Mechanism of phytochrome signaling mediated by protein interaction

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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.