Mechanism of phytochrome signaling mediated by protein interaction

固着植物和光自养植物已经发展出非常敏感和复杂的系统来正确识别光条件以使其存活和成功繁殖。光敏色素是植物中的主要光感受器。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。光敏色素具有特殊的光致可逆构象变化特性，蛋白质相互作用是光敏色素信号传导的重要机制。为了研究光敏色素与蛋白质的相互作用，需要光敏色素与发色团的结合。通过在一个操纵子中共表达血红素加氧酶和各种胆色素还原酶，并使用两个相容的质粒与脱辅基光敏色素一起，我们开发了一个在大肠杆菌中产生具有各种发色团的光敏色素的系统。利用细菌的代谢工程生产各种胆色素，组装成光敏色素，将有助于光感受器的分子分析。我们还产生了表达表位标记的光敏色素的转基因植物。导入的融合基因具有活性和功能，可拯救phyA和phyB的光敏色素突变表型。我们采用的另一种鉴定相互作用蛋白质的方法是酵母双杂交测定。鉴定了一种新的含锌指结构域的转录因子。利用体外表达的蛋白质验证了相互作用对波长的特异性。除了拟南芥的工作外，我们还从玉米、豌豆和番茄中分离了血红素加氧酶和植物色素合成酶的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以较高水平表达。

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.