Mechanism of phytochrome signaling mediated by protein interaction

无柄植物和光自养植物已经发展出非常敏感和复杂的系统来识别适当的光条件，以确保它们的生存和成功繁殖。光敏色素是植物中一种主要的光感受器。然而，光敏色素介导的信号转导机制在很大程度上是未知的。由于光敏色素具有非常特殊的特性，在光照下表现出光可逆的构象变化，因此蛋白质相互作用可能是光敏色素信号转导的关键机制。为了研究蛋白质与光敏色素的相互作用，需要光敏色素与发色团的光活性。通过在单个操纵子中共同表达血红素加氧酶和各种胆色素还原酶，利用两种兼容的质粒，我们开发了一种在大肠杆菌中生产具有多种发色团的光敏色素的系统。细菌代谢工程生产各种胆磷脂组装成光敏色素将促进光感受器的分子分析。我们也培育了表达表位标记光敏色素的转基因植物。引入的融合基因对挽救phyya和phyB光敏色素突变体表型具有活性和功能。我们采用的另一种鉴定相互作用蛋白的方法是酵母双杂交试验。鉴定出一种新的含有锌指结构域的转录因子。通过体外表达的蛋白验证了相互作用对波长的特异性。由于敲除突变体表现出延迟开花表型，因此鉴定的蛋白质是介导光生物学反应的良好候选者。除了拟南芥研究外，我们还从玉米、豌豆和番茄中分离到了用于植物色素发色团生物合成的血红素加氧酶和光敏色素合成酶基因和相应的突变体。特别是，番茄AUREA蛋白在大肠杆菌中以可溶性形式表达。

Sessile and photoautotrophic plants have developed very sensitive and sophisticated system to recognize light conditions properly for their survival and successful propagation. Phytochrome is a major photoreceptor in plants. However, the mechanisms of signal transduction mediated by phytochrome is largely unknown. The protein interaction should be a key mechanism for signaling for phytochrome since phytochrome have very specific character showing photoreversible conformation changes by illumination. To study protein interaction with phytochrome, the photoactive phytochrome with chromophore is required. By co-expression of heme oxygenase and various bilin reductase(s) in a single operon in conjunction with apophytochrome using two compatible plasmids, we developed a system to produce phytochromes with various chromophores in Escherichia coli. The metabolic engineering of bacteria for the production of various bilins for assembly into phytochromes will facilitate the molecular analysis of photoreceptors. We also generated transgenic plants that express epitope-tagged phytochromes. The introduced fusion genes were active and functional to rescue phytochrome mutant phenotype of phyA and phyB. Another approach we took to identify interacting proteins was yeast two-hybrid assays. A novel transcription factor containing zinc finger domain was identified. The specificity of interaction for wavelength was verified by using expressed proteins in vitro. The identified protein is a good candidate to mediated photobiological response since the knock-out mutant showed delayed flowering phenotype.In addition to Arabidopsis works, we isolated several genes and corresponding mutants for heme oxygenase and phytochromobilin synthase for phytocrome chromophore biosynthesis from maize, pea and tomato. In particular, the tomato AUREA protein was expressed at higher levels in soluble form in E.coli.