证实它们在转基因梨愈伤组织以及过表达和基因编辑的番茄（Solanum lycopersicum）果实中抑制花青素生物合成。同时，PyERF4.1/PyERF4.2 与 PyERF3-PyMYB114-PybHLH3 的共转化抑制了梨果实和草莓（Fragaria vesca）托盘中的花青素生物合成。进一步的实验表明，PyMYB114 激活了 PyERF4.1/PyERF4.2 的转录；PyERF4.1/PyERF4.2 随后与 PyERF3 相互作用，影响 PyERF3-PyMYB114-PybHLH3 复合物的稳定性，从而抑制花青素生物合成基因 Pyrus anthocyanidin synthase（PyANS）的转录。此外，缺失 ERF 相关的两亲性抑制（EAR）基序消除了 PyERF4.1/PyERF4.2 对花青素生物合成的抑制作用，而 PyERF4.2-EAR 基序（LxLxM 到 LxLxL）的突变则增强了抑制作用，表明 EAR 基序是 PyERF4.1/PyERF4.2 抑制梨中花青素生物合成所必需的。我们的研究揭示了一个反馈调节环机制，该机制平衡了红皮梨中花青素的过度积累，为花青素生物合成的调控机制和红皮梨颜色调控网络提供了新的见解。
