Arabidopsis phytochrome A signal transduction: characterization and identifcation of cytoplasmic events and components

植物使用光敏色素作为红色/远红传感光感受器来监测其光环境并相应地调节发育的许多方面。光敏色素 A (phyA) 是拟南芥中的五种光敏色素之一，可调节早期幼苗发育以及其他反应。 phyA 激活后，易位至细胞核，调节基因表达。两种信号中间体，远红延长下胚叶醇 (FHY1) 和 FHY1 样 (FHL)，对于核易位至关重要，因为如果没有它们，细胞核中就检测不到 phyA。利用fhl/fhy1双突变体中唯一的细胞质phyA，先前可以揭示phyA的新细胞质功能。在后续项目中，进一步研究了phyA导入的调控和FHY1结合对phyA特性的影响，以及其他细胞质成分在早期phyA信号传导中的作用。从而详细研究了FHY1-phyA复合物转位至细胞核的机制。还研究了在具有功能性 phyA 的光依赖性酵母二杂交和三杂交测定中已经鉴定的潜在其他信号成分。从而发现了细胞质磷酸酶对phyA极低能量密度响应(VLFR)的影响。此外，还对异源表达的 phyA 与关键信号传导成分的相互作用进行了生化和生物物理分析。在该项目的拟议扩展中，将进一步研究易位过程中 FHY1 二次修饰的体内状态、phyA-FHY1 相互作用的结构基础以及发现的磷酸酶和小泛素样修饰剂 (SUMO) 连接酶的功能。拟议的项目将是与马尔堡埃森实验室的合作项目，该实验室将提供该项目的结构、生物物理和质谱任务，而蔡德勒实验室将致力于蛋白质相互作用、复杂成员的功能以及核易位的动力学和调节。拟议的项目将增进对这种光感受器功能的理解。核易位的调节在多种生物体中受到普遍关注，对受体的诱导性和定向易位的详细了解可能对于许多应用中基因表达的靶向操作有价值，甚至超出了植物领域。

Plants use phytochromes as red/far red sensing photoreceptors to monitor their light environment and regulate many aspects of development accordingly. Phytochrome A (phyA) is one of five phytochromes in Arabidopsis, regulating early seedling development among other responses. Following its activation, phyA translocates to the nucleus where it regulates gene expression. Two signaling intermediates, far red elongated hypocotol (FHY1) and FHY1-like (FHL), are crucial for nuclear translocation, as in their absence no phyA is detectable in the nucleus. Taking advantage of exclusively cytoplasmic phyA in the fhl/fhy1 double mutant, novel cytoplasmic functions of phyA could be revealed previously. In the follow-up project, the regulation of phyA import and the impact of FHY1 bindingon phyA properties, as well as the action of other cytoplasmatic components in early phyA signaling was further investigated. Thereby the mechanism by which the FHY1-phyA complex is translocated to the nucleus has been investigated in detail. Potential other signalling components which have already been identified in light-dependent yeast two- and three-hybrid assays with functional phyA have also been investigated. Thereby the influence of a cytoplasmic phosphatase on the phyA very low fluence response (VLFR) was discovered. Furthermore, heterologously expressed phyA wasbiochemically and biophysically analyzed concerning its interaction with the key signaling components. In the proposed extension of the project the in vivo status of FHY1s secondary modification during translocation, the structural basis of the phyA-FHY1 interaction and the function of the discovered phosphatase and small ubiquitin like modifier (SUMO) ligase will be investigated further. The proposed project will be a collaborative project with the Essen lab in Marburg which will provide the structural, biophysical and mass spectrometry tasks of the project while the Zeidler lab will work on Protein interaction, the function of the complex members and the kinetic and regulation of the nuclear translocation. The proposed project will advance the understanding of this photoreceptor’s function. The regulation of nuclear translocation is of general interest in a variety of organisms, and a detailed understanding of inducible and directional translocation of a receptor may prove valuable for targeted manipulations of gene expression in a number of applications,reaching beyond the plant field.