National Key Laboratory of Crop Biology, College of Horticulture Science and Engineering, Shandong Agricultural University, Tai-An, Shandong 271018, China.
State Key Laboratory of Crop Stress Biology for Arid Areas, College of Horticulture, Northwest A&F University, Yangling, Shaanxi 712100, China.
Sci Rep. 2017 Mar 20;7:44484. doi: 10.1038/srep44484.
In higher plants, jasmonate ZIM-domain (JAZ) proteins negatively regulate the biosynthesis of anthocyanins by interacting with bHLH transcription factors. However, it is largely unknown if and how other regulators are involved in this process. In this study, the apple MdJAZ2 protein was characterized in regards to its function in the negative regulation of anthocyanin accumulation and peel coloration. MdJAZ2 was used as a bait to screen a cDNA library using the yeast two-hybrid method. The hypersensitive induced reaction (HIR) proteins, MdHIR2 and MdHIR4, were obtained from this yeast two-hybrid. The ZIM domain of MdJAZ2 and the PHB domain of the MdHIR proteins are necessary for their interactions. The interactions were further verified using an in vitro pull-down assay. Subsequently, immunoblotting assays demonstrated that MdHIR4 enhanced the stability of the MdJAZ2-GUS protein. Finally, a viral vector-based transformation method showed that MdHIR4 inhibited anthocyanin accumulation and fruit coloration in apple by modulating the expression of genes associated with anthocyanin biosynthesis.
在高等植物中，茉莉酸 ZIM 结构域（JAZ）蛋白通过与 bHLH 转录因子相互作用，负调控花色苷的生物合成。然而，目前还不清楚是否以及如何有其他调控因子参与这一过程。本研究对苹果 MdJAZ2 蛋白的功能进行了研究，该蛋白在花色苷积累和果皮着色的负调控中发挥作用。MdJAZ2 被用作诱饵，通过酵母双杂交方法筛选 cDNA 文库。从该酵母双杂交中获得了过敏反应诱导蛋白 MdHIR2 和 MdHIR4。MdJAZ2 的 ZIM 结构域和 MdHIR 蛋白的 PHB 结构域是其相互作用所必需的。通过体外下拉实验进一步验证了它们的相互作用。随后，免疫印迹实验表明 MdHIR4 增强了 MdJAZ2-GUS 蛋白的稳定性。最后，基于病毒载体的转化方法表明，MdHIR4 通过调节与花色苷生物合成相关的基因的表达，抑制了苹果中花色苷的积累和果实着色。
